扶肝化纤汤含药血清对TGF-β1诱导HSC-T6细胞增殖及TGF-β1/Smad信号通路的影响

Effects of Serum Containing Fugan Huaxian Decoction(扶肝化纤汤) on TGF-β1 Induced HSC-T6 Proliferation and TGF-β 1/Smad Signaling Pathway

目的探讨扶肝化纤汤抗肝纤维化的可能作用机制。方法将40只雄性Wistar大鼠随机分为正常对照组、扶肝化纤汤组,每组20只,扶肝化纤汤组以16. 78g/kg扶肝化纤汤灌胃,正常对照组予等体积生理盐水灌胃,每日1次,连续7天,末次给药后于大鼠腹主动脉取血制备含药血清。培养大鼠肝星状细胞HSC-T6,设置空白对照组及1%、2%、5%、8%、10%、15%、20%扶肝化纤汤含药血清组,检测不同浓度扶肝化纤汤含药血清对转化生长因子β1 (TGF-β1)诱导的HSC-T6活化增殖的影响,确定15%扶肝化纤汤含药血清为最佳给药浓度。细胞实验分为空白对照组(HSC-T6细胞)、正常组(HSC-T6细胞+正常对照血清+TGF-β1)、中药组(HSC-T6细胞+15%扶肝化纤汤含药血清+TGF-β1);阻断组1 (HSC-T6细胞+SIS3)、阻断组2 (HSC-T6细胞+正常对照血清+TGF-β1+SIS3)及阻断组3 (HSC-T6细胞+15%扶肝化纤汤含药血清+TGF-β1+SIS3),培养24 h后比较各组HSC-T6细胞增殖抑制率及细胞中Smad 3、Smad 7、α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原mRNA表达。结果与空白对照组比较,正常组、中药组、阻断组2、阻断组3 HSC-T6细胞增殖抑制率差异有统计学意义(P <0. 01)。与正常组比较,中药组、阻断组2、阻断组3HSC-T6细胞增殖抑制率差异均有统计学意义(P <0. 01)。与中药组比较,阻断组3抑制率明显升高(P <0. 01)。与空白对照组比较,中药组、阻断组2、阻断组3均可下调Smad 3、Ⅰ型胶原mRNA表达,上调Smad 7、α-SMA mRNA表达(P <0. 01);正常组可上调Smad 3、α-SMA、Ⅰ型胶原mRNA表达,下调Smad 7mRNA表达(P <0. 01)。与正常组比较,中药组、阻断组2、阻断组3均可下调Smad 3、α-SMA、Ⅰ型胶原mRNA表达,上调Smad 7 mRNA表达(P <0. 01)。与中药组比较,阻断组3可下调Smad 3、α-SMA、Ⅰ型胶原mRNA表达,上调Smad 7 mRNA表达(P <0. 01)。结论扶肝化纤汤能够抑制TGF-β1诱导的HSC-T6细胞的活化增殖,其可能通过影响TGF-β1/Smad信号转导通路发挥抗肝纤维化作用。

Objective To explore the possible mechanism of anti-liver fibrosis in Fugan Huaxian Decoction(扶肝化纤汤).Methods A total of 40 male Wistar rats were randomly divided into a normal control group and a Fugan Huaxian Decoction group,with 20 rats in each group.Fugan Huaxian Decoction group was intragastrically administered with 16.78 g/kg Fugan Huaxian Decoction,and the normal control group was given equal volume of normal saline by gavage,once a day for 7 consecutive days.After the last administration,blood was taken from the abdominal aorta of the rats to prepare the drug-containing serum.Rat hepatic stellate cells HSC-T6 were cultured,and blank control group as well as 1%,2%,5%,8%,10%,15%and 20%Fugan Huaxian Decoction containing serum group were set up.The effects of different concentrations of Fugan Huaxian Decoction serum on the activation and proliferation of HSC-T6 induced by transforming growth factorβ1(TGF-β1)were determined.The serum containing15%Fugan Huaxian Decoction was determined as the optimal concentration.Cell experiments were divided into a blank control group(HSC-T6 cells),a normal group(HSC-T6 cells+normal control serum+TGF-β1),a Chinese medicine group(HSC-T6 cells+15%Fugan Huaxian Decoction serum+TGF-β1);a blocking group 1(HSC-T6 cells+SIS3),a blocking group 2(HSC-T6 cells+normal control serum+TGF-β1+SIS3)and a blocking group 3(HSC-T6 cells+15%Fugan Huaxian Decoction containing serum+TGF-β1+SIS3).After 24 hours of culture,the proliferation inhibition rate of HSC-T6 cells and Smad 3,Smad 7,αsmooth muscle actin(α-SMA)and type I collagen mRNA of each group were compared.Results Compared with the blank control group,the inhibition rate of HSC-T6 cell proliferation in the normal group,the Chinese medicine group,the blocking group and the blocking group had statistically significant difference(P<0.01).Compared with the normal group,the inhibition rate of HSC-T6 cell proliferation in the Chinese medicine group,the blocking group 2 and the blocking group 3 had statistically significant difference(P<0.01).Compared with the Chinese medicine group,the inhibition rate of blocking group 3 was significantly increased(P<0.01).Compared with the blank control group,the expression of Smad 3 and type I collagen mRNA was down-regulated in the Chinese medicine group,blocking group 2 and blocking group 3,and the expression of Smad 7 andα-SMA mRNA was up-regulated(P<0.01).The normal group could upregulate Smad 3 andα-SMA and type I collagen mRNA expression,down-regulated Smad 7 mRNA expression(P<0.01).Compared with the normal group,the Chinese medicine group,the blocking group 2 and the blocking group3 all down-regulated the expression of Smad 3,α-SMA and type I collagen and up-regulated the expression of Smad 7 mRNA(P<0.01).Compared with the Chinese medicine group,blocking group 3 could down-regulate the expression of Smad 3,α-SMA and type I collagen and up-regulate the expression of Smad 7 mRNA(P<0.01).Conclusion Fugan Huaxian Decoction could inhibit the activation and proliferation of HSC-T6 cells induced by TGF-β1,which may play an anti-fibrosis effect by affecting TGF-β1/Smad signal transduction pathway.