疏肝健脾活血方含药血清对TGF-β1诱导活化的LX-2中CUGBP1、IFN-γ、Smad7的影响

Effects of Shugan Jianpi Huoxue Recipe serum on CUGBP1,IFN-γand Smad 7 in the activated LX-2 induced by TGF-β1

目的:观察疏肝健脾活血方含药血清对TGF-β1诱导活化的人肝星状细胞株LX-2中CUGBP1、IFN-γ、Smad7的影响,探讨疏肝健脾活血方抗肝纤维化的可能作用机制。方法:20只SD大鼠制备含药血清,CCK8测定含药血清细胞毒性;慢病毒转染建立CUGBP1过表达和敲减LX-2细胞稳转株,在此基础上对CUGBP1过表达空载(LV空载)及敲减空载(sh空载)、CUGBP1过表达(LV-CUGBP1)及敲减(sh-CUGBP1)稳转株,分别予相应空白血清、TGF-β1、疏肝健脾活血方含药血清干预。RT-PCR检测各组α-SMA、CUGBP1、IFN-γ、Smad7 mRNA表达;ELISA检测各组上清液中IFN-γ含量。结果:CCK8法测定含药血清对LX-2的半数抑制浓度约为40%。完成CUGBP1过表达和敲减稳转株构建。与LV-CUGBP1组比较,LV-CUGBP1+TGF-β1组中α-SMA、CUGBP1 mRNA表达升高(P<0.01),IFN-γ、Smad7 mRNA表达降低(P<0.01,P<0.05);与LV-CUGBP1+TGF-β1组比较,LV-CUGBP1+TGF-β1+中药组中α-SMA、CUGBP1 mRNA表达降低(P<0.01,P<0.05);Smad7 mRNA表达升高(P<0.01)。与sh-CUGBP1组比较,sh-CUGBP1+TGF-β1组中α-SMA mRNA表达升高(P<0.01)、Smad7 mRNA表达降低(P<0.01);与sh-CUGBP1+TGF-β1组比较,sh-CUGBP1+TGF-β1+中药组中α-SMA、CUGBP1 mRNA表达降低(P<0.05),Smad7 mRNA表达升高(P<0.01)。疏肝健脾活血方含药血清干预后LX-2细胞上清液中IFN-γ含量增加,CUGBP1过表达后IFN-γ含量减少,CUGBP1敲减后IFN-γ含量增加。结论:疏肝健脾活血方含药血清可有效抑制LX-2细胞活化,可通过抑制CUGBP1对IFN-γ的降解发挥作用,并上调IFN-γ和Smad7表达。

Objective:To observe the effects of serum containing Shugan Jianpi Huoxue Recipe on CUGBP1,IFN-γand Smad7 in human hepatic stellate cell line(LX-2)activated by TGF-β1,and to explore the possible mechanism of Shugan Jianpi Huoxue Recipe on anti-hepatic fibrosis.Methods:Twenty SD rats were used to prepare drug-containing serum,and the cytotoxicity of drug-containing serum was determined by CCK8.CUGBP1 overexpression and knockdown LX-2 cell lines were established by lentivirus transfection.On this basis,CUGBP1 overexpression no-load and knockdown no-load,CUGBP1 overexpression and knockdown stable transfected cell lines were given or withheld the interventions of blank serum,TGF-β1,Shugan Jianpi Huoxue Recipe containing serum(SGJPHX).The expressions ofα-SMA,CUGBP1,IFN-γand Smad7 mRNA were detected by PCR,and the content of IFN-γin the cell supernatant was detected by ELISA.Results:The half inhibitory concentration of LX-2 in SGJPHX serum was about 40%as determined by CCK8 method.The construction of CUGBP1 overexpression and knockdown stable transfected cell lines were completed.PCR assay revealed that the expressions ofα-SMA and CUGBP1 mRNA were significantly elevated(P<0.01)and the expressions of IFN-γand Smad7 mRNA were significantly reduced(P<0.01,P<0.05)in the LV-CUGBP1+TGF-β1 group compared with the LV-CUGBP1 group.Compared with the LV-CUGBP1+TGF-β1 group,α-SMA and CUGBP1 mRNA expressions were significantly decreased(P<0.01,P<0.05),and Smad7 mRNA expression was significantly elevated in the LV-CUGBP1+TGF-β1+SGJPHX group(P<0.01).Compared with the sh-CUGBP1 group,α-SMA mRNA expression was elevated and Smad7 mRNA expression was significantly decreased in the sh-CUGBP1+TGF-β1 group(P<0.01).Compared with the sh-CUGBP1+TGF-β1 group,α-SMA and CUGBP1 mRNA expressions were significantly decreased in the sh-CUGBP1+TGF-β1+SGJPHX group(P<0.05)and Smad7 mRNA expression was significantly elevated(P<0.01).The content of IFN-γin the supernatant of LX-2 cells increased after the intervention of SGJPHX serum,the content of IFN-γdecreased after the overexpression of CUGBP1,and the content of IFN-γincreased after the knockdown of CUGBP1.Conclusion:Shugan Jianpi Huoxue Recipe could effectively inhibit the activation of LX-2 cells by inhibiting the degradation of IFN-γby CUGBP1 and up-regulating the expression of IFN-γand Smad7.