Effects Of HCPT On PDGF Stimulated Hepatic Stellate Cell Collagen Gene Expression And Its Intracellular Signal Transduction Via ERK

Objective:To investigate the effects of hydrocamptothecin(HCPT) on the expression of a-smooth muscle actin (a-SMA) andⅠ,Ⅲtype collagen mRNA in rat hepatic stellate cells (HSC) stimulating by platelet-derived growth factor(PDGF) in vitro, and to explore the regulation of a-SMA andⅠ,Ⅲcollagen mRNA expression with extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway of HSC cells controlled by HCPT, providing a experimental evidence to the clinical application of HCPT on hepatic fibrosis。Methods:Rat HSC line(HSC-T6) were cultured by high sugar Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FBS), The cells were divided into four groups after serum-starved for 24 hours as follows:control group (0.4%FBS group); PDGF (lOng/ml) stimulated group; PD98059+PDGF group (30μmol/LPD98059+10ng/mlPDGF); HCPT+PDGF group(0.5mg/l HCPT+10ng/ml PDGF)。After 15min,30min,60min, The western blot assay were used to detect the different expressions of phosphorylation ERK1/2 protein; After 12h,24h,48h, The proliferation rates of the HSC-T6 stimulated by PDGF on the effect of HCPT were determined by MTT assay. The effects of HCPT on the expression of a-SMA and typeⅠ,Ⅲcollagen mRNA in HSC-T6 stimulated by PDGF were determined by semi-quantitative RT-PCR assay。Results:1,MTT assay indicated 12h,24h,48h, the proliferation rates of HSC-T6 of PDGF-BB group were significantly increased; however, the proliferation rates of HSC-T6 induced with HCPT and PD98059 (the inhibition of ERK1/2), were reduced, Compared with the control group, was statistically significant (P<0.01) and proportional with the reaction time; 2,The results of RT-PCR showed 12h,24h,48h, PDGF group, HSC-T6 a-SMA andⅠ,Ⅲcollagen mRNA expression was significantly higher than the control group, compared with the control group, was statistically significant (P<0.01) and proportional with the reaction time; HCPT and PD98059 group, HSC-T6α-SMA andⅠ,Ⅲcollagen mRNA expression was significantly lower than the control group, compared with the control group, was statistically significant (P<0.01),and proportional with the reaction time; But between the two groups was no significant difference (P> 0.05); 3,The results of western Blot indicated 15min,30min,60min, PDGF effect of cultured HSC-T6 cell ERK1/2 signaling enhanced the expression of protein phosphorylation, peaked at 30 minutes,compared with the control group, was statistically significant (P<0.01); HCPT and PD98059 role of HSC-T6 cells cultured ERK1/2 signaling reduced the expression of protein phosphorylation, inhibition of the peak at 30 minutes, compared with the control group, was statistically significant (P<0.01); But between the two groups was no significant difference (P> 0.05)。Conclusion:HCPT may inhibit the proliferation and the expression of a-SMA, typeⅠ,Ⅲcollagen mRNA of HSC-T6 induced by PDGF, The mechanism was related to the ERK1/2 pathway.