Neuroprotective effect of ginkgolide B on bupivacaine ... · 2...

Title Neuroprotective effect of ginkgolide B on bupivacaine-inducedapoptosis in SH-SY5Y cells

Author(s) Li L Zhang QG Lai LY Wen XJ Zheng T Cheung CW ZhouSQ Xu SY

Citation Oxidative Medicine and Cellular Longevity 2013 v 2013 particle no 159864

Issued Date 2013

URL httphdlhandlenet10722194665

Rights Creative Commons Attribution 30 Hong Kong License

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 159864 11 pageshttpdxdoiorg1011552013159864

Research ArticleNeuroprotective Effect of Ginkgolide B on Bupivacaine-InducedApoptosis in SH-SY5Y Cells

Le Li1 Qing-guo Zhang1 Lu-ying Lai1 Xian-jie Wen1 Ting Zheng1

Chi-wai Cheung2 Shu-qin Zhou1 and Shi-yuan Xu1

1 Department of Anesthesiology Zhujiang Hospital Southern Medical University Guangzhou Guangdong 510282 China2Department of Anesthesiology The University of Hong Kong Hong Kong

Correspondence should be addressed to Shu-qin Zhou 154843189qqcom and Shi-yuan Xu xsy111yeahnet

Received 5 July 2013 Accepted 18 August 2013

Academic Editor Zhengyuan Xia

Copyright copy 2013 Le Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Local anesthetics are used routinely and effectively However many are also known to activate neurotoxic pathways We tested theneuroprotective efficacy of ginkgolide B (GB) an active component of Ginkgo biloba against ROS-mediated neurotoxicity causedby the local anesthetic bupivacaine SH-SY5Y cells were treated with different concentrations of bupivacaine alone or followingpreincubation with GB Pretreatment with GB increased SH-SY5Y cell viability and attenuated intracellular ROS accumulationapoptosis mitochondrial dysfunction and ER stress GB suppressed bupivacaine-induced mitochondrial depolarization andmitochondria complex I and III inhibition and increased cleaved caspase-3 and Htra2 expression which was strongly indicative ofactivation of mitochondria-dependent apoptosis with concomitantly enhanced expressions of Grp78 caspase-12 mRNA proteinand ER stress GB also improved ultrastructural changes indicative ofmitochondrial and ER damage induced by bupivacaineTheseresults implicate bupivacaine-induced ROS-dependent mitochondria ER dysfunction and apoptosis which can be attenuated byGB through its antioxidant property

1 Introduction

Local anesthetics are among the most common clinical drugsand are generally regarded as safe [1 2] However they havealso been shown to be neurotoxic even at normal clinicaldose [3 4] This neurotoxicity is mediated at least in partby activation of apoptotic pathways [5 6] In the caudaequina intrathecally administered local anesthetics inducedcell swelling atrophy edema axonal degeneration and theappearance of myelin ovoids as well as macrophage infiltra-tion [7] These morphological signs of degeneration indicatethat local anesthetics can initiate a complex cascade of directcytotoxic and ensuing inflammatory responses although themolecular mechanisms of local anesthetic toxicity are stilllargely unknown

Local anesthetics have been shown to induce neuraldysfunction and apoptosis in vitro [8ndash11] For example bupi-vacaine may inhibit mitochondrial respiratory complexes Iand III leading to decreased ATP production collapse of themitochondrial membrane potential (Δ120595m) overproduction

of reactive oxygen species (ROS) and ultimately liberationof cytochrome c and activation of the caspase-3-dependentapoptosis pathway [10ndash12] In fact ROS accumulation mito-chondrial uncoupling and depolarization of 120595m are amongthe earliest indicators of apoptosis induced by local anesthet-ics [13 14] In addition tomitochondrial damage dysfunctionof the endoplasmic reticulum (ER) stress has also beenimplicated in apoptosis Arai and Nonaka et al proposed thatoxidative stress associated with local anesthetics can induceCa2+ release from intracellular stores including the roughendoplasmic reticulum (rER) [11 15] Loss of intraluminalCa2+ may lead to ER stress [16] further ROS generation[17] and activation of ER-dependent apoptosis pathways [18]Thus mitochondrial and ER damage associated with ROSoverproduction may act synergistically to evoke cell deathin response to bupivacaine or other structurally related localanesthetics

Ginkgo biloba has been used in traditional Chinesemedicine for thousands of years Evidence accumulated overthe last decade suggests that concentrated and partially

2 Oxidative Medicine and Cellular Longevity

Control 05 10 15 20Concentration of bupivacaine (mmolL)

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Figure 1 Proliferation effects of bupivacaine on SH-SY5Y cells and GB weakened bupivacaine-induced cell injury in SH-SY5Y cells (a) Cellswere incubated in the presence or absence of various concentrations of bupivacaine for 24 h (lowast119875 lt 005 versus control group) Cell growthwas determined by the MTT assay (b) Cell viability was decreased by treatment with 1mmolL bupivacaine for 24 hours Decreased viabilitywas inhibited by GB pretreatment for 6 hours except for cells treated with 5120583molL GB (lowast119875 lt 005 versus nonpretreated group) Each datapoint represents the mean plusmn SD of 6 separate experiments

purified extracts of Ginkgo biloba leaves may afford pro-tection against certain neurological diseases [19] Indeedginkgolide B (GB) the major active component of Ginkgobiloba extract has been used to treat degenerative dementiaand neurosensory disorders [20] Even for children GB is asafe drug without adverse reactions [21] Furthermore GBreduced the level of ROS in vivo suggesting that the rawextract contains antioxidants [22]Therefore GBmay protectneurons against the neurotoxicity of local anesthetics likebupivacaine by reducing oxidative stress

The principal aims of this study were to examine themolecular mechanisms of bupivacaine toxicity and the neu-roprotective efficacy of GB in vitro

2 Materials and Methods

21 Materials The human neuroblastoma cell line SH-SY5Ywas purchased from the Shanghai Institutes for BiologicalSciences Bupivacaine hydrochloride (purity 999) waspurchased from Sigma (St Louis MO USA) Ginkgolide B(purityge 995)was obtained from theNational Institutes forFood and Drug Control and dissolved in dimethyl sulfoxide(DMSO) (KeyGEN China) Other reagents used includedDMEMF12 medium and fetal bovine serum (GibcoUSA) 551015840 661015840-tetrachloro-111015840 331015840-tetraethyl tetrethylbenzimidalyl carbocyanine iodide (JC-1) 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2-tetrazolium bromide (MTT) 2101584071015840-dichlorofluorescein diacetate (DCFH-DA) mitochondrialisolation agent and mitochondrial storage solution (all fromBeyotime China) anti-Grp78 and anti-caspase-12 (AbgentUSA) anti-cleaved caspase-3 and anti-HtrA2 (Abcam UK)anti-GAPDH antibody (Goodhere China) and AnnexinV-FITC and propidium iodide (KeyGEN China) Thecell counting Kit-8 (CCK8) was purchased from Dojindo

(Dojindo Kumamoto Japan) All reagents were obtainedfrom commercial suppliers and were of standard biochemicalquality

22 Cell Culture Cells of the SH-SY5Y line were culturedin DMEMF12 medium supplemented with 15 fetal bovineserum 100UmLpenicillin and 100120583gmL streptomycin andmaintained in a humidified 5 CO

2incubator at 37∘C The

media were changed every 2 days

23 MTT Assay The effect of bupivacaine on the numberof viable SH-SY5Y cells was determined by the MTT assayCells were seeded onto 96-well plates at 5 times 103 cellswellwith 100120583L of culture medium and treated with various con-centrations of bupivacaine as indicated below Treated cellswere incubated with 20120583L MTT at 37∘C for 4 h the mediumremoved and 150120583L DMSO added to dissolve the formazancrystals produced from MTT by viable cells The opticaldensity (OD) of the homogenous purple formazanDMSOsolutions was measured using a spectrophotometer (Bio-TekUSA) at 570 nm

24 CCK-8 Assay Cells were seeded onto 96-well plates at5 times 103 cellswell in 100120583L culture medium In pilot experi-ments to determine the working range of GB cultures werepretreated with 5ndash40 120583molL GB in new media for 6 h orsubjected to a control media change prior to treatment with1mmolL bupivacaine for 24 h (the half-maximal neurotoxicdose according to [23]) After bupivacaine treatment 10 120583L ofCCK-8was added to eachwell for another 3 h at 37∘CTheODwas read at 450 nm using a spectrophotometer

25 Apoptosis Assay by Flow Cytometry Cells were seededonto 24-well plates at 5 times 105 cellswell in 500120583L culture

Oxidative Medicine and Cellular Longevity 3

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(a) 0120583molL GB + 1mmolL bupivacaine

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(d) 20120583molL GB + 1mmolL bupivacaine

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(e) 40120583molL GB + 1mmolL bupivacaine

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Figure 2 GB pretreatment decreased the number of apoptotic cells induced by bupivacaine ((a)ndash(e)) Cells were treated with 0 5 10 20 and40120583molL GB for 6 hours respectively prior to treatment with 1mmolL bupivacaine for 24 h (f) Summarized data show apoptotic rate asdetected by flow cytometry Data represented are the mean plusmn SD of 6 separate experiments (lowast119875 lt 005 versus 0 120583molL GB)

medium After control or GB pretreatment and bupivacaineadministration as described the cells were rinsed with PBSharvested and resuspended in 500120583L binding buffer To thiscell suspensionwas added 5120583LAnnexinV-FITC (amarker ofearly apoptosis) and 5120583L propidium iodide (a marker of lateapoptosis) After 10min incubation cell apoptosis was deter-mined by flow cytometry (BD FACS Calibur USA)

26 Measurement of Reactive Oxygen Species Cells wereseeded onto 24-well plates at 5 times 105 cellswell in 500120583L

culture medium and divided into four treatment groups (i)untreated controls (Con) (ii) cells treated with 1mmolLbupivacaine for 24 h (Bup) (iii) cells pretreated with40 120583molL GB for 6 h and (iv) cells treated with 40120583molLGB for 6 h prior to 1mmolL bupivacaine exposure for 24 h(GB+Bup) Intracellular accumulation of ROSwas estimatedusing the redox-sensitive fluorescent dye DCFH-DA Thecells were incubated with 10120583molL DCFH-DA at 37∘C dur-ing the last 20min of Con Bup GB or GB + Bup treatmentTreated and DCFH-DA-stained cells were washed 3 times


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Figure 3 The levels of reactive oxygen species were measured by flow cytometry GB pretreatment decreased ROS overproduction inducedby bupivacaine Summarized data shows the Geo mean of ROS as detected by flow cytometry Data represented are mean plusmn SD of 6 separateexperiments (lowast119875 lt 001)

in PBS harvested and resuspended in PBS Fluorescentsignal intensitywas determined by flow cytometry to estimaterelative ROS accumulation

27 Mitochondrial Membrane Potentials Assay Mitochon-drialmembrane potential (120595m)depolarization an early eventin the mitochondrial apoptosis cascade was measured fluo-rometrically using JC-1 Briefly cells cultured in 6-well plates

and treated as described for the ROSmeasurementwere incu-batedwith JC-1 staining solution (5120583gmL) at 37∘C for 20minand rinsed twice with PBS Mitochondrial membrane poten-tial was estimated by measuring the fluorescence ratio of freeJC-1 monomers (green) to JC-1 aggregates in mitochondria(red) by dual emission fluorescence microscopy (NikonECLIPSE TE2000-u Japan) and flow cytometry Mitochon-drial depolarization is indicated by an increase in the propor-tion of cells emitting green fluorescence


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Figure 4 GB attenuated the bupivacaine-induced decline of mitochondrial membrane potential (Δ120595m) SH-SY5Y cells were treated withbupivacaine for 24 hours in the presence or absence of GB (a) SH-SY5Y cells were observed using fluorescent microscopy (b) Δ120595m weredetected by flow cytometry (c) Δ120595m expressed as the ratio of red fluorescence over green fluorescence Data represented are the mean plusmn SDof 6 separate experiments (lowast119875 lt 001)


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Figure 5 GB attenuated bupivacaine-induced decrease in theactivity of mitochondrial complexes I and III Experiments wererepeated 6 times and the data were presented as the mean plusmn SD(lowast119875 lt 001)

28 Isolation of Mitochondrial Mitochondria were isolatedfrom SH-SY5Y cells cultured in 6-well plates and treatedas described for ROS measurements After rinsing twice inPBS cells were harvested centrifuged at 600timesg for 5min at4∘C and then homogenized in 1mL mitochondria isolationreagent until 50 of the cells were lysed Homogenates werecentrifuged at 600timesg for 5min at 4∘C to remove large debrisand unlysed cells The supernatant containing mitochondriathen transferred to another centrifuge tube and mitochon-dria precipitated by centrifugation at 11000timesg for 10minat 4∘C After centrifugation the pellet was resuspended inmitochondrial storage solution

29 Measurement of Respiratory Complex I and Complex IIIActivities The activities of respiratory chain complexes I andIII were determined according to the methods as describedby Zhang et al [24] All assays were performed at 25∘Cin a final volume of 1mL using a spectrophotometer Torelease complexes from the mitochondrial membrane iso-latedmitochondriawere subjected to three freeze-thaw cycles(25∘C to minus25∘C) in hypotonic media (25mmolL potassiumphosphate 5mmolLMgCl

2 pH 72) before activitymeasure-

mentsThe enzyme activity was expressed in nanomolars perminute per milligram protein

210 Western Blotting Cells were incubated as described forthe ROS measurements harvested and lysed in lysis bufferAfter centrifugation the soluble protein concentration inthe supernatant was determined by a BCA Protein AssayKit (Beyotime China) Protein samples were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis

Table 1 qRT-PCR primers

Gene Primers

18srRNA Forward 51015840-CCT GGA TAC CGC AGC TAG GA-31015840

Reverse 51015840-GCG GCG CAA TAC GAA TGC CCC-31015840

GRP78 Forward 51015840-TGC AGC AGG ACA TCA AGT TC-31015840

Reverse 51015840-CGC TGG TCA AAG TCT TCT CC-31015840

Caspase-12 Forward 51015840-GGA GAA AGA GAG GCG AAC AT-31015840

Reverse 51015840-CCT GGA TAC CGC AGC TAG GA-31015840

(20120583glane) electrotransferred to polyvinylidene difluoride(PVDF) membranes Membranes were blocked with 5nonfat drymilk inTris-buffered saline and then immunoblot-ted with anti-Grp78 (1 500) anti-caspase-12 (1 500) anti-cleaved caspase-3 (1 500) anti-HtrA2 (1 500) or anti-GAPDH antibody (1 1000 as the gel loading control)overnight at 4∘C All antibodies were diluted in Tris-HCl-buffered saline containing 5 nonfat dry milk and 01Tween-20 After rinsing immunolabeled membranes wereincubated with horseradish peroxidase (HRP) conjugatedanti-rabbit immunoglobulin (1 1000) for 1 h Specific pro-teins were detected by enhanced chemiluminescence andexposure to X-ray film Bands were quantified by scanningthe films The expression levels of Grp78 caspase-12 cleavedcaspase-3 and HtrA2 protein were normalized to GAPDH

211 Quantitative Real Time PCR (qRT-PCR) To investigatethe effect of bupivacaine on ER stress we examined Grp78and caspase-12mRNA expression levels by qRT-PCR TotalRNA was isolated using an RNA Isolation Kit (QiagenUSA) according to the manufacturerrsquos instructions DNaseI (TAKARA Japan) was used to remove DNA from totalRNA cDNA was synthesized using a cDNA Synthesis Kit(Promega USA) and theMaxima SYBRGreen qPCRMasterMix (2X) (Fermentas USA) was used to quantify geneexpression Conditions for amplification and quantificationincluded initial denaturing (50∘C for 2 minutes and 95∘C for10 minutes) followed by 40 cycles of 2 amplification stages(95∘C for 15 seconds and 60∘C for 1 minute) for primerannealing and elongation A dissociation stage (95∘C for 15seconds 60∘C for 1 minute and 95∘C for 15 seconds) wasadded at the end of amplification stage to ensure that a singleamplicon was produced and to validate the primer pairsReactions were performed in triplicate Relative expressionlevels of caspase-12 and Grp78 mRNA were quantified usingthe 2-ΔΔCT method [25 26] and 18 srRNA as the normaliz-ing gene The primers used are listed in Table 1

212 Transmission Electron Microscopy (TEM) Neuroblas-toma cells treated as described for the ROS measurementswere harvested washed once with PBS fixed in 25 glu-taraldehyde at 4∘C for 1 h postfixed in 1 osmic acid for30min and stained with lead uranium Cell ultrastructurewas observed under a transmission electron microscope(Hitachi-600 Japan)

213 Statistical Analysis All values are expressed as means plusmnSD Multiple comparisons between groups were analyzed by


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Figure 6 Htra2 induction and cleaved caspase-3 activation were detected by Western blot Experiments were repeated three times and thedata were presented as mean plusmn SD (lowast119875 lt 001 119875 lt 005)

one-way ANOVA LSD was performed as post hoc analysisfor multiple comparisons between groups A 119875 lt 005 wasconsidered significant

3 Results

31 Bupivacaine Reduced Cell Viability The effect of bupi-vacaine on the viability of SH-SY5Y neuroblastoma cellswas first examined using the by MTT assay Bupivacaine(1 15 2mmolL) significantly reduced viable cell numbercompared to controls (Figure 1(a)) We then estimated viablecell number at multiple time points during treatment with1mmolL bupivacaine the LD50 measured in a previousstudy [23] Compared to controls bupivacaine reduced viablecell number at all time points between 24 and 48 h Thusbupivacaine reduced SHSY5Y cell proliferation induced celldeath or both

32 GB Attenuated Cell Toxicity Induced by Bupivacaine Inour pilot experiment treatment with 5ndash40120583molL GB for6 h did not affect cell proliferation The antiproliferative orcytotoxic effect of 1mmolL bupivacaine was then comparedbetween GB-pretreated (5ndash40120583molL) and GB-naıve cul-tures using the CCK-8 assay Cell counts were higher at all GBdoses except at 5 120583molL the lowest dose tested (Figure 1(b))To determine if GB actually protected SHSY5Y cells againstbupivacaine-mediated cytotoxicity apoptosis was examinedby flow cytometry

33 GB Attenuated Bupivacaine-Induced SH-SY5Y Cell Apop-tosis Ginkgolide B pretreatment decreased bupivacaine-induced apoptosis as evidenced by reduced Annexin V+PIminusand Annexin V+PI+ cell numbers (representing early or lateapoptosis resp) at 40 120583molL GB (Figure 2) The reductionwas similar for both Annexin V+PIminus and Annexin V+PI+cell populations indicating that GB blocked the initiation ofapoptosis

34 GB Attenuated ROS Production Induced by Bupiva-caine Treatment with 1mmolL bupivacaine increased theintracellular ROS accumulation indicated by DCFH-DAfluorescencewhileGBpretreatment significantly reduced theROS-dependent fluorescent signal (Figure 3) These resultssuggest that GB acts to preserve mitochondrial functionelevates endogenous antioxidant capacity and (or) possessesinherent antioxidant activity

35 GB Inhibited Mitochondrial Depolarization Induced byBupivacaine The mitochondrial membrane potential (120595m)is correlated with functional activity while loss of 120595m (depo-larization) is indicative of mitochondrial uncoupling and isan early sign of apoptosis We estimated 120595m in SH-SY5Ycells by the shift in JC-1 fluorescence (from red to green)Exposure to 1mmolL bupivacaine resulted in 120595m dissipa-tion while GB pretreatment preserved 120595m during bupiva-caine exposure (Figure 4) These results suggest that GBmay prevent apoptosis by preserving mitochondrial functionand by preventing activation of mitochondrial-dependentapoptosis


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Figure 7 Grp78 and caspase-12 mRNA expression as detected byqRT-PCR Experimentswere repeated three times and the datawerepresented as the mean plusmn SD (lowast119875 lt 001 119875 lt 005)

36 Preservation of Mitochondrial Respiratory Complex I andIII Activity by GB The activities of mitochondrial complexesI and III were significantly reduced by bupivacaine sug-gesting potential loss of oxidative phosphorylation Againdisruption of mitochondrial function was reversed by GBpretreatment (Figure 5)

37 The Level of Cleaved Caspase-3 and HtrA2 Mitochon-drial uncoupling and 120595m depolarization are associated witha dramatic increase in mitochondrial inner membrane per-meability and the release of factors (eg cytochrome c andHtrA2) that initiate or regulate apoptotic pathways West-ern blots (Figure 6) revealed significantly elevated cleaved(activated) caspase-3 and HtrA2 expression in bupivacaine-treated cultures compared to controls an effect that wassuppressed by GB pretreatment

38 GB Reduced ER Stress Induced by Bupivacaine Expres-sion of Grp78 and caspase-12 is indicative of ER stress Quan-titative RT-PCR andWestern blots revealed enhanced Grp78and caspase-12 mRNA (Figure 7) and protein expressionlevels (Figure 8) in BG-naıve bupivacaine-treated cells com-pared to controls responses that were reversed by GBpretreatment

39 Morphological Changes of Cells Normal healthy SH-SY5Y cells were round and regular with typically shapedER and mitochondrial membranes in TEM images (Fig-ure 9(a)) After exposure to bupivacaine for 24 h the ERappeared swollen and degranulated while mitochondriawere swollen with loss of internal membrane structure

(Figure 9(b)) Cells treated with 40 120583molL GB showed anearly normal ultrastructure (Figure 9(c)) indicating thatGB had little endogenous toxicity or physiological effectson ER or mitochondrial function Cells pretreated with GBprior to bupivacaine exposure resembled controls at theultrastructural level with only slight expansion of the ER(Figure 9(d)) This preservation of ER and mitochondrialstructure strongly suggests that GB protected SH-SY5Y cellsagainst bupivacaine-induced mitochondrial and ER damage

4 Discussion

Ginkgolide B (GB) an active component of the traditionalmedicinal herb Ginkgo biloba protected SH-SY5Y cells frombupivacaine-induced injury Pretreatment with 40120583molLGB suppressed bupivacaine-induced mitochondrial depolar-ization mitochondria complex I and III inhibition ROSaccumulation ER stress and apoptosis These results impli-catemitochondrial dysfunction and ER stress in bupivacaine-induced apoptosis and highlight GB as a potential neuropro-tectant against bupivacaine toxicity through its antioxidantproperty

The therapeutic time window is critical in definingthe potential clinical utility of any neuroprotective agentGinkgolide B has been shown to exert significant protectiveeffect in cerebral ischemia injury up to 2 h following intra-venous administration after reperfusion in rat [27] In ourpilot experiment pretreatment with 5ndash40120583mmolL GB for2 h 4 h could not protect SH-SY5Y cells from bupivacaineneurotoxicity which is different from other peoplersquos previousresearch Only 6 h duration of pretreatment with GB con-ferred protective effect therefore 6 h treatment protocol wasused in our study

Oxidoredox homeostasis is essential for cellular survivalOverproduction of ROS leads to oxidative stress and plays animportant role in the process of apoptosis in many cell types[28] which can be ameliorated by endogenous and exogenousantioxidants Bupivacaine was shown to induce ROS genera-tion in SH-SY5Y cells [6] while GB reduced ROS levels invivo [22] suggesting that GBmay protect against bupivacainetoxicity by suppressing ROS accumulation Bupivacaine didsubstantially increase ROS amajor initiator of apoptosis [29]while preincubation with GB suppressed ROS accumulationand many of the biochemical and morphological signs ofoxidative stress To investigate the potential reasons forincreased ROS production we measured the activities ofmitochondrial complexes I and III the main generators ofROS [30] The activity of both complexes decreased afterbupivacaine treatment while GB pretreatment partiallyreversed this effect By preserving oxidative phosphorylationGB maintained 120595m and decreased ROS production associ-ated with mitochondrial uncoupling Aside from mitochon-drial dysfunction however ROS may also be generated bycalcium-dependent protease activity nNOS and acidosisthe contributions of which were not examined and warrantfurther study as possible mechanisms of bupivacaine toxicity

Besides energy production via the electron transportchain mitochondria are responsible for several other impor-tant cellular functions including the initiation and regulation


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Figure 8 Grp78 and caspase-12 expression representing ER stress as detected by Western blot Experiments were repeated three times andthe data were presented as the mean plusmn SD (lowast119875 lt 001 119875 lt 005)

(a) (b)

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Figure 9 Morphologic changes of SH-SY5Y cells (a) Cells in the control group retained a normal ultrastructure (b) cells in the Bup groupcontained degranulated rER swollen Mt and hazy mitochondrial structures (c) treatment with GB resulted in and showed a nearly normalstructure (d) pretreatment with GB represented slight expansion of ER


of apoptosis [31] Local anesthetics may dissipate 120595m andactivate caspases leading to apoptotic cell death [6 32] Inour study apoptotic cell death induced by bupivacaine wasassociated with 120595m depolarization and both bupivacaine-induced apoptosis and 120595m dissipation were attenuated byGB The HtrA2 protein is a serine protease that acts as aproapoptotic factor following release from themitochondrialmatrix through large nonselective pores (permeability tran-sition pores mPTPs) that can be opened by overproductionof ROS [33 34] Release of mitochondrial HtrA2 into thecytoplasm was inhibited by the caspase inhibitor z-VAD-fmk[34 35] suggesting that caspase activation may precede andpossibly induce HtrA2 release Increased cleaved caspase-3 isassociatedwithmitochondria-dependent apoptosis followingsustained loss of 120595m [36] Thus bupivacaine likely inducedapoptosis by reducingmitochondria complex activity leadingto overproduction of ROS collapse of the mitochondrialmembrane potential release of proapoptotic factors from themitochondrial matrix and subsequent caspase-3 activation

ES stress may activate alternate apoptotic pathways orexacerbate mitochondria-dependent apoptosis The ER iscritical for protein synthesis and folding lipid and sterol syn-thesis and calcium homeostasis Stressors such as hypoxiaglucose deprivation and calcium depletion from the ERlumen lead to ER dysfunction [37] resulting in cellularcalcium dysregulation protein misfolding and aggregationand activation of proapoptotic effectors such as caspase-12Bupivacaine caused ER stress as evidenced by elevatedcaspase-12 and Grp78 expression and this stress may haveresulted from ROS accumulation as reported by Takahashiet al [17]

Grp78 is a well-characterized indicator of UPR activation(the unfolded protein response) and a critical protectantagainst ER stress by preventing protein aggregation [38]However when the ER stress is severe or prolonged theincrease in Grp78 is no longer sufficient to prevent apoptosisand theUPR switches from a cytoprotective to a proapoptoticresponse involving activation of specific effector proteinssuch as caspase-12 which is activated only by ER stress-initiated apoptotic pathways [37] In accordance with pre-vious studies [39 40] Grp78 expression was upregulated inparallel with caspase-12 indicative of ER stress and ER stress-specific apoptosis Ginkgolide B inhibited the overexpressionof Grp78 and caspase-12 suggesting that suppression ofcell death resulted from disruption of both mitochondrialand ER-dependent apoptotic pathways This conclusion wasfurther corroborated by TEM images showing reducedorganelle swelling andmaintenance of ER andmitochondrialmembrane integrity in cells pretreated with GB prior tobupivacaine

Some limitations of this study should be noted Firstwe examined doses of bupivacaine (1mmolL or 003)that are not clinically relevant as local injections often use025 or 05 although the duration of exposure was greatlyprolonged in this study Second these in vitro results fromtransformed neuroblastoma cells may not be applicable toneurons in vivo Nonetheless previous results have demon-strated morphological signs of oxidative stress and apop-tosis following local anesthetic administration in vivo and

exogenous antioxidants (like GB) have well-established neu-roprotective efficacy

In summary the current study suggests that bupivacaineelicits ROS production which in turn triggers mitochondrialdepolarization mitochondria-dependent apoptosis and ERstress These pathological responses were reduced or ame-liorated by pretreatment with ginkgolide B These resultsprovide novel insights into themolecularmechanisms under-lying the neurotoxicity of bupivacaine and highlight GB as aprototype treatment for the neurotoxicity elicited by this classof local anesthetics

Acknowledgments

This study was supported by the Grants from the NationalNatural Science Foundation of China (no 81271390) andNatural Science Foundation of Guangdong Province China(no 2011010004056) All of the authors have no financialrelationship with a biotechnology manufacturer a pharmac-eutical company or other commercial entities with an interestin subject matter or materials discussed in the papers

References

[1] Y Auroy D Benhamou L Bargues et al ldquoMajor complicationsof regional anesthesia in France the SOS regional anesthesiahotline servicerdquo Anesthesiology vol 97 no 5 pp 1274ndash12802002

[2] C J Park S A Park T G Yoon S J Lee K W Yum and H JKim ldquoBupivacaine induces apoptosis via ROS in the Schwanncell linerdquo Journal of Dental Research vol 84 no 9 pp 852ndash8572005

[3] N M Gibbs and P Rodoreda ldquoPrimary anaesthetic deaths inWestern Australia from 1985ndash2008 causationand preventabil-ityrdquo Anaesth Intensive Care vol 41 no 3 pp 302ndash310 2013

[4] J D Griffiths N V Le S Grant P Hebbard and C RoyseldquoSymptomatic local anaesthetic toxicity and plasma ropivacaineconcentrations after transversus abdominis plane block forCaesarean sectionrdquo British Journal of Anaesthesia vol 110 no6 pp 996ndash1000 2013

[5] Z Tan S Dohi J Chen Y Banno and Y Nozawa ldquoInvolvementof the mitogen-activated protein kinase family in tetracaine-induced PC12 cell deathrdquoAnesthesiology vol 96 no 5 pp 1191ndash1201 2002

[6] J Lu S Y Xu Q G Zhang R Xu and H Y Lei ldquoBupivacaineinduces apoptosis via mitochondria and p38 MAPK dependentpathwaysrdquo European Journal of Pharmacology vol 657 no 1ndash3pp 51ndash58 2011

[7] S Sakura Y Kirihara T Muguruma T Kishimoto and Y SaitoldquoThe comparative neurotoxicity of intrathecal lidocaine andbupivacaine in ratsrdquo Anesthesia and Analgesia vol 101 no 2pp 541ndash547 2005

[8] J W Russell K A Sullivan A J Windebank D N Herrmannand E L Feldman ldquoNeurons undergo apoptosis in animal andcell culture models of diabetesrdquo Neurobiology of Disease vol 6no 5 pp 347ndash363 1999

[9] E Lelkes B R Unsworth and P I Lelkes ldquoReactive oxygenspecies apoptosis and altered NGF-induced signaling in PC12pheochromocytoma cells cultured in elevated glucose an invitro cellular model for diabetic neuropathyrdquo NeurotoxicityResearch vol 3 no 2 pp 189ndash203 2001


[10] R Perez-Castro S Patel Z V Garavito-Aguilar et al ldquoCytotox-icity of local anesthetics in human neuronal cellsrdquo Anesthesiaand Analgesia vol 108 no 3 pp 997ndash1007 2009

[11] Y Arai T Kondo K Tanabe et al ldquoEnhancement of hyper-thermia-induced apoptosis by local anesthetics on human his-tiocytic lymphoma U937 cellsrdquo Journal of Biological Chemistryvol 277 no 21 pp 18986ndash18993 2002

[12] O Cela C Piccoli R Scrima et al ldquoBupivacaine uncouples themitochondrial oxidative phosphorylation inhibits respiratorychain complexes I and III and enhances ROSproduction resultsof a study on cell culturesrdquoMitochondrion vol 10 no 5 pp 487ndash496 2010

[13] S Bouderba M N Sanz C Sanchez-Martin et al ldquoHepaticmitochondrial alterations and increased oxidative stress innutritional diabetes-prone Psammomys obesus modelrdquo Exper-imental Diabetes Research vol 2012 Article ID 430176 8 pages2012

[14] D B Zorov C R Filburn L-O Klotz J L Zweier and S JSollott ldquoReactive oxygen species (ROS)-induced ROS release anew phenomenon accompanying induction of the mitochon-drial permeability transition in cardiac myocytesrdquo Journal ofExperimental Medicine vol 192 no 7 pp 1001ndash1014 2000

[15] I Nonaka A Takagi and S Ishiura ldquoPathophysiology ofmusclefiber necrosis induced by bupivacaine hydrochloride (Mar-caine)rdquo Acta Neuropathologica vol 60 no 3-4 pp 167ndash1741983

[16] A Devarajan V R Grijalva N Bourquard et al ldquoMacrophageparaoxonase 2 regulates calcium homeostasis and cell survivalunder endoplasmic reticulum stress conditions and is sufficientto prevent the development of aggravated atherosclerosis inparaoxonase 2 deficiencyapoE(minusminus) mice on a Western dietrdquoMolecular Genetics and Metabolism vol 107 pp 416ndash427 2012

[17] S Takahashi Y Izawa and N Suzuki ldquoAstroglial pentose phos-phate pathway rates in response to high-glucose environmentsrdquoASN Neuro vol 4 no 2 pp 71ndash88 2012

[18] W Abbas K A Khan M K Tripathy et al ldquoInhibitionof ER stress-mediated apoptosis in macrophages by nuclear-cytoplasmic relocalization of eEF1A by the HIV-1 Nef proteinrdquoCell Death amp Disease vol 3 p e368 2012

[19] C Zhang X Tian Y Luo and X Meng ldquoGinkgolide B attenu-ates ethanol-induced neurotoxicity through regulatingNADPHoxidasesrdquo Toxicology vol 287 no 1ndash3 pp 124ndash130 2011

[20] K M Maclennan C L Darlington and P F Smith ldquoThe CNSeffects of Ginkgo biloba extracts and ginkgolide Brdquo Progress inNeurobiology vol 67 no 3 pp 235ndash257 2002

[21] M Esposito andMCarotenuto ldquoGinkgolide B complex efficacyfor brief prophylaxis of migraine in school-aged children anopen-label studyrdquo Neurological Sciences vol 32 no 1 pp 79ndash81 2011

[22] M Huang Y Qian T Guan L Huang X Tang and YLi ldquoDifferent neuroprotective responses of Ginkgolide B andbilobalide the two Ginkgo components in ischemic rats withhyperglycemiardquo European Journal of Pharmacology vol 677 no1ndash3 pp 71ndash76 2012

[23] R Werdehausen S Fazeli S Braun et al ldquoApoptosis inductionby different local anaesthetics in a neuroblastoma cell linerdquoBritish Journal of Anaesthesia vol 103 no 5 pp 711ndash718 2009

[24] S Zhang J Fu and Z Zhou ldquoIn vitro effect of manganesechloride exposure on reactive oxygen species generation andrespiratory chain complexes activities of mitochondria isolatedfrom rat brainrdquoToxicology in Vitro vol 18 no 1 pp 71ndash77 2004

[25] T D Schmittgen and K J Livak ldquoAnalyzing real-time PCR databy the comparative CT methodrdquo Nature Protocols vol 3 no 6pp 1101ndash1108 2008

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] W Fang Y Deng Y Li et al ldquoBlood brain barrier permeabilityand therapeutic time window of Ginkgolide B in ischemia-reperfusion injuryrdquo European Journal of Pharmaceutical Sci-ences vol 39 no 1ndash3 pp 8ndash14 2010

[28] B Brodska and A Holoubek ldquoGeneration of reactive oxygenspecies during apoptosis induced by DNA-damaging agentsandor histone deacetylase inhibitorsrdquo Oxidative Medicine andCellular Longevity vol 2011 Article ID 253529 7 pages 2011

[29] N Zamzami P Marchetti M Castedo et al ldquoSequential reduc-tion of mitochondrial transmembrane potential and generationof reactive oxygen species in early programmed cell deathrdquoJournal of Experimental Medicine vol 182 no 2 pp 367ndash3771995

[30] T A Young C C Cunningham and S M Bailey ldquoReactiveoxygen species production by the mitochondrial respiratorychain in isolated rat hepatocytes and livermitochondria studiesusingmyxothiazolrdquoArchives of Biochemistry andBiophysics vol405 no 1 pp 65ndash72 2002

[31] G Nagy A Koncz D Fernandez and A Perl ldquoNitric oxidemitochondrial hyperpolarization and T cell activationrdquo FreeRadical Biology andMedicine vol 42 no 11 pp 1625ndash1631 2007

[32] M E Johnson C B Uhl K-H Spittler H Wang and G JGores ldquoMitochondrial injury and caspase activation by the localanesthetic lidocainerdquo Anesthesiology vol 101 no 5 pp 1184ndash1194 2004

[33] P Bernardi A Krauskopf E Basso et al ldquoThe mitochondrialpermeability transition from in vitro artifact to disease targetrdquoFEBS Journal vol 273 no 10 pp 2077ndash2099 2006

[34] D Arnoult B Gaume M Karbowski J C Sharpe F Cecconiand R J Youle ldquoMitochondrial release of AIF and EndoGrequires caspase activation downstream of BaxBak-mediatedpermeabilizationrdquoThe EMBO Journal vol 22 no 17 pp 4385ndash4399 2003

[35] M Zoratti I Szabo and U de Marchi ldquoMitochondrial perme-ability transitions how many doors to the houserdquo Biochimicaet Biophysica Acta vol 1706 no 1-2 pp 40ndash52 2005

[36] N J Waterhouse K A Sedelies V R Sutton et al ldquoFunctionaldissociation of Δ120595m and cytochrome c release defines thecontribution of mitochondria upstream of caspase activationduring granzyme B-induced apoptosisrdquo Cell Death and Differ-entiation vol 13 no 4 pp 607ndash618 2006

[37] M Schroder and R J Kaufman ldquoThe mammalian unfoldedprotein responserdquo Annual Review of Biochemistry vol 74 pp739ndash789 2005

[38] M Boyce and J Yuan ldquoCellular response to endoplasmicreticulum stress a matter of life or deathrdquo Cell Death andDifferentiation vol 13 no 3 pp 363ndash373 2006

[39] A Takada T Miki A Kuno et al ldquoRole of ER Stress inventricular contractile dysfunction in type 2 diabetesrdquo PLoSOne vol 7 no 6 Article ID e39893 2012

[40] E Araki S Oyadomari and M Mori ldquoEndoplasmic reticulumstress and diabetesmellitusrdquo InternalMedicine vol 42 no 1 pp7ndash14 2003

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

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 159864 11 pageshttpdxdoiorg1011552013159864

Research ArticleNeuroprotective Effect of Ginkgolide B on Bupivacaine-InducedApoptosis in SH-SY5Y Cells

Le Li1 Qing-guo Zhang1 Lu-ying Lai1 Xian-jie Wen1 Ting Zheng1

Chi-wai Cheung2 Shu-qin Zhou1 and Shi-yuan Xu1

1 Department of Anesthesiology Zhujiang Hospital Southern Medical University Guangzhou Guangdong 510282 China2Department of Anesthesiology The University of Hong Kong Hong Kong

Correspondence should be addressed to Shu-qin Zhou 154843189qqcom and Shi-yuan Xu xsy111yeahnet

Received 5 July 2013 Accepted 18 August 2013

Academic Editor Zhengyuan Xia

Copyright copy 2013 Le Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Local anesthetics are used routinely and effectively However many are also known to activate neurotoxic pathways We tested theneuroprotective efficacy of ginkgolide B (GB) an active component of Ginkgo biloba against ROS-mediated neurotoxicity causedby the local anesthetic bupivacaine SH-SY5Y cells were treated with different concentrations of bupivacaine alone or followingpreincubation with GB Pretreatment with GB increased SH-SY5Y cell viability and attenuated intracellular ROS accumulationapoptosis mitochondrial dysfunction and ER stress GB suppressed bupivacaine-induced mitochondrial depolarization andmitochondria complex I and III inhibition and increased cleaved caspase-3 and Htra2 expression which was strongly indicative ofactivation of mitochondria-dependent apoptosis with concomitantly enhanced expressions of Grp78 caspase-12 mRNA proteinand ER stress GB also improved ultrastructural changes indicative ofmitochondrial and ER damage induced by bupivacaineTheseresults implicate bupivacaine-induced ROS-dependent mitochondria ER dysfunction and apoptosis which can be attenuated byGB through its antioxidant property

1 Introduction

Local anesthetics are among the most common clinical drugsand are generally regarded as safe [1 2] However they havealso been shown to be neurotoxic even at normal clinicaldose [3 4] This neurotoxicity is mediated at least in partby activation of apoptotic pathways [5 6] In the caudaequina intrathecally administered local anesthetics inducedcell swelling atrophy edema axonal degeneration and theappearance of myelin ovoids as well as macrophage infiltra-tion [7] These morphological signs of degeneration indicatethat local anesthetics can initiate a complex cascade of directcytotoxic and ensuing inflammatory responses although themolecular mechanisms of local anesthetic toxicity are stilllargely unknown

Local anesthetics have been shown to induce neuraldysfunction and apoptosis in vitro [8ndash11] For example bupi-vacaine may inhibit mitochondrial respiratory complexes Iand III leading to decreased ATP production collapse of themitochondrial membrane potential (Δ120595m) overproduction

of reactive oxygen species (ROS) and ultimately liberationof cytochrome c and activation of the caspase-3-dependentapoptosis pathway [10ndash12] In fact ROS accumulation mito-chondrial uncoupling and depolarization of 120595m are amongthe earliest indicators of apoptosis induced by local anesthet-ics [13 14] In addition tomitochondrial damage dysfunctionof the endoplasmic reticulum (ER) stress has also beenimplicated in apoptosis Arai and Nonaka et al proposed thatoxidative stress associated with local anesthetics can induceCa2+ release from intracellular stores including the roughendoplasmic reticulum (rER) [11 15] Loss of intraluminalCa2+ may lead to ER stress [16] further ROS generation[17] and activation of ER-dependent apoptosis pathways [18]Thus mitochondrial and ER damage associated with ROSoverproduction may act synergistically to evoke cell deathin response to bupivacaine or other structurally related localanesthetics

Ginkgo biloba has been used in traditional Chinesemedicine for thousands of years Evidence accumulated overthe last decade suggests that concentrated and partially



120

100

080

060

040

020

000

Cel

l via

bilit

y (r

elat

ive t

o un

treat

men

t)

lowast

lowast

lowast


(a)

100

090

080

070

060

050

040

030

Cel

l via

bilit

y (r

elat

ive t

o un

treat

men

t)

lowast

lowast lowast



(b)














102

102

103

103

104

104

105

105PI

Per

CP-A

Annexin V FITC-A


102

102

103

103

104

104

105

105

PI P

erCP

-A

Annexin V FITC-A


102

102

103

103

104

104

105

105

PI P

erCP

-A

Annexin V FITC-A


102

103

104

105

PI P

erCP

-A

102 103 104 105

Annexin V FITC-A


102

103

104

105

PI P

erCP

-A

102 103 104 105

Annexin V FITC-A


lowast

lowast

lowast

B GB5 + B GB10+ B GB20 + B GB40 + B

Apop

totic

cells

()

5000

6000

4000

3000

2000

1000


(f)






GB

150

100

50

0

102 103 104 105

FITC-A

Cou

nt

150

100

50

0102 103 104 105

FITC-A

Cou

nt150

200

250

100

50

0102 103 104 105

FITC-A

Cou

nt

200

150

100

50

0

102 103 104 105

FITC-A

Cou

nt

BupCon

GB + Bup

(a)

5000

4000

3000

2000

1000

000

lowast lowast

Con Bup GB GB + Bup

Geo

mea

n (times100

)


(b)







Con

Bup

GB

GB +

Bup

(a)

Con

Bup

GB


0102 103 104 105 10

210

310

410

5

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

125150

100

5025

75

0

Cou

nt

150

100

50

0

Cou

nt

125100

5025

75

125100

5025

75

0

150

100

50

0

100

5025

75

0

Cou

ntC

ount

Cou

ntC

ount

100

5025

75

0

Cou

nt

125100

5025

75

0

Cou

nt

GB+

Bup

(b)

lowast

lowast

Con Bup GB GB + Bup

1200

1000

800

600

400

200

000

Ratio

of J

C-1

poly

mer

mon

omer


(c)



1000

8000

6000

4000

2000

000

lowastlowast

lowast

lowastlowast

Activ

ities

of m

itoch

ondr

ia co

mpl

exes

(nm

ol m

inminus1

mgminus

1pr

otei

n)

lowast

GB


ConBup GB + Bup









Gene Primers












Cleaved caspase-3

HtrA2

17kDa

38kDa

36kDa GAPDH

250

200

150

100

050

000Con Bup GB GB

lowast

lowastlowast

Clea

ved

casp

ase-

3G

APD

H p

rote

in

100

080

060

040


GB + Bup

Htr

A2

GA

PDH

pro

tein

Con GBBup




3 Results







250

200

150

100

050

000

Relat

ive e

xpre

ssio

n (C

on=1

)

Caspase-12 Grp78

lowast

lowastlowast

GBConBup GB + Bup








4 Discussion






Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















120

100

080

060

040

020

000

Cel

l via

bilit

y (r

elat

ive t

o un

treat

men

t)

lowast

lowast

lowast


(a)

100

090

080

070

060

050

040

030

Cel

l via

bilit

y (r

elat

ive t

o un

treat

men

t)

lowast

lowast lowast



(b)














102

102

103

103

104

104

105

105PI

Per

CP-A

Annexin V FITC-A


102

102

103

103

104

104

105

105

PI P

erCP

-A

Annexin V FITC-A


102

102

103

103

104

104

105

105

PI P

erCP

-A

Annexin V FITC-A


102

103

104

105

PI P

erCP

-A

102 103 104 105

Annexin V FITC-A


102

103

104

105

PI P

erCP

-A

102 103 104 105

Annexin V FITC-A


lowast

lowast

lowast

B GB5 + B GB10+ B GB20 + B GB40 + B

Apop

totic

cells

()

5000

6000

4000

3000

2000

1000


(f)






GB

150

100

50

0

102 103 104 105

FITC-A

Cou

nt

150

100

50

0102 103 104 105

FITC-A

Cou

nt150

200

250

100

50

0102 103 104 105

FITC-A

Cou

nt

200

150

100

50

0

102 103 104 105

FITC-A

Cou

nt

BupCon

GB + Bup

(a)

5000

4000

3000

2000

1000

000

lowast lowast

Con Bup GB GB + Bup

Geo

mea

n (times100

)


(b)







Con

Bup

GB

GB +

Bup

(a)

Con

Bup

GB


0102 103 104 105 10

210

310

410

5

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

125150

100

5025

75

0

Cou

nt

150

100

50

0

Cou

nt

125100

5025

75

125100

5025

75

0

150

100

50

0

100

5025

75

0

Cou

ntC

ount

Cou

ntC

ount

100

5025

75

0

Cou

nt

125100

5025

75

0

Cou

nt

GB+

Bup

(b)

lowast

lowast

Con Bup GB GB + Bup

1200

1000

800

600

400

200

000

Ratio

of J

C-1

poly

mer

mon

omer


(c)



1000

8000

6000

4000

2000

000

lowastlowast

lowast

lowastlowast

Activ

ities

of m

itoch

ondr

ia co

mpl

exes

(nm

ol m

inminus1

mgminus

1pr

otei

n)

lowast

GB


ConBup GB + Bup









Gene Primers












Cleaved caspase-3

HtrA2

17kDa

38kDa

36kDa GAPDH

250

200

150

100

050

000Con Bup GB GB

lowast

lowastlowast

Clea

ved

casp

ase-

3G

APD

H p

rote

in

100

080

060

040


GB + Bup

Htr

A2

GA

PDH

pro

tein

Con GBBup




3 Results







250

200

150

100

050

000

Relat

ive e

xpre

ssio

n (C

on=1

)

Caspase-12 Grp78

lowast

lowastlowast

GBConBup GB + Bup








4 Discussion






Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















102

102

103

103

104

104

105

105PI

Per

CP-A

Annexin V FITC-A


102

102

103

103

104

104

105

105

PI P

erCP

-A

Annexin V FITC-A


102

102

103

103

104

104

105

105

PI P

erCP

-A

Annexin V FITC-A


102

103

104

105

PI P

erCP

-A

102 103 104 105

Annexin V FITC-A


102

103

104

105

PI P

erCP

-A

102 103 104 105

Annexin V FITC-A


lowast

lowast

lowast

B GB5 + B GB10+ B GB20 + B GB40 + B

Apop

totic

cells

()

5000

6000

4000

3000

2000

1000


(f)






GB

150

100

50

0

102 103 104 105

FITC-A

Cou

nt

150

100

50

0102 103 104 105

FITC-A

Cou

nt150

200

250

100

50

0102 103 104 105

FITC-A

Cou

nt

200

150

100

50

0

102 103 104 105

FITC-A

Cou

nt

BupCon

GB + Bup

(a)

5000

4000

3000

2000

1000

000

lowast lowast

Con Bup GB GB + Bup

Geo

mea

n (times100

)


(b)







Con

Bup

GB

GB +

Bup

(a)

Con

Bup

GB


0102 103 104 105 10

210

310

410

5

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

125150

100

5025

75

0

Cou

nt

150

100

50

0

Cou

nt

125100

5025

75

125100

5025

75

0

150

100

50

0

100

5025

75

0

Cou

ntC

ount

Cou

ntC

ount

100

5025

75

0

Cou

nt

125100

5025

75

0

Cou

nt

GB+

Bup

(b)

lowast

lowast

Con Bup GB GB + Bup

1200

1000

800

600

400

200

000

Ratio

of J

C-1

poly

mer

mon

omer


(c)



1000

8000

6000

4000

2000

000

lowastlowast

lowast

lowastlowast

Activ

ities

of m

itoch

ondr

ia co

mpl

exes

(nm

ol m

inminus1

mgminus

1pr

otei

n)

lowast

GB


ConBup GB + Bup









Gene Primers












Cleaved caspase-3

HtrA2

17kDa

38kDa

36kDa GAPDH

250

200

150

100

050

000Con Bup GB GB

lowast

lowastlowast

Clea

ved

casp

ase-

3G

APD

H p

rote

in

100

080

060

040


GB + Bup

Htr

A2

GA

PDH

pro

tein

Con GBBup




3 Results







250

200

150

100

050

000

Relat

ive e

xpre

ssio

n (C

on=1

)

Caspase-12 Grp78

lowast

lowastlowast

GBConBup GB + Bup








4 Discussion






Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GB

150

100

50

0

102 103 104 105

FITC-A

Cou

nt

150

100

50

0102 103 104 105

FITC-A

Cou

nt150

200

250

100

50

0102 103 104 105

FITC-A

Cou

nt

200

150

100

50

0

102 103 104 105

FITC-A

Cou

nt

BupCon

GB + Bup

(a)

5000

4000

3000

2000

1000

000

lowast lowast

Con Bup GB GB + Bup

Geo

mea

n (times100

)


(b)







Con

Bup

GB

GB +

Bup

(a)

Con

Bup

GB


0102 103 104 105 10

210

310

410

5

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

125150

100

5025

75

0

Cou

nt

150

100

50

0

Cou

nt

125100

5025

75

125100

5025

75

0

150

100

50

0

100

5025

75

0

Cou

ntC

ount

Cou

ntC

ount

100

5025

75

0

Cou

nt

125100

5025

75

0

Cou

nt

GB+

Bup

(b)

lowast

lowast

Con Bup GB GB + Bup

1200

1000

800

600

400

200

000

Ratio

of J

C-1

poly

mer

mon

omer


(c)



1000

8000

6000

4000

2000

000

lowastlowast

lowast

lowastlowast

Activ

ities

of m

itoch

ondr

ia co

mpl

exes

(nm

ol m

inminus1

mgminus

1pr

otei

n)

lowast

GB


ConBup GB + Bup









Gene Primers












Cleaved caspase-3

HtrA2

17kDa

38kDa

36kDa GAPDH

250

200

150

100

050

000Con Bup GB GB

lowast

lowastlowast

Clea

ved

casp

ase-

3G

APD

H p

rote

in

100

080

060

040


GB + Bup

Htr

A2

GA

PDH

pro

tein

Con GBBup




3 Results







250

200

150

100

050

000

Relat

ive e

xpre

ssio

n (C

on=1

)

Caspase-12 Grp78

lowast

lowastlowast

GBConBup GB + Bup








4 Discussion






Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Con

Bup

GB

GB +

Bup

(a)

Con

Bup

GB


0102 103 104 105 10

210

310

410

5

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

102 103 104 105

PE-A

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

102

103

104

105

FITC-A

125150

100

5025

75

0

Cou

nt

150

100

50

0

Cou

nt

125100

5025

75

125100

5025

75

0

150

100

50

0

100

5025

75

0

Cou

ntC

ount

Cou

ntC

ount

100

5025

75

0

Cou

nt

125100

5025

75

0

Cou

nt

GB+

Bup

(b)

lowast

lowast

Con Bup GB GB + Bup

1200

1000

800

600

400

200

000

Ratio

of J

C-1

poly

mer

mon

omer


(c)



1000

8000

6000

4000

2000

000

lowastlowast

lowast

lowastlowast

Activ

ities

of m

itoch

ondr

ia co

mpl

exes

(nm

ol m

inminus1

mgminus

1pr

otei

n)

lowast

GB


ConBup GB + Bup









Gene Primers












Cleaved caspase-3

HtrA2

17kDa

38kDa

36kDa GAPDH

250

200

150

100

050

000Con Bup GB GB

lowast

lowastlowast

Clea

ved

casp

ase-

3G

APD

H p

rote

in

100

080

060

040


GB + Bup

Htr

A2

GA

PDH

pro

tein

Con GBBup




3 Results







250

200

150

100

050

000

Relat

ive e

xpre

ssio

n (C

on=1

)

Caspase-12 Grp78

lowast

lowastlowast

GBConBup GB + Bup








4 Discussion






Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1000

8000

6000

4000

2000

000

lowastlowast

lowast

lowastlowast

Activ

ities

of m

itoch

ondr

ia co

mpl

exes

(nm

ol m

inminus1

mgminus

1pr

otei

n)

lowast

GB


ConBup GB + Bup









Gene Primers












Cleaved caspase-3

HtrA2

17kDa

38kDa

36kDa GAPDH

250

200

150

100

050

000Con Bup GB GB

lowast

lowastlowast

Clea

ved

casp

ase-

3G

APD

H p

rote

in

100

080

060

040


GB + Bup

Htr

A2

GA

PDH

pro

tein

Con GBBup




3 Results







250

200

150

100

050

000

Relat

ive e

xpre

ssio

n (C

on=1

)

Caspase-12 Grp78

lowast

lowastlowast

GBConBup GB + Bup








4 Discussion






Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cleaved caspase-3

HtrA2

17kDa

38kDa

36kDa GAPDH

250

200

150

100

050

000Con Bup GB GB

lowast

lowastlowast

Clea

ved

casp

ase-

3G

APD

H p

rote

in

100

080

060

040


GB + Bup

Htr

A2

GA

PDH

pro

tein

Con GBBup




3 Results







250

200

150

100

050

000

Relat

ive e

xpre

ssio

n (C

on=1

)

Caspase-12 Grp78

lowast

lowastlowast

GBConBup GB + Bup








4 Discussion






Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















250

200

150

100

050

000

Relat

ive e

xpre

ssio

n (C

on=1

)

Caspase-12 Grp78

lowast

lowastlowast

GBConBup GB + Bup








4 Discussion






Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con GBBup

30kDa

78kDa

36kDa

Caspase-12

Grp78

GAPDH

200

150

100

050


lowast lowast

Con Bup GB GB + Bup

GB + Bup

Casp

ase-

12G

APD

H p

rote

in

200

150

100

050

000

Grp

78G

APD

H p

rote

in



(a) (b)

(c) (d)









Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Neuroprotective effect of ginkgolide B on bupivacaine ... · 2...

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Transcript of Neuroprotective effect of ginkgolide B on bupivacaine ... · 2...