Research Article NLRP3 Inflammasome Activation in the Brain...

Research ArticleNLRP3 Inflammasome Activation in the Brain after GlobalCerebral Ischemia and Regulation by 17120573-Estradiol

Roshni Thakkar1 Ruimin Wang1 Gangadhara Sareddy2 Jing Wang1

Dharma Thiruvaiyaru3 Ratna Vadlamudi2 Quanguang Zhang1 and Darrell Brann1

1Department of Neuroscience and Regenerative Medicine Medical College of Georgia Augusta University Augusta GA USA2Department of Obstetrics and Gynecology University of Texas Health Science Center San Antonio TX USA3Department of Mathematics Augusta University Augusta GA USA

Correspondence should be addressed to Quanguang Zhang qzhangaugustaedu and Darrell Brann dbrannaugustaedu

Received 30 June 2016 Revised 23 August 2016 Accepted 7 September 2016

Academic Editor Marta C Monteiro

Copyright copy 2016 Roshni Thakkar et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

17120573-Estradiol (E2) is a well-known neuroprotective factor in the brain Recently our lab demonstrated that the neuroprotective andcognitive effects of E2 requiremediation by the estrogen receptor (ER) coregulator protein and proline- glutamic acid- and leucine-rich protein 1 (PELP1) In the current study we examined whether E2 acting via PELP1 can exert anti-inflammatory effects in theovariectomized rat and mouse hippocampus to regulate NLRP3 inflammasome activation after global cerebral ischemia (GCI)Activation of the NLRP3 inflammasome pathway and expression of its downstream products cleaved caspase-1 and IL-1120573 wererobustly increased in the hippocampus after GCI with peak levels observed at 6-7 days Expression of P2X7 receptor an upstreamregulator of NLRP3 was also increased after GCI E2 markedly inhibited NLRP3 inflammasome pathway activation caspase-1and proinflammatory cytokine production as well as P2X7 receptor expression after GCI (at both the mRNA and protein level)Intriguingly the ability of E2 to exert these anti-inflammatory effects was lost in PELP1 forebrain-specific knockoutmice indicatinga key role for PELP1 in E2 anti-inflammatory signaling Collectively our study demonstrates that NLRP3 inflammasome activationand proinflammatory cytokine production are markedly increased in the hippocampus after GCI and that E2 signaling via PELP1can profoundly inhibit these proinflammatory effects

1 Introduction

The steroid hormone 17120573-estradiol (E2) is produced pri-marily by the ovaries in females and has multiple actionsthroughout the body and brain In the brain there is a signif-icant body of work demonstrating that E2 is neuroprotectivein both acute and chronic neurodegenerative disorders suchas cerebral ischemia traumatic brain injury Alzheimerrsquosdisease and Parkinsonrsquos disease [1ndash4] For instance in 1997Simpkins et al demonstrated for the first time that E2was neuroprotective in an ovariectomized rat focal cerebralischemia (FCI) model [5] Since then numerous studies haveconfirmed the neuroprotective effects of E2 in both focaland global cerebral ischemia (GCI) models and implicatedboth genomic and extranuclear signaling mechanisms inmediation of the E2 effects [4 6ndash11] In the brain the neuro-protective actions of E2 have been implicated to be mediated

by the classical estrogen receptors estrogen receptor-120572 (ER-120572) and estrogen receptor-120573 (ER-120573) [4 9] However there isalso evidence that a newputative ER calledG-protein coupledreceptor 1 (GPER1) may also contribute to neuroprotection[6 12 13]

Upon E2 binding to ER several coregulatory proteinsassociate with the ER to regulate E2 transcriptional activityThe ER-coregulator protein association leads to formation ofan ldquoER signalosomerdquo which facilitates both ER genomic andextranuclear actions [14] Recent work by our group led tothe cloning and characterization of a novel ER coregulatorcalled proline- glutamic acid- and leucine-rich protein 1(PELP1) [15 16] PELP1 is amultidomain scaffold protein thatcan interact with ERs using the nuclear receptor interactionmotif LXXLL and with Src kinase and PI3K kinase usingSH2 and PXXPmotifs [16] PELP1 is expressed in a variety oftissues with highest expression in the brain ovaries testes

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 8309031 17 pageshttpdxdoiorg10115520168309031

2 Oxidative Medicine and Cellular Longevity

and uterus [16ndash20] In addition PELP1 exhibits both nuclearand cytoplasmic localization [20] and phosphorylation ofPELP1 can control its localization interaction with otherproteins and stability in cells [16 17] To understand itsimportance in E2 actions in the brain our group recentlycreated a PELP1 forebrain-specific knockout (PELP1 FBKO)mouse model [16] Using the PELP1 FBKO mouse modelwe demonstrated a critical role for PELP1 in mediating E2regulation of extranuclear and genomic signaling as well asE2-induced neuroprotection and cognitive function in thehippocampus following ischemic injury [16]

While the role of E2 as a neuroprotective factor has beenwell established its role in regulation of neuroinflamma-tion has been less studied Growing evidence suggests thatneuroinflammation can contribute significantly to neuronaldamage and cell death in neurodegenerative disorders andthat attenuation of neuroinflammation can be neuropro-tective [21ndash23] With respect to E2 several studies haveshown that it can suppress microglial activation and proin-flammatory cytokine production in vivo and in vitro [24ndash28] although the mechanisms underlying the effects remainunclear A significant advance to the neuroinflammation fieldoccurred in 2002 when Martinon and coworkers identifiedldquoinflammasomesrdquo as key proteins that trigger the inflamma-tory response [29] Inflammasomes are multiprotein com-plexes that mediate activation of caspase-1 and promotesecretion of proinflammatory cytokines Inflammasomes areactivated in response to either pathogen associatedmolecularpatterns (PAMPs) derived from invading pathogens ordamage-associated molecular patterns (DAMPs) released bydying cells [30] Inflammasomes belong to the NOD-likereceptor family (NLRs) NLRs are encoded by a family of 22genes and are mainly divided as NLRP (NOD-like receptorwith a pyrin domain) and NLRC (NOD-like receptors withCARD domain) family members [31]

The structure of NLRP includes a carboxy-terminalleucine rich repeat (LRR) a nucleotide binding domaincalled the NACHT domain and an N-terminal pyrin domain(PYD) The PYD domain of inflammasome binds to a PYDdomain of apoptosis associated speck like protein (ASC)which is an adaptor protein [31] ASC also has a caspaseactivation and recruitment (CARD) domain which binds tothe CARD domain of the procaspase-1 enzyme NLRP3 isthemost abundantly found inflammasome and contributes tothemajority of production of proinflammatory cytokines [2331 32] Inflammasomes are primed and activated by triggerslike pathogens and metabolic and genotoxic stressors Forinstance the P2X7 receptor is activated by elevation ofextracellular ATP which then leads to activation of theNLRP3 inflammasome complex [33] Once activated inflam-masomes enhance downstream activation of caspase-1 andrelease of proinflammatory cytokines such as IL-1120573 and IL-18[29 34] which initiate a cascade of detrimental inflammatoryevents and apoptosis [35]

Of all of the inflammasome proteins NLRP3 is byfar the most studied inflammasome in cerebral ischemiastudies NLPR3 expression has been described in multiplebrain cell types including astrocytes microglia neuronsand endothelial cells [22 36ndash39] Numerous studies have

shown that NLRP3 is elevated in the brain of human strokepatients [40] and experimental stroke animals [40ndash44]Furthermore NLRP3 appears to have an important role inischemic pathology as NLRP3 knockout animals have signif-icantly reduced infarct size and neurovascular damage afterfocal cerebral ischemia [38] Additionally immunoglobulintreatment suppresses NLRP3 activity and strongly protectsneurons in ischemic animals [40] In addition Aim2 andNLRC4 knockout animals have also been reported to havereduced inflammation and infarct size after focal cerebralischemia but the effects appear to be independent of IL-1120573production [45]

With respect to E2 regulation of NLRP3 inflammasomeactivation E2 has been reported in one study to suppressNLRP3 inflammasome gene expression in the cerebral cortexafter focal cerebral ischemia (FCI) [46] while in a secondstudy an ER-120573 agonist suppressed caspase-1 ASC and IL-1120573expression in the hippocampus after global cerebral ischemia(GCI) [47]These studies suggest that E2 can regulate inflam-masome activation however several important questionsremain unanswered including the following (1) What isthe temporal pattern of NLRP3 inflammasome expressionin the hippocampus after GCI (2) In which cell type inthe hippocampus does NLRP3 inflammasome activationoccur (3) Does E2 only affect expression of the NLRP3inflammasome or does it also regulate NLRP3 inflammasomecomplex formation (4)What are themechanisms underlyingE2 regulation of the NLRP3 inflammasome (5) Does theER coregulator PELP1 mediate E2 regulation of the NLRP3inflammasome in the brain The goal of the current studywas to address these key questions The results of the studyreveal that both expression and complex formation of theNLRP3 inflammasome as well as activated caspase-1 and IL-1120573 are robustly increased in the hippocampus of both therat and mouse after GCI and this effect is strongly inhibitedby E2 replacement The results also demonstrate that NLRP3inflammasome activation occurs in bothmicroglia and astro-cytes after GCI and that E2 inhibits expression of both P2X7and TXNIP two well-known upstream inducers of NLRP3inflammasome activation Finally the results reveal that theER coregulator protein PELP1 is essential for mediation of E2regulatory effects upon activation of the NLRP3 inflamma-some

2 Materials and Methods

21 Animals and Surgical Procedures

211 OvariectomizedRat Studies AugustaUniversity Institu-tional Animal Care and Use Committee approved all animalprocedures and the studies were conducted in accordancewith National Institutes of Health guidelines for animalresearch Three-month-old young adult Sprague Dawley ratswere bilaterally ovariectomized under isoflurane anesthesiaand separated into shams ischemia-reperfusion injury andinjury with estrogen (E2) treatment groupsThe E2 treatmentgroup animals were immediately administered with 17120573-estradiol dissolved in 20 120573-cyclodextrin added to mini-pumps (05 120583Lhr 14-day release Alzet Cupertino CA)

Oxidative Medicine and Cellular Longevity 3

Pumps were placed in the upper mid-back region to allowsubcutaneous administration of E2 The dose of E2 used ledto production of physiological Diestrus I levels of circulatingE2 (10ndash15 pgmL) All rats except for sham controls weresubjected to global cerebral ischemia (GCI) via 4-vesselocclusion method [48] after 7 days of ovariectomy Oneday prior to occlusion that is 6 days after ovariectomy allanimals were anesthetized using ketaminexylazine and theirvertebral arteries were electrocauterized and the commoncarotid arteries were exposed Twenty-four hours later thecommon carotid arteries were transiently occluded withhemostatic clips for 12 minutes for all animals except theshams Sham animals had their arteries exposed but notoccluded Ischemia-reperfusion was allowed to occur andanimals were sacrificed using transcardial perfusion anddecapitation at 1 3 and 7 days after GCI

212 PELP1 Forebrain-Specific Knockout Mouse StudiesYoung adult femaleC27BL6PELP1 forebrain-specific knock-outmice were generated as described by our group previously[16] FLOX control as well as PELP1 knockout mice werebilaterally ovariectomized and implanted with either placeboor 17120573-estradiol (E2) subcutaneous mini-pumps immedi-ately following ovariectomy After 7 days of ovariectomyall animals except the shams were subjected to two-vesselocclusion for global cerebral ischemia (GCI) Animals wereanesthetized brieflywith ketaminexylazine and the two com-mon carotid arteries were exposed and occluded transientlywith hemostatic clips for 40minutes Sham animals had theirarteries exposed but not occluded All animals were sacrificedusing transcardial perfusion at 6 days after GCI

22 Tissue Collection All animals were transcardially per-fused and decapitated at the desired time point after GCIBrains were dissected in the midsagittal plane and fixedin 4 paraformaldehyde for 24 hours cryoprotected in30 sucrose and sectioned on a cryostat to obtain 20-micron-thick hippocampal sections These sections werethen used for immunofluorescence staining and proximityligation assay (Duolink) For RT-PCR and Western blotanalysis brains were collected and the hippocampal tissuewas dissected out frozen and either processed for RNAisolation via one-step RT-PCR or homogenized for detectionof proteins via Western blot analysis as described below

23 RT-PCR Hippocampal tissue sampleswere collected andRNA was isolated using the SV total RNA isolation system(Promega)The RNAwas then used for the reverse transcrip-tase PCR reaction using the Superscript III one-step RT-PCRsystem with platinum Taq DNA Polymerase (Invitrogen) andthe primers listed in Table 1 (Integrated DNA Technologies)The gene expression analyses were done using the compara-tive ΔΔCt method The mRNA level changes were expressedas fold change as compared to the sham animals All Ct valuesfor target genes were normalized to CypA gene [49]

24 Western Blot Analysis Hippocampal tissue was collected7 days afterGCI asmentioned above Individual hippocampal

sample from each animal was homogenized in RIPA bufferusing silica beads and a Mini-Beadbeater (Biospec ProductsOKUSA)Thehomogenatewas centrifuged at 13000 rpm for10 minutes at 4∘C and the supernatant was used for proteinestimation by Lowryrsquos Assay (Lowryrsquos Assay Kit Sigma) Fiftymicrograms of protein for each sample was separated on 15sodium dodecyl sulfate-polyacrylamide gel electrophoresistransferred on nitrocellulose membrane and blocked with5 bovine serum albumin for 1 hour at room temperaturewith gentle shaking Blocking was followed by incubationwith primary antibodies NLRP3 (Santa-Cruz Biotechnologysc-34408) ASC (Santa-Cruz Biotechnology sc-22514-R) cl-caspase-1 (Santa-Cruz Biotechnology sc-22165) and IL-1120573(Abcam ab9722) overnight at 4∘Cwith gentle shaking Glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH Santa-Cruz Biotechnology sc-32233) was used as a loading controlThe membrane was then washed with 1x TBST to removeunbound primary antibody and incubated with secondaryAlexa Fluor 680 or 800 anti-rabbitgoatmouse IgG for 1hour at room temperature with gentle shaking Blots werescanned usingOdyssey Imaging System (LI-CORBioscienceLincoln NB) The intensity of bands was quantified usingImageJ software The immunoblot data was corrected forcorresponding GAPDH values and presented as fold changein protein as compared to sham animals

25 Immunofluorescence Staining and Confocal MicroscopyAnalysis Twenty 120583m thick coronal sections were washedwith PBS and 04Triton-X PBS for 20minutesThe sectionswere then blocked with 10 normal donkey serum for 1 hourat room temperature in PBS containing 01 Triton X-100followed by incubation with primary antibody for 1ndash3 nightsat 4∘C in the same buffer Primary antibodies used for thisstudy included rabbit-NLRP3 (Santa-Cruz Biotechnology sc-66846) rabbit-ASC (Santa-Cruz Biotechnology sc-22514-R)goat-cleaved caspase-1 (Santa-Cruz Biotechnology sc-22165)rabbit-IL-1120573 (Abcam ab9722) goat-Iba1 (Abcam ab5076)rabbit-P2X7 receptor (Sigma P8232) and mouse-NeuN(Millipore MAB377) After primary antibody incubationsections were washed for 3 times 10 minutes at room temperature(RT) followed by incubation with the appropriate secondaryantibody Alexa-Fluor488568647 donkey anti-rabbitanti-mouseanti-goat (Invitrogen) RT1 hour Sections were thenwashedwith PBS containing 01Triton X-100 for 3times 10minfollowed by 2 times 5min with 1x PBS and briefly with waterThe sections were then mounted with water-based mountingmedium containing antifading agents and observed usingconfocal microscopy All images were captured on a confocallaser microscope (Carl Zeiss Germany) using the Zen soft-ware at 40x magnification and 50 120583m scale bar

26 Duolink Proximity Ligation Assay Tissue sections wereblocked in 5 (volvol) donkey serum for 1 hour at roomtemperature and incubated overnight with primary anti-bodies goat-NLRP3 (Santa-Cruz Biotechnology sc-34408)and rabbit-ASC (Santa-Cruz Biotechnology sc-22514-R) at4∘C These sections were then incubated with Duolink PLAprobes anti-Rabbit MINUS (Sigma-Aldrich DUO92005)


Table 1

Gene Forward primer Reverse primerNLRP3 51015840 AGAAGCTGGGGTTGGTGAATT 31015840 51015840 GTTGTCTAACTCCAGCATCTG 31015840

ASC 51015840 CCCATAGACCTCACTGATAAAC 31015840 51015840 AGAGCATCCAGCAAACCA 31015840

Caspase-1 51015840 AGGAGGGAATATGTGGG 31015840 51015840 AACCTTGGGCTTGTCTT 31015840

IL-1120573 51015840 TGACCCATGTGAGCTGAAAG 31015840 51015840 AGGGATTTTGTCGTTGCTTG 31015840

TXNIP 51015840 CTGATGGAGGCACAGTGAGA 31015840 51015840 CTCGGGTGGAGTGCTTAG 31015840

P2rX7 51015840 CTACTCTTCGGTGGGGGCTT 31015840 51015840 AACCCTGGTCAGAATGGCAC 31015840

CypA 51015840 TATCTGCACTGCCAAGACTGAGTG 31015840 51015840 CTTCTTGCTGGTCTTGCCATTCC 31015840

and anti-goat PLUS (Sigma-Aldrich DUO92003) for 1 h at37∘C Ligation and amplification were carried out at 37∘Cusing the Duolink in situ detection reagent kit (Sigma-Aldrich DUO92008) according to the manufacturerrsquos proto-col All sections were then mounted on a slide and all imageswere captured on a confocal laser microscope (Carl ZeissGermany) using the Zen software at 40x magnification and50 120583m scale bar

27 Antibody Specificity in Immunofluorescence StainingSpecificity of inflammasome pathway factor antibodies usedin immunofluorescence staining was tested by preabsorptionwith the antigen peptide or recombinant protein (IL-1120573)available from the manufacturer For preabsorption fivefoldconcentration of each peptide NLRP3 ASC cleaved caspase-1 and IL-1120573was incubatedwith primary antibody (at concen-trations used for immunofluorescence staining) overnight at4∘CThe immunofluorescence staining (withDAPI) was thenperformed as described above and images were captured ona confocal laser microscope (Carl Zeiss Germany) using theZen software at 40x magnification and 50 120583m scale bar

28 Quantification of Confocal Images The intensity of allconfocal images captured at 40xmagnificationwas quantifiedusing MATLAB software (version R2013a by MathworksNatick MA USA) as described previously [50] MATLAB isa programming environment with built-in image processingtools The intensity threshold for injured animals was identi-fied by applying amultilevel image threshold algorithm usingOtsursquos method in MATLAB [50] This value was then usedas an intensity threshold for sham and E2-treated animalsThe algorithm digitized each image into a 1024 times 1024matrixThe individual values contained in the matrix representedthe intensity value of pixels of a particular color that is redblue or green Using the threshold value obtained from thealgorithm the image was segmented into two regions oneabove the threshold value and one below Finally dividing thesegmented area with intensity above the threshold value bythe total image area enabled image quantification The datawere obtained as relative area of fluorescence as comparedto the entire area of the image The data was expressedas percentage of area activated in the entire captured fieldThe quantification of immunofluorescence images was alsoconfirmed by using ImageJ intensity analysis

29 Statistical Analysis The independent two-sample 119905-testwas conducted to investigate whether the mean fold changefor mRNA of factors under study for each of the three timepoints (1 3 and 7 days)was significantly different between theischemic and the estrogen-treated ratsThe one-way ANOVAtest was conducted to investigatewhether there is a significantdifference among sham ischemic and E2-treated rats onday 7 after injury for all proteins under study The two-wayANOVA test was conducted to investigate whether there is asignificant difference among sham ischemic and E2-treatedgroups of FLOX control versus the PELP1 KO mice as wellas whether there is a significant interaction between group(sham ischemic and E2) and type (FLOX and KO) for allmolecules under study Whenever the difference for either ofthe ANOVA tests was found to be significant post hoc testssuch as Bonferronirsquos test or Tamhanersquos test was conducted tomake pairwise comparisons of the groups of animals All testswere conducted at a 5 level of significance using the IBMSPSS software (version 23)

3 Results

31 Temporal Expression of NLRP3 Inflammasome PathwayFactors in theHippocampus after GCI andRegulation by Estra-diol We first examined the temporal expression of NLRP3inflammasome pathway factors and their regulation by E2in the hippocampus after GCI The first approach involveddetermining the temporal pattern of gene expression ofinflammasome pathway molecules NLRP3 ASC caspase-1and IL-1120573 As shown in Figure 1 mRNA levels for thesemark-ers were detected using RT-PCR analysis from hippocampustissue collected at days 1 3 and 7 after GCI The data arerepresented in terms of fold change as compared to the shamanimals that did not undergo ischemia The mRNA levelsfor NLRP3 (Figure 1(a)) were increased 2-fold and 15-fold atdays 1 and 3 respectively after injury E2 treatment stronglysuppressed NLRP3 gene expression at both days 1 and 3 afterinjury Likewise ASC mRNA levels were strongly elevated atdays 1 3 and 7 afterGCI and this effectwas strongly inhibitedby E2 treatment (Figure 1(b)) Caspase-1 mRNA levels wereincreased 2-fold at day 1 after injury and were significantlysuppressed by E2 (Figure 1(c)) However at days 3 and 7after GCI E2 paradoxically increased caspase-1mRNA levelsIt should be noted that the caspase-1 mRNA measured inthis study represents expression of the procaspase-1 and


1 3 7(Day)

GCI

NLRP3 mRNAlowastlowast

lowastlowast

0

05

1

15

2

25N

LRP3

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(a)

ASC mRNA

lowastlowast lowastlowast

lowastlowast

1 3 7(Day)

GCI

0

05

1

15

2

25

3

35

4

ASC

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(b)

Caspase-1 mRNA

lowastlowast

lowastlowast

lowastlowast

1 3 7(Day)

GCI

0

05

1

15

2

25

Casp

ase-

1 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

） + 2

(c)

lowastlowast

lowastlowastlowast

1 3 7(Day)

GCI

IL-1 mRNA

0

5

10

15

20

25

30

35

IL-1

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(d)

Figure 1 Temporal pattern of NLRP3 ASC caspase-1 and IL-1120573 gene expression after GCI and their suppression by E2 treatment mRNAsamples were collected from the hippocampus of adult female ovariectomized rats with andwithout E2 at various times afterGCI (a)NLRP3(b) ASC (c) caspase-1 and (d) IL-1120573 are all expressed temporally after GCI and their expression is robustly suppressed by E2 treatment atearly time points after GCI (lowastlowast119901 le 00001 lowast119901 lt 005 GCI versus GCI + E2) (119899 = 5-6 animals per group)

that cleavage of the procaspase-1 protein is required for itsactivationThe effects of GCI and E2 upon the cleaved (activeformof) caspase-1 are described below in a subsequent figureFinally IL-1120573 mRNA levels showed very high inductionin the hippocampal CA1 region after GCI with an 8ndash27-fold increase on days 1 3 and 7 after GCI (Figure 1(d))Similar to the results for the other NLRP3 inflammasomefactors E2 treatment robustly suppressed IL-1120573mRNA levels

at all three days after GCI We next examined GCI and E2regulation of protein levels of the NLRP3 inflammasomepathway molecules in the hippocampus using Western blotanalysis Representative Western blot results are shown inFigures 2(a)ndash2(d) while quantification of the results fromall samples is shown in Figures 2(e)ndash2(h) As shown inFigures 2(a)ndash2(h) the results demonstrate that protein levelsof NLRP3 ASC and ldquoactiverdquo cleaved caspase-1 and cleaved


NLRP3

GAPDH

Sham GCI

(100 kDa)

(37 kDa)

） + 2

(a)

ASC

GAPDH

(24 kDa)

(37 kDa)

(b)

Cl-caspase-1

GAPDH

Sham GCI

(37 kDa)

(20 kDa)

） + 2

(c)

GAPDH (37 kDa)

(17 kDa)Cl-IL-1

(d)

Sham GCI

NLRP3 lowast

0

1

2

3

4

NLR

P3 p

rote

in (f

old

chan

ge)

） + 2

(e)

ASC

Sham GCI

lowast

0

1

2

3

4

5

ASC

pro

tein

(fol

d ch

ange

)

） + 2

(f)

Cl-caspase-1

Sham GCI

lowast

0

05

1

15

2

25

3

Cl-c

aspa

se-1

pro

tein

(fol

d ch

ange

)

） + 2

(g)Sham GCI

lowast

Cleaved IL-1

0

05

1

15

2

25

Clea

ved

IL-1

prot

ein

(fold

chan

ge)

） + 2

(h)

Figure 2 Increase in NLRP3 ASC cleaved caspase-1 and cleaved IL-1120573 protein levels after GCI and its suppression by E2 Representativeimmunoblots from Western blot analysis of hippocampal samples collected 7 days after GCI show that (a) NLRP3 (b) ASC (c) cleavedcaspase-1 and (d) cleaved IL-1120573 are robustly increased after GCI as compared to shams and are significantly suppressed by E2 treatment(endashh) Quantification of immunoblots using ImageJ software showed that NLRP3 cleaved caspase-1 and cleaved IL-1120573 levels are increased25-fold after GCI and ASC is increased 6-fold This increase in protein levels is significantly suppressed by E2 treatment (119901 lt 005 shamversus GCI lowast119901 lt 005 GCI versus GCI + E2) (119899 = 4-5 animals per group)

IL-1120573 are significantly increased in the hippocampus at 7 daysafter GCI and that E2 treatment significantly attenuates theelevation of the NLRP3 inflammasome proteins

To further confirm the mRNA as well as Western blotresults we used immunofluorescence staining to detect the

protein expression of NLRP3 ASC ldquoactiverdquo cleaved caspase-1 (cl-caspase-1) and IL-1120573 at 7 days after GCI As shownin Figure 3(a) representative photomicrographs reveal thatthe expression of NLRP3 ASC ldquoactiverdquo cleaved caspase-1and IL-1120573 was significantly increased in the hippocampal


Sham

7-da

yG

CI7-

day

NLRP3 ASC Cl-caspase-1 IL-1

）+2

(a)NLRP3 ASC

Cl-caspase-1

Sham GCI

Sham GCI Sham GCI

Sham GCI

lowast

lowast lowast

lowast

IL-1

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

0

5

10

15

20

25

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

0

5

10

15

20

25

IL-1

expr

essio

n (

area

activ

ated

)

0

2

4

6

8

10

12

14A

SC ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

） + 2

） + 2

） + 2

(b)

Figure 3 Expression of ASC NLRP3 cleaved caspase-1 and IL-1120573 protein after GCI and suppression by E2 (a) Representative confocalimages show that ASC NLRP3 cleaved caspase-1 and IL-1120573 are activated in hippocampal CA1 region of the young adult female rat at day7 after GCI This expression is robustly suppressed by E2 as seen in the lower panel by reduced staining (magnification = 40x scale bar= 50120583m) (b) Quantification of fluorescence intensity using MATLAB software shows a statistically significant suppression of all of theseproteins at day 7 after GCI (119901 lt 005 sham versus GCI lowast119901 lt 005 GCI versus GCI + E2) (119899 = 5-6 animals per group)


Sham

NLR

P3-A

SC

inte

ract

ion

(PLA

)

7 d GCI 7 d ） + 2

(a)

lowast

Sham GCI0

2

4

6

8

10

12

NLR

P3-A

SC in

tera

ctio

n (

area

inte

ract

ion)

） + 2

(b)

Figure 4 Proximity ligation assay demonstrating NLRP3-ASC complex formation after GCI and its suppression by E2 (a) Representativeconfocal images of proximity ligation assay (Duolink in situ co-IP) show that NLRP3-ASC complex is formed in hippocampal CA1 region ofthe young adult female rat at day 7 after GCI This complex formation is robustly suppressed by E2 (magnification = 40x scale bar = 50120583m)(b) Quantification of fluorescence intensity usingMATLAB software shows a statistically significant suppression of complex formation at day7 after GCI (119901 lt 005 sham versus GCI lowast119901 lt 005 GCI versus GCI + E2) (119899 = 5-6 animals per group)

CA1 region of the rat hippocampus at 7 days after GCI ascompared to the sham controls Furthermore E2 treatmentrobustly suppressed the enhanced protein expression ofNLRP3 ASC ldquoactiverdquo cleaved caspase-1 and IL-1120573 in thehippocampus after GCI Figure 3(b) shows the results ofquantification of the immunofluorescence staining intensityusing MATLAB software As shown in Figure 3(b) all ofthe NLRP3 inflammasome pathway molecules were highlyexpressed at day 7 afterGCI andwere significantly suppressedby E2 The specificity of the NLRP3 inflammasome pathwayantibodies used in our studies was confirmed by preab-sorption studies with antigen (or when not available withthe recombinant protein eg for IL-1120573) As shown in Sup-plementary Figure 1 (see Supplementary Material availableonline at httpdxdoiorg10115520168309031) antigen orrecombinant protein preabsorption essentially completelyabolished staining for the NLRP3 inflammasome moleculesdemonstrating specificity of the antibodies Therefore cou-pled with the mRNA expression and Western blot data inFigures 1 and 2 the results in Figure 3 further confirm thatexpression of NLRP3 inflammasome pathway molecules isincreased in the rat hippocampus after GCI and suppressedby E2 treatment

Using triple immunofluorescent staining we next deter-mined the cell type of expression of NLRP3 inflammasomemolecules in the hippocampal CA1 region at 7 days after GCIAs shown in Supplementary Figure 2 triple immunofluores-cence staining of each of the NLRP3 inflammasome pathwaymolecules with either GFAP (astrocyte marker) or CD11b(microglial marker) and NeuN (neuronal marker) or DAPIrevealed thatNLRP3 ASC ldquoactiverdquo cleaved caspase-1 and IL-1120573 are colocalized with CD11b and GFAP in the hippocampalCA1 region at 7 days after GCI This finding suggests thatNLRP3 inflammasome pathway activation occurs primarily

in microglia and activated astrocytes in the hippocampus at7 days after GCI

32 Estradiol Suppresses NLRP3 Inflammasome Complex For-mation in the Hippocampus after GCI In the above studieswe examined E2 regulation of expression of NLRP3 inflam-masome pathway factors after GCI To further confirm acti-vation of the NLRP3 inflammasome we examined NLRP3inflammasome complex formation after GCI as assembly ofthe inflammasome complex is known to be essential forits activation To examine NLRP3 inflammasome complexformation we performed an in situ coimmunoprecipitationassay also known as a proximity ligation assay (or Duolinkassay) to measure the protein-protein interaction (complexformation) of NLRP3 and ASC in the rat hippocampalCA1 region at 7 days after GCI ASC binding to NLRP3 isknown to be critical for recruitment of caspase-1 and forNLRP3 inflammasome activation [31] The protein-proteininteraction in the proximity ligation assay was detected asred immunofluorescence signal under a confocalmicroscopeRepresentative photomicrographs of the proximity ligationassay results are shown in Figure 4(a) while quantification ofthe results from all images from all animals for intensity usingMATLAB is shown in Figure 4(b) As shown in Figures 4(a)and 4(b) the results of the proximity ligation assay revealed asignificant increase in NLRP3-ASC complex formation in thehippocampus after GCI as compared to sham controls Fur-thermore E2 treatment strongly suppressed theGCI-inducedNLRP3-ASC complex formation in the hippocampus

33 Estradiol Regulates Expression of Upstream Activators ofthe NLRP3 Inflammasome We hypothesized that E2 maycontrol NLRP3 inflammasome activation by regulating the


Iba1

P2X7

rN

euN

Sham 7-day GCI 7-day ） + 2

(a)P2X7 intensity

lowast

0

1

2

3

4

5

6

P2X7

expr

essio

n (

area

activ

ated

)

Sham GCI ） + 2

(b)

P2Xr7 mRNA

lowastlowast

lowastlowast

0

05

1

15

2

P2Xr

7 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

1 3 7(Day)

GCI） + 2

(c)

Figure 5 Increased expression of P2X7 receptor after GCI and its suppression by E2 treatment (a) Representative confocal images stainedfor Iba1 (red) P2X7r (green) and NeuN (blue) show the activation of microglia and increased expression of P2X7r in the hippocampal CA1region of young adult female rats 7 days after GCIThe P2X7 receptor colocalized very strongly with microglia (yellow color)This expressionand microglial activation is suppressed by E2 treatment (magnification = 40x scale bar = 50 120583m) (b) Quantification of fluorescence intensityusing MATLAB software shows a statistically significant suppression of P2X7 receptor by E2 at day 7 after GCI (119901 lt 005 sham versus GCIlowast119901 lt 005GCI versus GCI + E2) (119899 = 5-6 animals per group) (c) mRNA collected from rat hippocampus after GCI shows induction of P2rX7gene and its significant suppression by E2 treatment at days 3 and 7 after GCI (lowastlowast119901 lt 00001 GCI versus GCI + E2) (119899 = 5-6 animalsgroup)

expression of key upstream activators of the NLRP3 inflam-masome A well-known upstream activator of the NLRP3inflammasome is the ionotropic purinergic P2X7 receptorThe P2X7 receptor becomes activated by extracellular ATPwhich is a danger-associated molecular pattern (DAMP)released from damageddying neurons after injury [51] Wethus examined the ability of E2 to regulate protein andmRNAexpression levels of the P2X7 receptor in the hippocampususing triple immunofluorescent staining and quantitativeRT-PCR Representative photomicrographs of the tripleimmunofluorescent staining results for P2X7 receptor NeuN(neuronal marker) and Iba1 (microglial marker) are shownin Figure 5(a) while quantification of the results from allimages from all animals for intensity using MATLAB isshown in Figure 5(b) As shown in Figures 5(a) and 5(b) triple

immunofluorescence colocalization results with specific anti-bodies to the P2X7 receptor NeuN (neuronal marker)and Iba1 (microglial marker) revealed that immunoreactiveprotein levels of the P2X7 receptor in hippocampal CA1region of rat are strongly upregulated at 7 days after GCIFurthermore the P2X7 receptor immunostaining signal washighly colocalized with the microglia marker Iba1 indicatingits upregulation occurs in microglia Intriguingly P2X7receptor immunoreactive protein levels in the hippocampalCA1 region were profoundly suppressed by E2 treatment Wenext examined whether E2 could regulate gene expressionof the P2X7 receptor in the hippocampus at various timepoints after GCI As shown in Figure 5(c) P2X7 receptormRNA levels were significantly increased (15ndash2-fold) in thehippocampus from days 1 3 and 7 after GCI as compared


to sham controls E2 treatment had no effect upon the P2X7receptor mRNA levels at day 1 after GCI but significantlyattenuated its expression at days 3 and 7 after GCI We alsoexamined whether E2 could regulate another upstream reg-ulator of NLRP3 thioredoxin interacting protein (TXNIP)Following increased production of reactive oxygen species(such aswhat occurs after ischemic injury) TXNIP is releasedfrom its complex with thioredoxin (TRX) and can bindto the LRR region of NLRP3 and activate it [52 53] Asshown in Supplementary Figure 3A immunofluorescencestaining revealed that TXNIP expression was increased inrat hippocampal CA1 region at 7 days after GCI and thatthis expression is robustly suppressed by E2 treatmentQuantification of intensity of TXNIP for all images from allanimals is shown in Supplementary Figure 3B As shownin Supplementary Figure 3C mRNA levels of TXNIP werealso increased after GCI and significantly suppressed byE2 treatment at days 3 and 7 after GCI These resultssuggest that E2 may potentially regulate the inflammasomecomplex activation via its ability to suppress expression of theNLRP3 inflammasome upstream regulators P2X7 receptorand TXNIP

34 The Estrogen Receptor Coregulator PELP1 Is Essential forthe Ability of Estradiol to Regulate the NLRP3 Inflammasomeafter GCI To enhance understanding of the mechanismsinvolved in the E2 anti-inflammatory effects after GCI weexamined the role of the ER coregulator PELP1 Prior workby our group using a PELP1 forebrain-specific KO (PELP1FBKO) mouse model that we generated revealed that PELP1is required for E2-induced rapid and genomic signaling aswell as the neuroprotective and cognitive-enhancing effectsof E2 in the hippocampus after GCI [16] Moreover RNA-seqdata comparing hippocampal gene expression in E2-treatedFLOX control and PELP1 FBKO mice at 24 hours after GCIrevealed that a number of inflammatory pathway genes wereupregulated in the PELP1 FBKO mouse [16] We thereforeexamined whether the ability of E2 to suppress expression ofNLRP3 inflammasome pathway factors in the hippocampalCA1 region after GCI required PELP1 mediation We utilizedFLOX control and PELP1 FBKO mice and examined E2regulation ofNLRP3 inflammasome expression at 6 days afterGCI as this time point showed robust microglia activationand neuronal loss after GCI [16] Representative photomicro-graphs of immunofluorescent staining results for NLRP3 andASC are shown in Figures 6(a) and 6(c) respectively whilequantification of the results from all images from all animalsfor intensity using MATLAB is shown in Figures 6(b) and6(d) As shown in Figures 6(a)ndash6(d) immunofluorescencestaining of hippocampal CA1 region sections revealed thatNLRP3 and ASC were markedly elevated at day 6 after GCIin FLOX control mice and that E2 suppressed this effectIntriguingly the ability of E2 to suppress elevation of NLRP3and ASC in the hippocampus was lost in PELP1 FBKO mice(Figures 6(a)ndash6(d))

We next examined the downstream products of theNLRP3 inflammasome pathway ldquoactiverdquo cleaved caspase-1 and IL-1120573 using immunofluorescence staining of hip-pocampal sections Representative photomicrographs of

immunofluorescent staining results for ldquoactiverdquo cleavedcaspase-1 and IL-1120573 are shown in Figures 7(a) and 7(c)respectively while quantification of the results from allimages from all animals for intensity using MATLAB isshown in Figures 7(b) and 7(d) As shown in Figures7(a)ndash7(d) immunoreactive protein levels of ldquoactiverdquo cleavedcaspase-1 and IL-1120573 were significantly enhanced in the hip-pocampal CA1 region at 6 days after GCI in FLOX controlmice and E2 strongly suppressed the elevation of both ofthese factors Further the ability of E2 to suppress elevationof ldquoactiverdquo cleaved caspase-1 and IL-1120573 in the hippocampuswas lost in PELP1 FBKO mice (Figures 7(a)ndash7(d))

We next used the in situ proximity ligation assay todetermine whether the ability of E2 to suppress NLRP3-ASC complex formation after GCI is compromised in PELP1FBKO mice Representative photomicrographs of the prox-imity ligation assay results are shown in Figure 8(a) whilequantification of the results from all images from all animalsfor intensity of using MATLAB is shown in Figure 8(b)As shown in Figure 8 FLOX control mice demonstrated asignificantly increasedNLRP3-ASC complex formation in thehippocampal CA1 region at 6 days after GCI as detected bythe proximity ligation assay and E2 treatment completelyattenuated the complex formation However the ability of E2to inhibit the GCI-induced NLRP3-ASC complex formationwas lost in PELP1 FBKO mice (Figures 8(a) and 8(b))

Finally we examined the effect of PELP1 deletion uponthe ability of E2 to suppress expression of the P2X7 receptor inhippocampal CA1 region after GCI Representative photomi-crographs of triple immunofluorescent staining results for theP2X7 receptor Iba1 (microglia marker) and NeuN (neuronalmarker) are shown in Figure 9(a) while quantification ofthe results from all images from all animals for intensityusingMATLAB is shown in Figure 9(b) As shown in Figures9(a) and 9(b) triple immunofluorescence staining for theP2X7 receptor Iba1 (microglia marker) and NeuN (neuronalmarker) revealed that there was increased expression of theP2X7 receptor in the hippocampal CA1 region of FLOXcontrol as well as PELP1 knockout mice after GCI The P2X7receptor immunoreactive signal colocalized with Iba1 indi-cating that the enhanced P2X7 expression occurred primarilyin microglia As was the case in the ovariectomized rat P2X7receptor expression was suppressed by E2 treatment in FLOXcontrol mice that were subjected to GCI The suppressiveeffect of E2 on P2X7 receptor expression required PELP1mediation as evidenced by the loss of the suppressive effectof E2 in PELP1 FBKO mice (Figures 9(a) and 9(b))

4 Discussion

The current study provides several important findings Firstit demonstrates that the NLRP3 inflammasome is robustlyactivated inmicroglia and astrocytes in the hippocampal CA1region of both the rat and mouse after GCI Secondly itdemonstrates that low dose E2 treatment profoundly sup-pressesNLRP3 inflammasome activation in the hippocampusafter GCI Thirdly it provides novel insight into the mecha-nisms of E2 anti-inflammatory effects by demonstrating thatE2 suppresses induction of the upstream activators of the


FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO

FLOX sham FLOX GCI FLOX ） + 2

） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)

Figure 6 The ability of E2 to attenuate expression of NLRP3 and ASC after GCI is lost in PELP1 forebrain-specific knockout mice (a and c)Representative confocal images show that NLRP3 and ASC are induced in hippocampal CA1 region at day 6 after GCI in FLOX control aswell as PELP1 knockout mice The expression of these markers is suppressed by E2 in the FLOX control mice but not in the PELP1 KO micesections (KO = PELP1 knockout) (magnification = 40x scale bar = 50 120583m) (b and d) Quantification of fluorescence intensity usingMATLABsoftware shows a statistically significant suppression of all of these proteins by E2 in FLOX but not in KO mice (119901 lt 005 sham versus GCIlowast119901 lt 005 GCI versus GCI + E2 $119901 lt 005 FLOX GCI + E2 versus KO GCI + E2 NS not significant KO GCI versus KO GCI + E2) (119899 = 5-6animalsgroup)

NLRP3 inflammasome (P2X7 and TXNIP) after GCI andthat the ER coregulator protein PELP1 is essential for theanti-inflammatory actions of E2 in the hippocampus

A unique strength of our study is that we used a novelgenetic PELP1 forebrain-specific knockoutmodel to elucidate

the role of PELP1 in E2 anti-inflammatory actions Based onthe results of our study we propose that E2 binds to ER andrecruits the coregulator PELP1 to form an active transcrip-tional complex which leads to attenuation of expression ofthe upstream inducers of the NLRP3 inflammasome P2X7

12 Oxidative Medicine and Cellular LongevityCl

-cas

pase

-1Cl

-cas

pase

-1

FLOX sham FLOX GCI FLOX

KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)

Figure 7The ability of E2 to attenuate expression of cleaved caspase-1 and IL-1120573 after GCI is lost in PELP1 forebrain-specific knockout mice(a and c) Representative confocal images show that cleaved caspase-1 and IL-1120573 are induced in hippocampal CA1 region at day 6 after GCI inFLOX as well as PELP1 knockout mice The expression of these markers is suppressed by E2 in the FLOX control mice but not in the PELP1KOmice sections (KO = PELP1 knockout) (magnification = 40x scale bar = 50 120583m) (b and d) Quantification of fluorescence intensity usingMATLAB software shows a statistically significant suppression of all of these proteins by E2 in FLOX but not in KO mice (119901 lt 005 shamversus GCI lowast119901 lt 005 GCI versus GCI + E2 $119901 lt 005 FLOX GCI + E2 versus KO GCI + E2 NS not significant KO GCI versus KO GCI+ E2) (119899 = 5-6 animalsgroup)

and TXNIP in the hippocampus after GCI Suppression ofTXNIP and NLRP3 activation could also be due to E2 sup-pression of GCI-induced ROS generation as we previouslydemonstrated that E2 profoundly suppresses NOX2 NADPHoxidase activity and superoxide induction after GCI [7]

Furthermore in support of this possibility NOX2 knockoutmice have been shown to have a significantly reduced NLRP3activation after FCI that correlated with reduced infarct sizereduced edema and improved neurological outcome [38]While we believe that E2 effects involve mediation by nuclear

Oxidative Medicine and Cellular Longevity 13N

LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)

Figure 8 The ability of E2 to suppress NLRP3-ASC complex formation after GCI is lost in PELP1 forebrain-specific knockout mice (a)Representative confocal images of Duolink in situ co-IP show that ASC-NLRP3 complex is formed in hippocampal CA1 region at day 6 afterGCI This complex formation is robustly suppressed by E2 in the FLOX but not in KO (KO = PELP1 knockout) (magnification = 40x scalebar = 50 120583m) (b)MATLAB analysis of the images shows a statistically significant suppression of complex formation by E2 in FLOX but not inKOmice (119901 lt 005 sham versus GCI lowast119901 lt 005 GCI versus GCI + E2 $119901 lt 005 FLOX GCI + E2 versus KO GCI + E2 NS not significantKO GCI versus KO GCI + E2) (119899 = 5-6 animalsgroup)

ER we cannot rule out the possibility that extranuclear ERsignaling could be involved as well as we previously demon-strated that PELP1 also mediates extranuclear ER-mediatedrapid signaling effects in the hippocampus after GCI [16]

While our study did not address which ER mediates E2effects upon the NLRP3 inflammasome after GCI PELP1 caninteract with and mediate the effects of both ER-120572 and ER-120573 in various cells throughout the body [54] Intriguinglyboth ER-120572 and ER-120573 have been implicated to mediate anti-inflammatory actions of E2 in the brain Most relevant toour study ER-120573 antisense oligonucleotide knockdown hasbeen reported to attenuate E2 suppression of ASC caspase-1 and IL-1120573 after GCI [47] This study did not examinewhich inflammasomemediated the increase in IL-1120573 and wasregulated by E2 but our work provides strong evidence thatthe NLRP3 inflammasome is activated in the hippocampusafter GCI and strongly regulated by E2 ER-120572 has alsobeen implicated in mediating some of the anti-inflammatoryeffects of E2 in the brain For instance Vegeto et al [28] foundthat the ability of E2 to suppress lipopolysaccharide-inducedmicroglial activation and monocyte recruitment in the brainis lost in ER-120572 knockout mice However this study did notassess the role for ER-120572 in inflammasome regulation Finallyto our knowledge no study has examined the role of GPER1in the regulation of inflammasome activation in the brainStudies are ongoing in our laboratory to further address thisissue

Of significant note our study found that NLRP3 pathwayfactors were induced in both microglia and astrocytes afterGCI A similar multicell induction of NLRP3 pathway factors

has been reported previously in the cerebral cortex after focalcerebral ischemia [55] or traumatic brain injury [22] demon-strating that NLPR3 factors are expressed in neurons astro-cytes andmicroglia after brain injury Likewise expression ofP2X7 receptors has been reported inmultiple brain cell typesincluding microglia astrocytes and neurons [56ndash59] and isincreased after cerebral ischemia [60ndash62] Thus it appearsthat multiple cell types can express inflammasomes and beinvolved in neuroinflammation While less is known aboutthe role of NLRP3 inflammasome activation in astrocytesas compared to microglia recent studies have found thatastrocytes can express NLRP3 and ASC and display caspase-1 cleavage and release of IL-1120573 after induction with inflam-matory agents A120573 or in vitro oxygenglucose deprivation[36 37 63] This suggests that inflammasome activation inastrocytes may also contribute to neuroinflammation afterbrain injury In contrast NLRP1 and Aim2 expression hasbeen primarily reported in neurons in the ischemic or injuredbrain [64ndash66]

Finally previous work by our laboratory and others hasshown that E2 is profoundly neuroprotective and improvesfunctional outcome after GCI and FCI [5 9ndash11 16 67] Basedon our current results we propose that E2 inhibitionof NLRP3 inflammasome activation is an important con-tributing mechanism for E2 beneficial effects on neuronalsurvival and functional outcome after GCI In support ofthis possibility NLRP3 knockout mice have been shown tohave significantly reduced infarct size after FCI suggestingthat NLRP3 inflammasome activation contributes to neu-ronal damage and cell death after cerebral ischemia [38]


Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)

Figure 9 The ability of E2 to attenuate expression of P2X7 receptor after GCI is lost in PELP1 forebrain-specific knockout mice (a)Representative confocal images stained for Iba1 (red) P2X7 receptor (green) andNeuN (blue) show the activation of microglia and increasedexpression of P2X7 receptor in the hippocampal CA1 region of young adult female FLOX control as well as PELP1 KOmice 6 days after GCIThe P2X7 receptor colocalized very strongly with microglia This expression and microglial activation are suppressed by E2 treatment inFLOX control mice but not in PELP1 KO mice (KO = PELP1 knockout) (magnification = 40x scale bar = 50 120583m) (b) MATLAB analysis ofthe images shows a statistically significant suppression of all of these proteins by E2 in FLOX control but not in KO mice (119901 lt 005 shamversus GCI lowast119901 lt 005 GCI versus GCI + E2 NS not significant KO GCI versus KO GCI + E2) (119899 = 5-6 animalsgroup)

Furthermore intravenous immunoglobulin has been shownto suppress NLRP3 inflammasome-mediated neuronal celldeath after ischemic stroke and to improve neurologicaloutcome [40] Finally caspase-1 knockout mice as well ascaspase-1 inhibitor- or dominant negative-treated mice havebeen all reported to have significantly decreased neuronal celldeath and brain deficits following cerebral ischemia [68ndash71]Collectively these findings suggest that NLRP3 inflamma-some activation and resultant neuroinflammation contribute

significantly to the neuronal cell death and functional deficitsthat occur after cerebral ischemia and that E2 inhibitionof NLRP3 inflammasome activation may contribute signif-icantly to its beneficial protective effects following cerebralischemia

In conclusion the results of our study demonstrate thatNLRP3 inflammasome activation is strongly increased in thehippocampus after GCI and that E2 strongly suppresses bothexpression and activation of the NLRP3 inflammasome The


effects of E2 require the ER-coregulator PELP1 and involveattenuation of P2X7 and TXNIP two well-known upstreaminducers of NLRP3 inflammasome activationThese findingsprovide new insight into the anti-inflammatory effects ofE2 in the brain and suggest that therapeutic targeting ofthe NLRP3 inflammasome for inhibition by E2 analogues orNLRP3 inflammasome selective inhibitors may have efficacyin the treatment of GCI as well as other neurodegenerativedisorders that involve NLRP3 inflammasome activation andneuroinflammation

Abbreviations

ASC Apoptosis associated speck like proteinE2 17120573-EstradiolER-120572 Estrogen receptor alphaER-120573 Estrogen receptor-betaFCI Focal cerebral ischemiaGCI Global cerebral ischemiaIL-1120573 Interleukin-1betaNLRP3 NOD-like receptor with a pyrin domain-3PELP1 Proline- glutamic acid- and leucine-rich protein 1TXNIP Thioredoxin interacting protein

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

RoshniThakkar and RuiminWang contributed equally to thestudies and should be considered as co-first authors

Acknowledgments

The research presented in this article was conducted inpartial fulfillment of the PhD degree requirements set forthfor Roshni Thakkar and was supported by Research GrantsNS050730 NS088058 and NS086929 from the NationalInstitute of Neurological Disorders and Stroke NationalInstitutes of Health

References

[1] D Lebesgue V Chevaleyre R S Zukin and A M EtgenldquoEstradiol rescues neurons from global ischemia-induced celldeath multiple cellular pathways of neuroprotectionrdquo Steroidsvol 74 no 7 pp 555ndash561 2009

[2] J W Simpkins E Perez Xiaofei Wang Shaohua Yang YiWen and M Singh ldquoThe potential for estrogens in prevent-ing Alzheimerrsquos disease and vascular dementiardquo TherapeuticAdvances in Neurological Disorders vol 2 no 1 pp 31ndash49 2009

[3] J W Simpkins P S Green K E Gridley M Singh N C DeFiebre and G Rajakumar ldquoRole of estrogen replacement ther-apy in memory enhancement and the prevention of neuronalloss associated with Alzheimerrsquos diseaserdquoThe American Journalof Medicine vol 103 no 3 pp 19sndash25s 1997

[4] E B Engler-Chiurazzi C Brown J Povroznik and J Simp-kins ldquoEstrogens as neuroprotectants estrogenic actions in

the context of cognitive aging and brain injuryrdquo Progress inNeurobiology 2016

[5] J W Simpkins G Rajakumar Y-Q Zhang et al ldquoEstrogensmay reduce mortality and ischemic damage caused by middlecerebral artery occlusion in the female ratrdquo Journal of Neuro-surgery vol 87 no 5 pp 724ndash730 1997

[6] H Tang Q Zhang L Yang et al ldquoGPR30 mediates estrogenrapid signaling and neuroprotectionrdquo Molecular and CellularEndocrinology vol 387 no 1-2 pp 52ndash58 2014

[7] Q-G Zhang L Raz R Wang et al ldquoEstrogen attenuatesischemic oxidative damage via an estrogen receptor 120572-mediatedinhibition of NADPH oxidase activationrdquoThe Journal of Neuro-science vol 29 no 44 pp 13823ndash13836 2009

[8] L-C Yang Q-G Zhang C-F Zhou et al ldquoExtranuclear estro-gen receptors mediate the neuroprotective effects of estrogenin the rat hippocampusrdquo PLoS ONE vol 5 no 5 article e98512010

[9] D W Brann K Dhandapani C Wakade V B Mahesh andM M Khan ldquoNeurotrophic and neuroprotective actions ofestrogen basic mechanisms and clinical implicationsrdquo Steroidsvol 72 no 5 pp 381ndash405 2007

[10] D B Dubal M L Kashon L C Pettigrew et al ldquoEstradiolprotects against ischemic injuryrdquo Journal of Cerebral Blood Flowand Metabolism vol 18 no 11 pp 1253ndash1258 1998

[11] T Jover H Tanaka A Calderone et al ldquoEstrogen protectsagainst global ischemia-induced neuronal death and preventsactivation of apoptotic signaling cascades in the hippocampalCA1rdquo Journal of Neuroscience vol 22 no 6 pp 2115ndash2124 2002

[12] M Bourque M Morissette and T Di Paolo ldquoNeuroprotectionin Parkinsonian-treated mice via estrogen receptor 120572 activationrequires G protein-coupled estrogen receptor 1rdquoNeuropharma-cology vol 95 pp 343ndash352 2015

[13] E R Prossnitz and M Barton ldquoEstrogen biology new insightsinto GPER function and clinical opportunitiesrdquo Molecular andCellular Endocrinology vol 389 no 1-2 pp 71ndash83 2014

[14] M-A Arevalo I Azcoitia and L M Garcia-Segura ldquoTheneuroprotective actions of oestradiol and oestrogen receptorsrdquoNature Reviews Neuroscience vol 16 no 1 pp 17ndash29 2015

[15] R K Vadlamudi R-A Wang A Mazumdar et al ldquoMolecularcloning and characterization of PELP1 a novel human co-regulator of estrogen receptor 120572rdquo The Journal of BiologicalChemistry vol 276 no 41 pp 38272ndash38279 2001

[16] G R Sareddy Q Zhang R Wang et al ldquoProline- glutamicacid- and leucine-rich protein 1 mediates estrogen rapid sig-naling and neuroprotection in the brainrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 112 no 48 pp E6673ndashE6682 2015

[17] G R Sareddy and R K Vadlamudi ldquoPELP1 structure biologi-cal function and clinical significancerdquo Gene vol 585 no 1 pp128ndash134 2016

[18] M M Khan M Hadman C Wakade et al ldquoCloning expres-sion and localization of MNARPELP1 in rodent brain colo-calization in estrogen receptor-120572- but not in gonadotropin-releasing hormone-positive neuronsrdquo Endocrinology vol 146no 12 pp 5215ndash5227 2005

[19] M M Khan M Hadman L M De Sevilla et al ldquoCloningdistribution and colocalization of MNARPELP1 with gluco-corticoid receptors in primate and nonprimate brainrdquoNeuroen-docrinology vol 84 no 5 pp 317ndash329 2007


[20] R K Vadlamudi R-A Wang A Mazumdar et al ldquoMolecularcloning and characterization of PELP1 a novel human coreg-ulator of estrogen receptor 120572rdquo Journal of Biological Chemistryvol 276 no 41 pp 38272ndash38279 2001

[21] M THenekaM P Kummer A Stutz et al ldquoNLRP3 is activatedin Alzheimerrsquos disease and contributes to pathology in APPPS1micerdquo Nature vol 493 no 7434 pp 674ndash678 2013

[22] H-D Liu W Li Z-R Chen et al ldquoExpression of the NLRP3inflammasome in cerebral cortex after traumatic brain injury ina rat modelrdquo Neurochemical Research vol 38 no 10 pp 2072ndash2083 2013

[23] G Singhal E J Jaehne F Corrigan C Toben and B T BauneldquoInflammasomes in neuroinflammation and changes in brainfunction a focused reviewrdquo Frontiers in Neuroscience vol 8article 315 2014

[24] E Vegeto V Benedusi and A Maggi ldquoEstrogen anti-inflammatory activity in brain a therapeutic opportunity formenopause and neurodegenerative diseasesrdquo Frontiers in Neu-roendocrinology vol 29 no 4 pp 507ndash519 2008

[25] J Dang B Mitkari M Kipp and C Beyer ldquoGonadal steroidsprevent cell damage and stimulate behavioral recovery aftertransient middle cerebral artery occlusion in male and femaleratsrdquo Brain Behavior and Immunity vol 25 no 4 pp 715ndash7262011

[26] C M Brown T A Mulcahey N C Filipek and P M WiseldquoProduction of proinflammatory cytokines and chemokinesduring neuroinflammation novel roles for estrogen receptors120572 and 120573rdquo Endocrinology vol 151 no 10 pp 4916ndash4925 2010

[27] E Vegeto S Belcredito S Ghisletti C Meda S Etteri andA Maggi ldquoThe endogenous estrogen status regulates microgliareactivity in animal models of neuroinflammationrdquo Endocrinol-ogy vol 147 no 5 pp 2263ndash2272 2006

[28] E Vegeto S Belcredito S Etteri et al ldquoEstrogen receptor-alphamediates the brain antiinflammatory activity of estradiolrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 100 no 16 pp 9614ndash9619 2003

[29] F Martinon K Burns and J Tschopp ldquoThe inflammasomea molecular platform triggering activation of inflammatorycaspases and processing of proIL-120573rdquoMolecular Cell vol 10 no2 pp 417ndash426 2002

[30] H Guo J B Callaway and J P-Y Ting ldquoInflammasomesmechanism of action role in disease and therapeuticsrdquo NatureMedicine vol 21 no 7 pp 677ndash687 2015

[31] J G Walsh D A Muruve and C Power ldquoInflammasomes inthe CNSrdquoNature Reviews Neuroscience vol 15 no 2 pp 84ndash972014

[32] J P De Rivero Vaccari W D Dietrich and R W KeaneldquoActivation and regulation of cellular inflammasomes gaps inour knowledge for central nervous system injuryrdquo Journal ofCerebral Blood Flow andMetabolism vol 34 no 3 pp 369ndash3752014

[33] M J Leo Bours P C Dagnelie A L Giuliani A Wesseliusand F Di Virgilio ldquoP2 receptors and extracellular ATP a novelhomeostatic pathway in inflammationrdquo Frontiers in Bioscience(Scholar Edition) vol 3 no 4 pp 1443ndash1456 2011

[34] F Martinon A Mayor and J Tschopp ldquoThe inflammasomesguardians of the bodyrdquo Annual Review of Immunology vol 27pp 229ndash265 2009

[35] L C Freeman and J P-Y Ting ldquoThe pathogenic role ofthe inflammasome in neurodegenerative diseasesrdquo Journal ofNeurochemistry vol 136 supplement 1 pp 29ndash38 2016

[36] Z Jian S Ding H Deng et al ldquoProbenecid protects againstoxygen-glucose deprivation injury in primary astrocytes byregulating inflammasome activityrdquoBrain Research vol 1643 pp123ndash129 2016

[37] S Alfonso-Loeches J R Urena-Peralta M J Morillo-BarguesJ O-D La Cruz and C Guerri ldquoRole of mitochondria ROSgeneration in ethanol-induced NLRP3 inflammasome activa-tion and cell death in astroglial cellsrdquo Frontiers in CellularNeuroscience vol 8 article 216 2014

[38] F Yang Z Wang X Wei et al ldquoNLRP3 deficiency amelioratesneurovascular damage in experimental ischemic strokerdquo Jour-nal of Cerebral Blood Flow and Metabolism vol 34 no 4 pp660ndash667 2014

[39] L Lammerding A Slowik S Johann C Beyer and A Zend-edel ldquoPoststroke inflammasome expression and regulation inthe peri-infarct area by gonadal steroids after transient focalischemia in the rat brainrdquo Neuroendocrinology vol 103 no 5pp 460ndash475 2016

[40] D Yang-Wei Fann S-Y Lee S Manzanero et alldquoIntravenous immunoglobulin suppresses NLRP1 and NLRP3inflammasome-mediated neuronal death in ischemic strokerdquoCell Death and Disease vol 4 no 9 article e790 2013

[41] D Y-W Fann T Santro S Manzanero et al ldquoIntermittentfasting attenuates inflammasome activity in ischemic strokerdquoExperimental Neurology vol 257 pp 114ndash119 2014

[42] Y Tong Z H Ding F X Zhan et al ldquoThe NLRP3 inflam-masome and strokerdquo International Journal of Clinical andExperimental Medicine vol 8 no 4 pp 4787ndash4794 2015

[43] XWang R Li XWang Q Fu and SMa ldquoUmbelliferone ame-liorates cerebral ischemia-reperfusion injury via upregulatingthe PPAR gamma expression and suppressing TXNIPNLRP3inflammasomerdquo Neuroscience Letters vol 600 pp 182ndash1872015

[44] Y Li J Li S Li et al ldquoCurcumin attenuates glutamate neurotox-icity in the hippocampus by suppression of ER stress-associatedTXNIPNLRP3 inflammasome activation in a manner depen-dent onAMPKrdquoToxicology andApplied Pharmacology vol 286no 1 pp 53ndash63 2015

[45] A Denes G Coutts N Lenart et al ldquoAIM2 and NLRC4inflammasomes contribute with ASC to acute brain injuryindependently of NLRP3rdquo Proceedings of the National Academyof Sciences vol 112 no 13 pp 4050ndash4055 2015

[46] A Slowik and C Beyer ldquoInflammasomes are neuroprotectivetargets for sex steroidsrdquo Journal of Steroid Biochemistry andMolecular Biology vol 153 pp 135ndash143 2015

[47] J P De Rivero Vaccari H H Patel F J Brand M A Perez-Pinzon H M Bramlett and A P Raval ldquoEstrogen receptorbeta signaling alters cellular inflammasomes activity after globalcerebral ischemia in reproductively senescence female ratsrdquoJournal of Neurochemistry vol 136 no 3 pp 492ndash496 2016

[48] W A Pulsinelli and J B Brierley ldquoA new model of bilateralhemispheric ischemia in the unanesthetized ratrdquo Stroke vol 10no 3 pp 267ndash272 1979

[49] K Langnaese R John H Schweizer U Ebmeyer and GKeilhoff ldquoSelection of reference genes for quantitative real-timePCR in a rat asphyxial cardiac arrest modelrdquo BMC MolecularBiology vol 9 article 53 2008

[50] C Kozlowski and R M Weimer ldquoAn automated method toquantify microglia morphology and application to monitoractivation state longitudinally in vivordquo PLoS ONE vol 7 no 2Article ID e31814 2012


[51] A Gombault L Baron and I Couillin ldquoATP release andpurinergic signaling in NLRP3 inflammasome activationrdquoFrontiers in Immunology vol 3 article 414 2012

[52] R Zhou A Tardivel B Thorens I Choi and J TschoppldquoThioredoxin-interacting protein links oxidative stress toinflammasome activationrdquo Nature Immunology vol 11 no 2pp 136ndash140 2010

[53] L Minutoli D Puzzolo M Rinaldi et al ldquoROS-mediatedNLRP3 inflammasome activation in brain heart kidney andtestis ischemiareperfusion injuryrdquo Oxidative Medicine andCellular Longevity vol 2016 Article ID 2183026 10 pages 2016

[54] D W Brann Q-G Zhang R-M Wang V B Mahesh andR K Vadlamudi ldquoPELP1-A novel estrogen receptor-interactingproteinrdquoMolecular and Cellular Endocrinology vol 290 no 1-2pp 2ndash7 2008

[55] L Lammerding A Slowik S Johann C Beyer and A Zend-edel ldquoPost-stroke inflammasome expression and regulation inthe peri-infarct area by gonadal steroids after transient focalischemia in the rat brainrdquo Neuroendocrinology vol 103 no 52016

[56] Y Yu S Ugawa T Ueda et al ldquoCellular localization of P2X7receptor mRNA in the rat brainrdquo Brain Research vol 1194 pp45ndash55 2008

[57] G Collo S Neidhart E Kawashima M Kosco-Vilbois R ANorth and G Buell ldquoTissue distribution of the P2X7 receptorrdquoNeuropharmacology vol 36 no 9 pp 1277ndash1283 1997

[58] K Nagasawa C Escartin and R A Swanson ldquoAstrocytecultures exhibit P2X7 receptor channel opening in the absenceof exogenous ligandsrdquo Glia vol 57 no 6 pp 622ndash633 2009

[59] A Ohishi Y Keno A Marumiya et al ldquoExpression level ofP2X7 receptor is a determinant of ATP-induced death of mousecultured neuronsrdquo Neuroscience vol 319 pp 35ndash45 2016

[60] X-J Li R-F He S Li X-J Li and D-L Li ldquoEffects ofprogesterone on learning and memory and P2X7 receptorexpression in the hippocampus after global cerebral ischemiareperfusion injury in ratsrdquo Zhongguo Ying Yong Sheng Li Xue ZaZhi vol 28 no 5 pp 472ndash475 2012

[61] Y-M Lu R-R Tao J-Y Huang et al ldquoP2X 7 signaling pro-motes microsphere embolism-triggeredmicroglia activation bymaintaining elevation of Fas ligandrdquo Journal of Neuroinflamma-tion vol 9 article 172 2012

[62] A Melani S Amadio M Gianfriddo et al ldquoP2X7 receptormodulation on microglial cells and reduction of brain infarctcaused by middle cerebral artery occlusion in ratrdquo Journal ofCerebral Blood Flow andMetabolism vol 26 no 7 pp 974ndash9822006

[63] J Couturier I-C Stancu O Schakman et al ldquoActivation ofphagocytic activity in astrocytes by reduced expression of theinflammasome component ASC and its implication in a mousemodel ofAlzheimer diseaserdquo Journal of Neuroinflammation vol13 article 20 2016

[64] Y-C Wang W-Z Li Y Wu et al ldquoAcid-sensing ion channel1a contributes to the effect of extracellular acidosis on NLRP1inflammasome activation in cortical neuronsrdquo Journal of Neu-roinflammation vol 12 no 1 article 246 2015

[65] S E Adamczak J P De Rivero Vaccari G Dale et al ldquoPyrop-totic neuronal cell deathmediated by theAIM2 inflammasomerdquoJournal of Cerebral Blood Flow andMetabolism vol 34 no 4 pp621ndash629 2014

[66] X Wang G Chu Z Yang et al ldquoEthanol directly inducedHMGB1 release through NOX2NLRP1 inflammasome in neu-ronal cellsrdquo Toxicology vol 334 pp 104ndash110 2015

[67] Q-G Zhang R Wang M Khan V Mahesh and D WBrann ldquoRole of Dickkopf-1 an antagonist of theWnt120573-cateninsignaling pathway in estrogen-induced neuroprotection andattenuation of tau phosphorylationrdquo Journal of Neurosciencevol 28 no 34 pp 8430ndash8441 2008

[68] R M Friedlander V Gagliardini H Hara et al ldquoExpressionof a dominant negative mutant of interleukin-1120573 convertingenzyme in transgenicmice prevents neuronal cell death inducedby trophic factor withdrawal and ischemic brain injuryrdquo Journalof Experimental Medicine vol 185 no 5 pp 933ndash940 1997

[69] H Hara R M Friedlander V Gagliardini et al ldquoInhibitionof interleukin 1120573 converting enzyme family proteases reducesischemic and excitotoxic neuronal damagerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 94 no 5 pp 2007ndash2012 1997

[70] M Rabuffetti C Sciorati G Tarozzo E Clementi A AManfredi and M Beltramo ldquoInhibition of caspase-1-like activ-ity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosisreduction and decrease of proinflammatory cytokinesrdquo Journalof Neuroscience vol 20 no 12 pp 4398ndash4404 2000

[71] G P Schielke G-Y Yang B D Shivers and A L BetzldquoReduced ischemic brain injury in interleukin-1120573 convertingenzyme- deficient micerdquo Journal of Cerebral Blood Flow andMetabolism vol 18 no 2 pp 180ndash185 1998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and uterus [16ndash20] In addition PELP1 exhibits both nuclearand cytoplasmic localization [20] and phosphorylation ofPELP1 can control its localization interaction with otherproteins and stability in cells [16 17] To understand itsimportance in E2 actions in the brain our group recentlycreated a PELP1 forebrain-specific knockout (PELP1 FBKO)mouse model [16] Using the PELP1 FBKO mouse modelwe demonstrated a critical role for PELP1 in mediating E2regulation of extranuclear and genomic signaling as well asE2-induced neuroprotection and cognitive function in thehippocampus following ischemic injury [16]

While the role of E2 as a neuroprotective factor has beenwell established its role in regulation of neuroinflamma-tion has been less studied Growing evidence suggests thatneuroinflammation can contribute significantly to neuronaldamage and cell death in neurodegenerative disorders andthat attenuation of neuroinflammation can be neuropro-tective [21ndash23] With respect to E2 several studies haveshown that it can suppress microglial activation and proin-flammatory cytokine production in vivo and in vitro [24ndash28] although the mechanisms underlying the effects remainunclear A significant advance to the neuroinflammation fieldoccurred in 2002 when Martinon and coworkers identifiedldquoinflammasomesrdquo as key proteins that trigger the inflamma-tory response [29] Inflammasomes are multiprotein com-plexes that mediate activation of caspase-1 and promotesecretion of proinflammatory cytokines Inflammasomes areactivated in response to either pathogen associatedmolecularpatterns (PAMPs) derived from invading pathogens ordamage-associated molecular patterns (DAMPs) released bydying cells [30] Inflammasomes belong to the NOD-likereceptor family (NLRs) NLRs are encoded by a family of 22genes and are mainly divided as NLRP (NOD-like receptorwith a pyrin domain) and NLRC (NOD-like receptors withCARD domain) family members [31]

The structure of NLRP includes a carboxy-terminalleucine rich repeat (LRR) a nucleotide binding domaincalled the NACHT domain and an N-terminal pyrin domain(PYD) The PYD domain of inflammasome binds to a PYDdomain of apoptosis associated speck like protein (ASC)which is an adaptor protein [31] ASC also has a caspaseactivation and recruitment (CARD) domain which binds tothe CARD domain of the procaspase-1 enzyme NLRP3 isthemost abundantly found inflammasome and contributes tothemajority of production of proinflammatory cytokines [2331 32] Inflammasomes are primed and activated by triggerslike pathogens and metabolic and genotoxic stressors Forinstance the P2X7 receptor is activated by elevation ofextracellular ATP which then leads to activation of theNLRP3 inflammasome complex [33] Once activated inflam-masomes enhance downstream activation of caspase-1 andrelease of proinflammatory cytokines such as IL-1120573 and IL-18[29 34] which initiate a cascade of detrimental inflammatoryevents and apoptosis [35]

Of all of the inflammasome proteins NLRP3 is byfar the most studied inflammasome in cerebral ischemiastudies NLPR3 expression has been described in multiplebrain cell types including astrocytes microglia neuronsand endothelial cells [22 36ndash39] Numerous studies have

shown that NLRP3 is elevated in the brain of human strokepatients [40] and experimental stroke animals [40ndash44]Furthermore NLRP3 appears to have an important role inischemic pathology as NLRP3 knockout animals have signif-icantly reduced infarct size and neurovascular damage afterfocal cerebral ischemia [38] Additionally immunoglobulintreatment suppresses NLRP3 activity and strongly protectsneurons in ischemic animals [40] In addition Aim2 andNLRC4 knockout animals have also been reported to havereduced inflammation and infarct size after focal cerebralischemia but the effects appear to be independent of IL-1120573production [45]

With respect to E2 regulation of NLRP3 inflammasomeactivation E2 has been reported in one study to suppressNLRP3 inflammasome gene expression in the cerebral cortexafter focal cerebral ischemia (FCI) [46] while in a secondstudy an ER-120573 agonist suppressed caspase-1 ASC and IL-1120573expression in the hippocampus after global cerebral ischemia(GCI) [47]These studies suggest that E2 can regulate inflam-masome activation however several important questionsremain unanswered including the following (1) What isthe temporal pattern of NLRP3 inflammasome expressionin the hippocampus after GCI (2) In which cell type inthe hippocampus does NLRP3 inflammasome activationoccur (3) Does E2 only affect expression of the NLRP3inflammasome or does it also regulate NLRP3 inflammasomecomplex formation (4)What are themechanisms underlyingE2 regulation of the NLRP3 inflammasome (5) Does theER coregulator PELP1 mediate E2 regulation of the NLRP3inflammasome in the brain The goal of the current studywas to address these key questions The results of the studyreveal that both expression and complex formation of theNLRP3 inflammasome as well as activated caspase-1 and IL-1120573 are robustly increased in the hippocampus of both therat and mouse after GCI and this effect is strongly inhibitedby E2 replacement The results also demonstrate that NLRP3inflammasome activation occurs in bothmicroglia and astro-cytes after GCI and that E2 inhibits expression of both P2X7and TXNIP two well-known upstream inducers of NLRP3inflammasome activation Finally the results reveal that theER coregulator protein PELP1 is essential for mediation of E2regulatory effects upon activation of the NLRP3 inflamma-some

2 Materials and Methods

21 Animals and Surgical Procedures

211 OvariectomizedRat Studies AugustaUniversity Institu-tional Animal Care and Use Committee approved all animalprocedures and the studies were conducted in accordancewith National Institutes of Health guidelines for animalresearch Three-month-old young adult Sprague Dawley ratswere bilaterally ovariectomized under isoflurane anesthesiaand separated into shams ischemia-reperfusion injury andinjury with estrogen (E2) treatment groupsThe E2 treatmentgroup animals were immediately administered with 17120573-estradiol dissolved in 20 120573-cyclodextrin added to mini-pumps (05 120583Lhr 14-day release Alzet Cupertino CA)











Table 1












3 Results



1 3 7(Day)

GCI


lowastlowast

0

05

1

15

2

25N

LRP3

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(a)

ASC mRNA


lowastlowast

1 3 7(Day)

GCI

0

05

1

15

2

25

3

35

4

ASC

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(b)

Caspase-1 mRNA

lowastlowast

lowastlowast

lowastlowast

1 3 7(Day)

GCI

0

05

1

15

2

25

Casp

ase-

1 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

） + 2

(c)

lowastlowast

lowastlowastlowast

1 3 7(Day)

GCI

IL-1 mRNA

0

5

10

15

20

25

30

35

IL-1

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(d)





NLRP3

GAPDH

Sham GCI

(100 kDa)

(37 kDa)

） + 2

(a)

ASC

GAPDH

(24 kDa)

(37 kDa)

(b)

Cl-caspase-1

GAPDH

Sham GCI

(37 kDa)

(20 kDa)

） + 2

(c)

GAPDH (37 kDa)

(17 kDa)Cl-IL-1

(d)

Sham GCI

NLRP3 lowast

0

1

2

3

4

NLR

P3 p

rote

in (f

old

chan

ge)

） + 2

(e)

ASC

Sham GCI

lowast

0

1

2

3

4

5

ASC

pro

tein

(fol

d ch

ange

)

） + 2

(f)

Cl-caspase-1

Sham GCI

lowast

0

05

1

15

2

25

3

Cl-c

aspa

se-1

pro

tein

(fol

d ch

ange

)

） + 2

(g)Sham GCI

lowast

Cleaved IL-1

0

05

1

15

2

25

Clea

ved

IL-1

prot

ein

(fold

chan

ge)

） + 2

(h)






Sham

7-da

yG

CI7-

day


）+2

(a)NLRP3 ASC

Cl-caspase-1

Sham GCI

Sham GCI Sham GCI

Sham GCI

lowast

lowast lowast

lowast

IL-1

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

0

5

10

15

20

25

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

0

5

10

15

20

25

IL-1

expr

essio

n (

area

activ

ated

)

0

2

4

6

8

10

12

14A

SC ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

） + 2

） + 2

） + 2

(b)



Sham

NLR

P3-A

SC

inte

ract

ion

(PLA

)

7 d GCI 7 d ） + 2

(a)

lowast

Sham GCI0

2

4

6

8

10

12

NLR

P3-A

SC in

tera

ctio

n (

area

inte

ract

ion)

） + 2

(b)








Iba1

P2X7

rN

euN


(a)P2X7 intensity

lowast

0

1

2

3

4

5

6

P2X7

expr

essio

n (

area

activ

ated

)

Sham GCI ） + 2

(b)

P2Xr7 mRNA

lowastlowast

lowastlowast

0

05

1

15

2

P2Xr

7 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

1 3 7(Day)

GCI） + 2

(c)











4 Discussion



FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Table 1












3 Results



1 3 7(Day)

GCI


lowastlowast

0

05

1

15

2

25N

LRP3

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(a)

ASC mRNA


lowastlowast

1 3 7(Day)

GCI

0

05

1

15

2

25

3

35

4

ASC

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(b)

Caspase-1 mRNA

lowastlowast

lowastlowast

lowastlowast

1 3 7(Day)

GCI

0

05

1

15

2

25

Casp

ase-

1 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

） + 2

(c)

lowastlowast

lowastlowastlowast

1 3 7(Day)

GCI

IL-1 mRNA

0

5

10

15

20

25

30

35

IL-1

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(d)





NLRP3

GAPDH

Sham GCI

(100 kDa)

(37 kDa)

） + 2

(a)

ASC

GAPDH

(24 kDa)

(37 kDa)

(b)

Cl-caspase-1

GAPDH

Sham GCI

(37 kDa)

(20 kDa)

） + 2

(c)

GAPDH (37 kDa)

(17 kDa)Cl-IL-1

(d)

Sham GCI

NLRP3 lowast

0

1

2

3

4

NLR

P3 p

rote

in (f

old

chan

ge)

） + 2

(e)

ASC

Sham GCI

lowast

0

1

2

3

4

5

ASC

pro

tein

(fol

d ch

ange

)

） + 2

(f)

Cl-caspase-1

Sham GCI

lowast

0

05

1

15

2

25

3

Cl-c

aspa

se-1

pro

tein

(fol

d ch

ange

)

） + 2

(g)Sham GCI

lowast

Cleaved IL-1

0

05

1

15

2

25

Clea

ved

IL-1

prot

ein

(fold

chan

ge)

） + 2

(h)






Sham

7-da

yG

CI7-

day


）+2

(a)NLRP3 ASC

Cl-caspase-1

Sham GCI

Sham GCI Sham GCI

Sham GCI

lowast

lowast lowast

lowast

IL-1

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

0

5

10

15

20

25

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

0

5

10

15

20

25

IL-1

expr

essio

n (

area

activ

ated

)

0

2

4

6

8

10

12

14A

SC ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

） + 2

） + 2

） + 2

(b)



Sham

NLR

P3-A

SC

inte

ract

ion

(PLA

)

7 d GCI 7 d ） + 2

(a)

lowast

Sham GCI0

2

4

6

8

10

12

NLR

P3-A

SC in

tera

ctio

n (

area

inte

ract

ion)

） + 2

(b)








Iba1

P2X7

rN

euN


(a)P2X7 intensity

lowast

0

1

2

3

4

5

6

P2X7

expr

essio

n (

area

activ

ated

)

Sham GCI ） + 2

(b)

P2Xr7 mRNA

lowastlowast

lowastlowast

0

05

1

15

2

P2Xr

7 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

1 3 7(Day)

GCI） + 2

(c)











4 Discussion



FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 3 7(Day)

GCI


lowastlowast

0

05

1

15

2

25N

LRP3

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(a)

ASC mRNA


lowastlowast

1 3 7(Day)

GCI

0

05

1

15

2

25

3

35

4

ASC

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(b)

Caspase-1 mRNA

lowastlowast

lowastlowast

lowastlowast

1 3 7(Day)

GCI

0

05

1

15

2

25

Casp

ase-

1 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

） + 2

(c)

lowastlowast

lowastlowastlowast

1 3 7(Day)

GCI

IL-1 mRNA

0

5

10

15

20

25

30

35

IL-1

mRN

A (f

old

chan

ge v

ersu

s sha

m)

） + 2

(d)





NLRP3

GAPDH

Sham GCI

(100 kDa)

(37 kDa)

） + 2

(a)

ASC

GAPDH

(24 kDa)

(37 kDa)

(b)

Cl-caspase-1

GAPDH

Sham GCI

(37 kDa)

(20 kDa)

） + 2

(c)

GAPDH (37 kDa)

(17 kDa)Cl-IL-1

(d)

Sham GCI

NLRP3 lowast

0

1

2

3

4

NLR

P3 p

rote

in (f

old

chan

ge)

） + 2

(e)

ASC

Sham GCI

lowast

0

1

2

3

4

5

ASC

pro

tein

(fol

d ch

ange

)

） + 2

(f)

Cl-caspase-1

Sham GCI

lowast

0

05

1

15

2

25

3

Cl-c

aspa

se-1

pro

tein

(fol

d ch

ange

)

） + 2

(g)Sham GCI

lowast

Cleaved IL-1

0

05

1

15

2

25

Clea

ved

IL-1

prot

ein

(fold

chan

ge)

） + 2

(h)






Sham

7-da

yG

CI7-

day


）+2

(a)NLRP3 ASC

Cl-caspase-1

Sham GCI

Sham GCI Sham GCI

Sham GCI

lowast

lowast lowast

lowast

IL-1

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

0

5

10

15

20

25

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

0

5

10

15

20

25

IL-1

expr

essio

n (

area

activ

ated

)

0

2

4

6

8

10

12

14A

SC ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

） + 2

） + 2

） + 2

(b)



Sham

NLR

P3-A

SC

inte

ract

ion

(PLA

)

7 d GCI 7 d ） + 2

(a)

lowast

Sham GCI0

2

4

6

8

10

12

NLR

P3-A

SC in

tera

ctio

n (

area

inte

ract

ion)

） + 2

(b)








Iba1

P2X7

rN

euN


(a)P2X7 intensity

lowast

0

1

2

3

4

5

6

P2X7

expr

essio

n (

area

activ

ated

)

Sham GCI ） + 2

(b)

P2Xr7 mRNA

lowastlowast

lowastlowast

0

05

1

15

2

P2Xr

7 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

1 3 7(Day)

GCI） + 2

(c)











4 Discussion



FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NLRP3

GAPDH

Sham GCI

(100 kDa)

(37 kDa)

） + 2

(a)

ASC

GAPDH

(24 kDa)

(37 kDa)

(b)

Cl-caspase-1

GAPDH

Sham GCI

(37 kDa)

(20 kDa)

） + 2

(c)

GAPDH (37 kDa)

(17 kDa)Cl-IL-1

(d)

Sham GCI

NLRP3 lowast

0

1

2

3

4

NLR

P3 p

rote

in (f

old

chan

ge)

） + 2

(e)

ASC

Sham GCI

lowast

0

1

2

3

4

5

ASC

pro

tein

(fol

d ch

ange

)

） + 2

(f)

Cl-caspase-1

Sham GCI

lowast

0

05

1

15

2

25

3

Cl-c

aspa

se-1

pro

tein

(fol

d ch

ange

)

） + 2

(g)Sham GCI

lowast

Cleaved IL-1

0

05

1

15

2

25

Clea

ved

IL-1

prot

ein

(fold

chan

ge)

） + 2

(h)






Sham

7-da

yG

CI7-

day


）+2

(a)NLRP3 ASC

Cl-caspase-1

Sham GCI

Sham GCI Sham GCI

Sham GCI

lowast

lowast lowast

lowast

IL-1

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

0

5

10

15

20

25

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

0

5

10

15

20

25

IL-1

expr

essio

n (

area

activ

ated

)

0

2

4

6

8

10

12

14A

SC ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

） + 2

） + 2

） + 2

(b)



Sham

NLR

P3-A

SC

inte

ract

ion

(PLA

)

7 d GCI 7 d ） + 2

(a)

lowast

Sham GCI0

2

4

6

8

10

12

NLR

P3-A

SC in

tera

ctio

n (

area

inte

ract

ion)

） + 2

(b)








Iba1

P2X7

rN

euN


(a)P2X7 intensity

lowast

0

1

2

3

4

5

6

P2X7

expr

essio

n (

area

activ

ated

)

Sham GCI ） + 2

(b)

P2Xr7 mRNA

lowastlowast

lowastlowast

0

05

1

15

2

P2Xr

7 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

1 3 7(Day)

GCI） + 2

(c)











4 Discussion



FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sham

7-da

yG

CI7-

day


）+2

(a)NLRP3 ASC

Cl-caspase-1

Sham GCI

Sham GCI Sham GCI

Sham GCI

lowast

lowast lowast

lowast

IL-1

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

0

5

10

15

20

25

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

0

5

10

15

20

25

IL-1

expr

essio

n (

area

activ

ated

)

0

2

4

6

8

10

12

14A

SC ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

） + 2

） + 2

） + 2

(b)



Sham

NLR

P3-A

SC

inte

ract

ion

(PLA

)

7 d GCI 7 d ） + 2

(a)

lowast

Sham GCI0

2

4

6

8

10

12

NLR

P3-A

SC in

tera

ctio

n (

area

inte

ract

ion)

） + 2

(b)








Iba1

P2X7

rN

euN


(a)P2X7 intensity

lowast

0

1

2

3

4

5

6

P2X7

expr

essio

n (

area

activ

ated

)

Sham GCI ） + 2

(b)

P2Xr7 mRNA

lowastlowast

lowastlowast

0

05

1

15

2

P2Xr

7 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

1 3 7(Day)

GCI） + 2

(c)











4 Discussion



FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sham

NLR

P3-A

SC

inte

ract

ion

(PLA

)

7 d GCI 7 d ） + 2

(a)

lowast

Sham GCI0

2

4

6

8

10

12

NLR

P3-A

SC in

tera

ctio

n (

area

inte

ract

ion)

） + 2

(b)








Iba1

P2X7

rN

euN


(a)P2X7 intensity

lowast

0

1

2

3

4

5

6

P2X7

expr

essio

n (

area

activ

ated

)

Sham GCI ） + 2

(b)

P2Xr7 mRNA

lowastlowast

lowastlowast

0

05

1

15

2

P2Xr

7 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

1 3 7(Day)

GCI） + 2

(c)











4 Discussion



FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Iba1

P2X7

rN

euN


(a)P2X7 intensity

lowast

0

1

2

3

4

5

6

P2X7

expr

essio

n (

area

activ

ated

)

Sham GCI ） + 2

(b)

P2Xr7 mRNA

lowastlowast

lowastlowast

0

05

1

15

2

P2Xr

7 m

RNA

(fol

d ch

ange

ver

sus s

ham

)

1 3 7(Day)

GCI） + 2

(c)











4 Discussion



FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4 Discussion



FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















FLOX sham FLOX GCI

NLR

P3N

LRP3

KO sham KO GCI KO

FLOX ） + 2

） + 2

(a)

FLOX KO

NS

NLRP3

ShamGCI

$

lowast

0

2

4

6

8

10

12

NLR

P3 ex

pres

sion

( ar

ea ac

tivat

ed)

） + 2

(b)

ASC

ASC

KO sham KO GCI KO


） + 2

(c)

FLOX KO

NSASC

$

lowast

ShamGCI

0

2

4

6

8

10

12

14

16

ASC

expr

essio

n (

area

activ

ated

)

） + 2

(d)






-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















-cas

pase

-1Cl

-cas

pase

-1


KO sham KO GCI KO

） + 2

） + 2

(a)

Cl-caspase-1

FLOX KO

ShamGCI

0

2

4

6

8

10

Cl-c

aspa

se-1

expr

essio

n (

area

activ

ated

)

NS

$

lowast

） + 2

(b)

FLOX sham FLOX GCI

KO sham KO GCI

IL-1

IL-1

FLOX ） + 2

KO ） + 2

(c)

FLOX KO

ShamGCI

NS

$

lowast

IL-1

0

5

10

15

20

25

30IL

-1

expr

essio

n (

area

activ

ated

)

） + 2

(d)





LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LRP3

-ASC

inte

ract

ion

(PLA

)N

LRP3

-ASC

inte

ract

ion

(PLA

)

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

NS

ShamGCI

$

lowast

mdash

200

400

600

800

1000

NLR

P3-A

SC co

mpl

ex (

area

activ

ated

)

） + 2

(b)








Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Iba1

P2X7

Neu

NIb

a1P2

X7N

euN

FLOX sham FLOX GCI

KO sham KO GCI

FLOX ） + 2

KO ） + 2

(a)

FLOX KO

P2X7

NS

ShamGCI

0

2

4

6

8

10

12

P2X7

expr

essio

n (

area

activ

ated

)

lowast

） + 2

(b)







Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Abbreviations


Competing Interests




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article NLRP3 Inflammasome Activation in the Brain...

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Transcript of Research Article NLRP3 Inflammasome Activation in the Brain...