The immunomodulator ginsan induces resistance toThe immunomodulator ginsan induces resistance to

experimental sepsis by inhibiting Toll-like receptor mediatedexperimental sepsis by inhibiting Toll-like receptor mediated

inflammatory signalsinflammatory signals

Eur. J. Immunol. 2006. 36: 37–45Eur. J. Immunol. 2006. 36: 37–45

Ji-Yeon Ahn1,2, In-Soo Choi3, Ji-Young Shim1, Eun-Kyung Ji-Yeon Ahn1,2, In-Soo Choi3, Ji-Young Shim1, Eun-Kyung Yun1,Yun1,

Yeon-Sook Yun1, Gajin Jeong2 and Jie-Young Song1Yeon-Sook Yun1, Gajin Jeong2 and Jie-Young Song1

Ginseng contains multiple phytochemicals:Ginseng contains multiple phytochemicals:

Two major classes:Two major classes:

-Ginsenosides-Ginsenosides

-Polysaccharides-Polysaccharides Organic extract contains mostly ginsenosidesOrganic extract contains mostly ginsenosides

Aqueous extract contains less ginsenosides and more Aqueous extract contains less ginsenosides and more polysaccharidesspolysaccharidess

What are ginsenosides?What are ginsenosides?

What are polysaccharides?What are polysaccharides?

Basic structure of ginsenosides: Basic structure of ginsenosides: Glycoside = aglycone + sugar chain Glycoside = aglycone + sugar chain (glucose, maltose,fructose, (glucose, maltose,fructose,

saccharose saccharose attached at attached at C3, C6 and C20) C3, C6 and C20)

3 major groups depending on their aglycones: 3 major groups depending on their aglycones: Group I - protopanaxadiol type Group I - protopanaxadiol type Group II - protopanaxtriol type Group II - protopanaxtriol type

These are C-27 sterols (with a dammarane skeleton aglycone) These are C-27 sterols (with a dammarane skeleton aglycone)

Group III - Group III - oleanolic acid-type: C-30 triterpenoids. oleanolic acid-type: C-30 triterpenoids.

Water-soluble and acidic polysaccharidesWater-soluble and acidic polysaccharides

Ginsan, acidic polysaccharide with a M.W. of Ginsan, acidic polysaccharide with a M.W. of 150,000 150,000

Cold- fX: poly-furanosyl-pyranosyl-saccharidesCold- fX: poly-furanosyl-pyranosyl-saccharides

GinsenosidesGinsenosides PS PS -immunomodulatory -immunomodulatory + + + + + +

TNFTNF TNF TNF

IL-1, IL-2, IL-1, IL-2, TNFTNF

-anti-inflammatory ? -anti-inflammatory ? ++ -radioprotective + -radioprotective +

-antioxidative-antioxidative

+ + + + reduce ROS and increase anti-oxidant levelsreduce ROS and increase anti-oxidant levels -anti-tumor/ metastasis -anti-tumor/ metastasis + + + + -angiogenesis +/- ?-angiogenesis +/- ?

Pharmacological activities

Sepesis:Sepesis: Microbial infection in blood---Microbial infection in blood--- excessive inflammatory response-- excessive inflammatory response--

systemic organ failuresystemic organ failure

proinflammatory cytokines as mediatorsproinflammatory cytokines as mediators

TNF, IL-1, IL-6TNF, IL-1, IL-6 IL-12 + IL-18 –are important because they produce IL-12 + IL-18 –are important because they produce IFN-gamma IFN-gamma IFN + TNF- --IFN + TNF- -- synergistic effects in LPS effect synergistic effects in LPS effect

Gram-ve bacteria E. Coli--Gram-ve bacteria E. Coli-- LPS from cell wall: mediating LPS from cell wall: mediating agentagent

Gram +ve bacteria: S. aureus-Gram +ve bacteria: S. aureus- lipoteichoic acid, peptidoglycan lipoteichoic acid, peptidoglycan

from cell wall from cell wall

Toll like-receptors: Toll like-receptors: --Single membrane-spanning non-catalytic receptors, Innate immune system, Single membrane-spanning non-catalytic receptors, Innate immune system, -recognize threat, recognize molecules shared by pathogens but different from -recognize threat, recognize molecules shared by pathogens but different from host moleculeshost molecules

Function as dimersNeed co-receptors

Adaptor proteins

ligands e.g. lipo-peptides, glycolipids.lipoteichoic acids,LPS, HSP70, zymosan, single or double-stranded RNA, fibrinogen, small xenobiotics

Require kinases activation for signaling and modulation of gene expression

Rationale:Rationale:

Reducing a particular cytokine not effective in sepsisReducing a particular cytokine not effective in sepsis Ginsan as effective Biological Response Modifier Ginsan as effective Biological Response Modifier Stimulate NK & T cells, induce cytokines, induce Stimulate NK & T cells, induce cytokines, induce

tumoricidal & antimicrobial activity in macrophages.tumoricidal & antimicrobial activity in macrophages. Stimulate NO production in macrophages-in vitro Stimulate NO production in macrophages-in vitro

antisepticemic activity… by extension!antisepticemic activity… by extension! But these mediators also enhance septic symptoms!But these mediators also enhance septic symptoms!

Question: how does ginseng modulate plasma Question: how does ginseng modulate plasma cytokine profile in septic animals and whether there cytokine profile in septic animals and whether there are other mechanisms that protect animals from are other mechanisms that protect animals from sepsissepsis

ResultsResults

1. Ginsan (1. Ginsan (IVIV) 24 hr pretreatment protected mice from acute ) 24 hr pretreatment protected mice from acute sepsis (3 models)sepsis (3 models)

- S. aureus induced lethality (10% to 88% survival)- S. aureus induced lethality (10% to 88% survival)

-E.coli induced lethality-E.coli induced lethality

-CLP induced lethality-CLP induced lethality

25 ug/kg effective, quite low dose25 ug/kg effective, quite low dose

2. Ginsan enhanced clearance of bacteria from blood, spleen and 2. Ginsan enhanced clearance of bacteria from blood, spleen and kidneykidney

3. Ginsan increased bactericidal activity of peritoneal-3. Ginsan increased bactericidal activity of peritoneal-macrophages.macrophages.

Normal miceNormal mice isolated PM- isolated PM- incubated with ginsan in incubated with ginsan in vitro 3 hr -vitro 3 hr - partially killed labeled bacteria-- partially killed labeled bacteria-- analysed by analysed by FACS for uptake of bacteria by macrophages -FACS for uptake of bacteria by macrophages - index of index of phagocytosis.phagocytosis.

But PM isolated from 24 hr pretreatment with ginsan with or But PM isolated from 24 hr pretreatment with ginsan with or without infection showed samall if any increase of without infection showed samall if any increase of phagocytosis. Major weakness of lack of in vivo effect! phagocytosis. Major weakness of lack of in vivo effect!

May be PM is not the major site of bacterial clearanceMay be PM is not the major site of bacterial clearance

Ginsan enhances phagocytosis in S. aureus-infectedmacrophages. (A) Phagocytic activity was evaluated in PM isolated from intact mice and incubated with ginsan for 3 h. (B) PM were obtained from non-treated (dotted line)and S. aureus-infected mice treated with or without ginsan(25 lg/kg, bold and solid lines, respectively), and were thenstimulated with heat-killed S. aureus for 30 min at 37C.

4. Ginsan attenuates pro- and anti-inflammatorycytokine production in S. aureus-infected mice. Serum cytokine levels were determined at 0, 2, 4, 8, 11, and 24 h after the challenge with 1.5 108 CFU S. aureus

(No effect on Th2 cytokines IL-2 and IL-4 Ginsan also reduced anti-inflammatory IL-10 Note: -Cytokines are not detectable in control animals and ginsan did not stimulate any! -Not consistent with hypothesis.

5. Ginsan suppresses the expression of TLR and theadaptor MyD88 molecule in PM activated by S. aureus.

In vitro onlyPM isolated and treated with ginsan (0.1 ug/ml) for 6 hrs. Cells were washed, treated heat-killed S.aureus for 6 hrs.Measured by RT-PCR for RNA transcripts for TLR2, 4, 9 and MyD88

6. Ginsan inhibits S. aureus-induced MAPK and NF-kB activation in PM. PM were pretreated with ginsan (0.1 ug/mL) for 6 h and were subsequently treated with heat-killed S. aureus for 40 min. (A)MAPK phosphorylation was detected using Western blot analysis with antibodies specific for JNK 1/2, p38, and ERK1/2. (B) The concentration of NF-kB in nuclear extracts was determined using electrophoretic mobility shift assay.

S.aureus stimulate phosphorylation of JUN1/2 and MAPK in PM and suppressed by 0.1 ug/ml ginsan (detected by Western Blot using antibodies for JNK1/2, P38, and ERK1/2 ) D-R study to look for correlation?

Points for discussionPoints for discussion

1. Low effective dose: 25 ug/kg1. Low effective dose: 25 ug/kg

Given by IV!Given by IV!

2. Explanation of changes in pro-inflammatory and anti-inflammatory 2. Explanation of changes in pro-inflammatory and anti-inflammatory cytokines not very convincing. cytokines not very convincing.

“ “ ginsan eventually restore the balance between the proinflammatory and anti-ginsan eventually restore the balance between the proinflammatory and anti-inflammatory arms of the cytokine network in sepsis. Different time of inflammatory arms of the cytokine network in sepsis. Different time of infection is critical to the profiles but they did not show that.infection is critical to the profiles but they did not show that.

(No effect on Th2 cytokines IL-2 and IL-4

Ginsan also reduced anti-inflammatory IL-10

Note: -Cytokines are not detectable in control animals and ginsan did not stimulate any!

-Not consistent with hypothesis.

Back to the rationale: Back to the rationale:

Stimulate NO production in macrophages-in vitro Stimulate NO production in macrophages-in vitro antisepticemic antisepticemic activity… by extension!activity… by extension!

But these mediators also enhance septic symptoms!But these mediators also enhance septic symptoms! Question: how does ginseng modulate plasma cytokine profile in septic Question: how does ginseng modulate plasma cytokine profile in septic

animals and whether there are other mechanisms that protect animals from animals and whether there are other mechanisms that protect animals from sepsissepsis

Discussion:Discussion: Unpublished data from author:Unpublished data from author: Ginsan stimulated TLR in normal macrophages (Ginsan stimulated TLR in normal macrophages (not shown in present not shown in present

studystudy) but down regulate them in septic macrophages….suggest ginsan ) but down regulate them in septic macrophages….suggest ginsan could induce tolerance against septic challenges.could induce tolerance against septic challenges.

Other: sublethal dose LPS pretreatment protect subsequent LPS induced Other: sublethal dose LPS pretreatment protect subsequent LPS induced lethality by reducing proinflammatory cytokines lethality by reducing proinflammatory cytokines

Major weakness; not show effect of ginsan in present study to test the Major weakness; not show effect of ginsan in present study to test the hypothesishypothesis

Tripterygium Wifordii Extracts [EA & PS] on LPS-induced NO production in macrophages

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EA also inhibited COX-2 m-RNA expression and PGE2 production

Others showed inhibition of cytokines production