Effects Of HGF On Proliferation And Apoptosis Of Rat Primary Cultured HSC Treated With PDGF-BB And The Mechanism

Background: Liver fibrosis is characterized by hepatic dysfunction with extensive accumulation of fibrous tissue. In response to chronic hepatic injury, activated hepatic stellate cell (HSC) played an important role in liver fibrosis. Recently, it was indicated that hepatic fibrosis was reversible, in the process of hepatic fibrosis resolution HSC undergone spontaneous apoptosis. Although administration or gene expression of hepatocyte growth factor (HGF) leaded to improvement in hepatic fibrosis, the related mechanisms were not fully understood. We investigated the machanisms of HGF on HSC, focusing on proliferation and apoptosis in HSC.Methods: Rat HSC was isolated by an improved collagenase-pronase digested method and identified as HSC by immunocytochemical stain. First, passage cells were used to investigate the effect of HGF on proliferation of HSC, the cells were divided into four groups: normal control group, PDGF-BB treated group, PDGF-BB+HGF treated group and HGF treated group. Five concentrations of PDGF-BB: 2,5,10,20,40ng/ml and four concentrations of HGF: 0.5,1,2,5μg/ml were used, cells were treated with 12, 24 and 48 hour respectively. The effect of HGF on proliferation of HSC was detected by MTT. After selected the optimized concentration and treatment time, apoptosis of HSC was observed. The cells were divided into three groups: normal control group, PDGF-BB+HGF treated group and HGF treated group. After treated with 10ng/ml PDGF-BB and 2μg/ml HGF for 6, 12 and 24 hour, apoptosis was detected by AO/EB stain, TUNEL and ANNEXINⅤFITC/PI, expression level of P65 were detected by immunocytochemical stain, and DNA-protein binding complex of NF-κB were detected by electrophoretic mobility shift assay.Results:1. The yield of HSC was 2-5×107 per rat, purity exceed 98% and survival rate exceed 90%.2. HGF had a dose-dependent increased inhibtion effect on PDGF-BB induced proliferation in HSC at the concentrations of 0.5μg/ml,1μg/ml,2μg/ml and 5μg/ml, respectively. There were no differences of inhibition ratio between 2μg/ml treated group and 5μg/ml HGF treated group(P>0.05), also between 0.5μg/ml HGF treated group and PDGF-BB treated group(P>0.05). In other groups,the differences of inhibition rate were found (P<0.05). Proliferation of HSC was respectively reduced from PDGF-BB treated group,PDGF-BB+HGF treated group,normal control group to HGF treated group(P<0.05).3. The apoptosis rate was gradually increased from normal control group, PDGF-BB+HGF treated group to HGF treated group by AO/EB stain. It was observed by our study that apoptosis rate increased in a longer intervention time. Compared with 12 hour,differences of apoptosis rate of 6 hour were considered to be statistically significant(P<0.05),but differences was no statistically significant between 12 hour and 24 hour(P>0.05). Treated with 12 hour, apoptosis rate was respectively increased from normal control group, PDGF-BB+HGF treated group to HGF treated group(P<0.05),were respectively 1.64%,3.22%,6.66% detected by ANNEXINⅤFITC/PI stain.4. DNA-protein binding activity of NF-κB was respectively attenuated from normal control group, PDGF-BB+HGF treated group to HGF treated group detected by electrophoretic mobility shift assay. The expression of P65 was mainly in nucelus in normal control group, then nucelus expression was reduced in PDGF-BB+HGF treated group and HGF treated group compared to normal control group, and the expression levels were attenuated.Conclusion:1. It is an important model to use primary cultured rat HSC to investigate liver fibrosis in vitro.2. HGF inhibits proliferation of HSC directly via its tyrosine kinase receptor c-Met, and also blockade proliferation of HSC induced by PDGF-BB. This double suppression of proliferation of HSC may be one of the mechanisms of HGF improved hepatic fibrosis. 3. HGF can induce actived HSC apoptosis. It may be the other aspect of the mechanisms of HGF improved hepatic fibrosis.4. It is observed in our study that the nucelus expression of P65 and DNA-protein binding activity of NF-κB attenuated accompanied to the apoptosis rate of HSC induced by HGF, which indicate that HGF induces HSC apoptosis by repressed NF-κB signal pathway.