Regulating VEGF/VEGFR Expression In HSC/SEC Co-culture System By Shugan Jianpi Huoxue Recipe

Objective Based on the pathological mechanism of angiogenesis in hepatic fibrosis,the co-culture of hepatic sinusoidal endothelial cells(SECs)at different hepatic fibrosis stage and VEGF over expressed hepatic stellate cells(HSCs)was used as experimental material to explore the anti-fibrosis mechanism of Shugan Jianpi Huoxue recipe in regulating VEGF expression and determine the optimal time of intervention.Methods 1.HSC and SEC were isolated from normal rats,and rats were subcutaneously injected with CCl4 to prepare liver fibrosis model,isolated model rats for model SEC after 2/4/8 weeks injection respectively.2.Constructing VEGF sh RNA recombinant lentivirus to transfect HSC in vitro,detecting the expression of green fluorescent protein and VEGF protein in transfected HSC by Western Blot.3.Medium concentration of Shugan Jianpi Huoxue recipe was used for rat intragastric administration to prepare the medicine for drug containing serum.MTT was used to detect the effect of different concentrations of the drug containing serum on the HSCs' proliferation,and determine the best effective concentration.4.Normal HSC and SEC,the liver fibrosis model SEC were isolated from the three time points of 2W,4W,8W,along with the VEGF over-expressed HSC cells were divided into 6 groups: H group: normal HSC cultured by blank serum medium;HS group: normal HSC and normal SEC co-cultured by blank serum medium;HM group: normal HSC and model SEC cocultured by blank serum medium;VM group: VEGF over-expressed HSC and model SEC co-cultured by blank serum medium;HMZ group: normal HSC and model SEC co-cultured by 10% drug-containing serum medium;HVM group: VEGF overexpressed HSC and model SEC co-cultured by 10% drug-containing serum medium(18 groups in total)5.Western Blot was used to detect the protein expression of collagen IV,VEGF,VEGFR-2 in HSC and CD31,v WF in SEC.The m RNA expression of VEGF,VEGFR-1,VEGFR-2 in HSC was detected by Real time Quantitative PCR(RT-PCR).Results 1.10% drug-containing serum had the best inhibitory effect;2.Model SEC cocultured with HSC stimulated the proliferation of HSC(P<0.01);3.The protein expression of CD31 and v WF in SEC was up-regulated in VM group(P<0.01)compared with H group and HS group(P<0.05 or P<0.01),lasted to 8w;the expression of collagen IV,VEGF,VEGFR-2 protein and VEGF m RNA up-regulated in co-cultured HSC(P<0.05 or P<0.01).4.Compared with H group and HS group,the protein expression of CD31 and v WF in SEC of HM group increased(P<0.05 or P<0.01),the expression of collagen IV,VEGF,VEGFR-2 protein and VEGF m RNA up-regulated in co-cultured HSC(P<0.05 or P<0.01).Compared with HM group,the expression of CD31 and v WF protein in SEC of HMZ group was decreased(P<0.05 or P<0.01),the expression of collagen IV,VEGF,VEGFR-2 protein was lower in co-cultured HSC at 4w(P<0.05 or P<0.01),the VEGF m RNA expressed less as well(P < 0.01).Conclusion The inhibitory effect of 10% drug containing serum on the proliferation of HSC in vitro is the most significant.Hepatic fibrosis model SEC promoted the proliferation of HSC,with the over-expression of VEGF the proliferation enhanced,but with the effect of drug containing serum the proliferation inhibited.The overexpression of VEGF in the early stage(2w)of hepatic fibrosis has promoted the defenestrae of SEC,which may be caused by binding with VEGFR-2.Shugan Jianpi Huoxue recipe could reverse the defenestrae of SEC at the early stage of liver fiber for 2w and 4 w.The mechanism may be related to the down-regulation of VEGF/VEGFR-2 expression.