白藜芦醇对小鼠非酒精性脂肪性肝炎相关肝细胞癌的治疗作用及机制研究

Therapeutic effect of resveratrol on nonalcoholic steatohepatitis associated hepatocellular carcinoma in mice

目的探讨白藜芦醇(RSV)在非酒精性脂肪性肝炎相关肝细胞癌(NASH-HCC)小鼠模型中的治疗作用及分子机制。方法选取出生后4d C57BL/6J小鼠,每只背部皮下注射STZ 200μg,4周龄时选取雄性小鼠,随机分为STZ+普通饲料组、STZ+高脂高胆固醇饲料(HFHC)组和STZ+HFHC+RSV(RSV 400mg/kg,1次/d灌胃)组,每组10只。于16周龄处死实验小鼠,测定血清生化指标,采用RT-PCR法检测肝组织肿瘤坏死因子(TNF)-α、单核细胞趋化蛋白(MCP)-1、I型胶原蛋白(Collagen Type I)、转化生长因子(TGF)-β、磷脂酰肌醇蛋白聚糖3(GPC3)和胆汁酸相关受体法尼醇X受体(FXR)及其下游靶基因小异二聚体伴侣(SHP)和肝细胞膜上胆汁酸盐输出泵(BSEP)mRNA的表达水平,采用超高效液相色谱串联质谱联用仪(UPLC-MS/MS)检测鹅去氧胆酸(CDCA)、去氧胆酸(DCA)、牛磺β鼠胆酸(TβMCA)、甘氨胆酸(GCA)、胆酸(CA)、牛磺胆酸(TCA)、牛磺鹅去氧胆酸(TCDCA)、牛磺石胆酸(TLCA)、石胆酸(LCA)等胆汁酸及总胆汁酸的含量,观察肝组织病理变化及进行非酒精性脂肪性肝病活动度积分(NAS)评定。结果与STZ+普通饲料组比较,STZ+HFHC组肝指数、AST、HDL、LDL、TC、TNF-α、MCP-1、Collagen Type I、TGF-β、GPC3 mRNA表达水平,总胆汁酸、CDCA、DCA、TβMCA含量以及脂肪变、小叶内炎症、NAS评分均明显升高,FXR mRNA表达水平明显下降(均P<0.05);与STZ+HFHC组比较,STZ+HFHC+RSV组肝指数、FPG、ALT、AST、HDL、LDL、TC、TG,MCP-1、Collagen Type I、TGF-β和GPC3 mRNA表达水平,总胆汁酸、CDCA、DCA、TβMCA、GCA、CA、TCA、TCDCA、TLCA含量以及脂肪变、NAS评分均明显下降,FXR、SHP和BSEP mRNA表达水平均明显升高(均P<0.05)。结论RSV具有抗NASH相关HCC的作用,可以作为治疗NASH-HCC的候选药物。

Objective To investigate the therapeutic effect and molecular mechanism of resveratrol(RSV)on nonalcoholic steatohepatitis-hepatocellular carcinoma(NASH-HCC)in mice.Methods Thirty C57BL/6J male mice were injected with STZ 200μg and randomly divided into three groups with 10 mice in each group:STZ+standard chow,STZ+high fat high cholesterol(HFHC)and STZ+HFHC+RSV groups.The mice were sacrificed in the 16th week.The body weight,liver index and serum biochemical indicators were determined.The expression of tumor necrosis factor(TNF)-α,monocyte chemoattractant protein(MCP)-1,Collagen Type I,transforming growth factor(TGF)-β,glypican(GPC)3,Farnixide X receptor(FXR),small heterodimer partner(SHP)and bile salt export pump(BSEP)mRNA was determined by RT-PCR.The absolute concentration of bile acids was measured by means of UPLC-MS/MS,such as chenodeoxycholic acid(CDCA),deoxycholic acid(DCA),Tauroβmuricholic acid(TβMCA),glycocholic acid(GCA),cholic acid(CA),taurocholic acid(TCA),taurochenodeoxycholic acid(TCDCA),taurolithocholic acid(TLCA),lithocholic acid(LCA)and total bile acid.The liver tissues were examined histopathologically with HE,Oil Red stain and NAS scores were assessed.Results Compared to STZ+standard chow group,the liver index,AST,HDL,LDL,TC,TNF-α,MCP-1,collagen type I,TGF-β,GPC3 mRNA expression,total bile acid,CDCA,DCA,TβMCA,steatosis,intralobular inflammation and NAS scores were significantly increased,and FXR mRNA expression was significantly decreased in the STZ+HFHC group(all P<0.05).Liver index,FPG,ALT,AST,HDL,LDL,TC,TG,MCP-1,collagen type I,TGF-βand GPC3 mRNA expression level,total bile acid,CDCA,DCA,TGF-β,GCA,CA,TCA,TCDCA,TLCA,steatosis and NAS scores in the STZ+HFHC+RSV group were decreased,the expression of FXR,SHP and BSEP mRNA increased significantly compared to STZ+HFHC group(all P<0.05).Conclusion RSV exerts an anti-NASH-HCC effect and may be a candidate agent for the treatment of NASH-HCC.