Effects Of Lipopolysaccharide On The Apoptosis Of Activated Hepatic Stellate Cells

BackgroundHepatic fibrosis is characterized by the intrahepatic connective tissue dysplasiadue to the deposition of excessive extracellular matrix, as a result from chronic liverdiseases including HBV, HCV, autoimmune hepatitis and primary sclerosingcholangitis. The activation of hepatic stellate cell(HSC) is the key of pathogenesis ofhepatic fibrosis. Chronic inflammatory reaction promotes the activation of HSC andthe activated HSC secretes excessive extracellular matrix, finally hepatic fibrosisoccurs. Previous studies show hepatic fibrosis is reversible pathologically, in whichthe apoptosis of activated HSC plays an essential role.Nerve growth factor(NGF), a main regulator of apoptosis of activated HSC, hasbeen demonstrated secreted from hepatocytes and binds to the receptor of p75NTR toinduce apoptosis of activated HSC. It has been well established that the TLR4-NF-κBaxis is pivotal in switching inflammatory reaction into profibrotic effects in thepathogenesis of hepatic frosis. Lipopolysaccharide(LPS), a main ligand of TLR4orTLR2, was proved increased markedly in circulation. And in other cellular studies, theexpression of p75NTR was regulated by the TLR4-MyD88pathway. It is thereforeplausible to hypothesize that during the pathogenesis of hepatic fibrosis, one handLPS inhibites secretion of NGF from hepatocytes and on the other hand attenuated theapoptosis of activated HSC in a TLR-MyD88-NF-κB dependent way.In the present study, rat models of hepatic fibrosis were established to observethe serum concentration of lipopolysaccharide and cellular apoptosis in liver sinusoidin vivo. HSC was isolated and stimulated by LPS to activate the expression of TLR4signaling pathway, followed by the treatment with MyD88siRNA to block the MyD88signal pathway as well as with BAY to inhibit NF-κB pathway, and the apoptosis ofHSC induced by NGF was observed. At the same time, isolated rat hepatocytes weretreated with LPS, and then the NGF secretion was detected. The hepatocytes treatedwith LPS were co-cultured with activated HSC, and the apoptosis of activated HSCwas examined.MethodsRat hepatic fibrosis models were induced by4-week continuous intraperitonealinjection with dimethylnitrosamines (DMN)(10μg/kg,continuous3days/week) andby bile duct ligation (BDL)(about for4weeks), and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB) and conjugatedbilirubin (DB), prothrombin time(PT) and γ-glutamyl transpeptidase(γ-GGT) wereexamined. HE staining, Masson staining and VG staining were used for assessment ofliver fibrosis. LPS concentration was analysized. Rat heapatic stellate cell wasisolated by using enzymatic digestion-density gradient centrifugation. In order to findthe optima stimulate time of LPS and the exact stimulate concentration of NGF, theapoptosis of HSC was examined under different conditions that LPS(10μg/ml) treatedto HSCs at diverse time intervals (30min,1h,6h,12h) and nerve growth factor (NGF)of different concentration(50ng/ml,100ng/ml,200ng/ml) treated to HSC for24hours.Cellular growth was detected by using CCK-8. Activated HSC was stimulated by LPSfor1h, followed by NGF for24h, and the flow cytometry and TUNEL methods wereused to detect cellular apoptosis, Real time-PCR and Western-blot were used to detectmolecules mRNA and protein expressions involved in cellular apoptosis. Under theconditions that MyD88SiRNA and BAY were used to inhibit MyD88pathways andblock NF-kappa B signaling pathway respectively, HSC was treated with LPS for1hand then with NGF for another24h, and then cellular apoptosis and gene and proteinexpressions in cellular apoptosis was detected, and the p75NTR protein expressionwas examined, too. Rat hepatocytes were isolated to explore the effects of LPS onNGF secretion from hepatocytes and to determine the effects of LPS-pretreatedhepatocytes on the apoptosis of activated HSC.ResultsCorrelation between plasma LPS and liver fibrosis1. Compared to normal rats, in BDL-and DMN-induced hepatic fibrotic rats, serumPT, AST, TB, DB and γ-GGT of BDL concentration were all higher(P<0.05or P<0.01),ALB was significantly lower(P<0.05or P<0.01), and ALT levels were similar.2. Plasma LPS concentration was increased signifcantly in BDL model rats comparedto normal ones(P<0.05). In DMN model group, LPS concentrations were higher thanthat in the normal(at2w,3w,4w), and the LPS concentration in model rats at4weekswas higher markedly in that in the normal(P<0.05or P<0.01).Effects of LPS on the apoptosis of activated HSC1. HSC was isolated and identified. Acitvated HSC was treated with LPS(10μg/ml) fordifferent time intervals(30min,1h,6h,12h), and LPS for1h increased TLR4mRNA,MyD88mRNA and TRAF mRNA expression significantly(P<0.01). Acitvated HSCwas treated with NGF for24h with different concentrations(50ng/ml,100ng/ml, 200ng/ml), and NGF at200ng/ml for24h promoted markedly the apoptosis ofactivated HSC(P<0.05), accompanied by the elevated mRNA expressions of fas, bax,p53and p75NTR and the decreased bcl-2mRNA(P<0.05).2.NGF(200ng/ml,24h)inhibited the prolifertion significantly(P<0.01), LPS(10μg/ml,1h)promoted the prolifertion significantly(P<0.05).3. LPS(10μg/ml,1h) inhibited the apoptosis of HSCs induced by NGF(200ng/ml,24h)by using flow cytometry and FITC-TUNEL assays(P<0.05)..4.LPS(10μg/ml,1h) inhibited expressions of bax mRNA, p53mRNA,p75NTR mRNAand NGF mRNA that were induced by NGF, and markedly elevated the bcl-2mRNAexpression that was inhibited by NGF(P<0.05).4. LPS(10μg/ml,1h) could inhibite the increased protein expressions of bax, p75NTR,NGF,cleaved-caspase3,cleaved-caspase9and PARP that were induced byNGF(200ng/ml,24h), and significantly increased bcl-2protein expression that wasinhibited by NGF(P<0.05).Roles of NF-κB dependent MyD88pathway in the apoptosis of HSC1. Activated HSC was stimulated by anti-TLR2or anti-TLR4antibodies, the proteinexpressions involved in cellular apoptosis(cleaved-caspase3, cleaved-caspase9, PARP,Bax and p75NTR). Anti-TLR4antibody but anti-TLR2antibody significantlyantagonized the inhibitory effects of LPS on these proteins expressions induced byNGF(P<0.05).2. LPS and MyD88siRNA had no effect on the apoptosis of HSCs by using flowcytometry(P<0.05). MyD88siRNA attenuated the inhibitory effects of LPS onapoptosis of HSC induced by NGF, with significantly antagonizing the inhibition ofLPS on the protein expressions involved in cellular apoptosis(cleaved-caspase3,cleaved-caspase9, PARP, Bax and p75NTR)(P<0.05). MyD88siRNA inhibitedp-IκB,p50and nuclear p65protein expressions that were increased by LPS(P<0.05).3. Stimulated by BAY, the NF-κB signaling pathway in activated HSC was blocked.Then NGF was treated to LPS-pretreated HSC and apoptosis was examined. AndBAY attenuated the inhibitory effects of LPS on HSC apoptosis(P<0.05) and on theelevated protein expressions (cleaved-caspase3, cleaved-caspase9, PARP, Bax andp75NTR) induced by NGF(P<0.05or P<0.01).Effects of LPS on hepatocyte-induced apoptosis of activated HSC1. Hepatocytes were isolated and stimulated by LPS for1h, and the NGF mRNA andprotein expressions were significant decreased than that in untreated cells(P<0.05). 2, Activated HSC could induced the secretion of NGF from hepatocytes(P<0.05).And by using transwell co-culture model, hepatocytes induced the apoptosis ofactivated HSC(P<0.05), however, pre-treated hepatocytes had no effects on theapoptosis of activated HSC(P<0.05).Conclusion1. Increased plasma LPS presented in the development of hepatic fibrosis andpositively correlated to the degree of hepatic fibrosis.2. LPS inhibited NGF-induced the apoptosis of activated HSC partly in aLPS-TLR4-MyD88-NF-κB manner.3. LPS inhibited the secretion of NGF from hepatocytes to attenuate the apoptosis ofactivated HSC.