Effects of Shugan Jianpi Huoxue Recipe serum on CUGBP1,IFN-γ and Smad 7 in the activated LX-2 induced by TGF-β1
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-β 1,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 of α-SMA,CUGBP1,IFN-γ and Smad7 mRNA were detected by PCR,and the content of IFN-γ in 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 of α-SMA and CUGBP1 mRNA were significantly elevated(P＜0.01)and the expressions of IFN-γ and 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-γ in the supernatant of LX-2 cells increased after the intervention of SGJPHX serum,the content of IFN-γ decreased after the overexpression of CUGBP1,and the content of IFN-γ increased 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-γ by CUGBP1 and up-regulating the expression of IFN-γ and Smad7.
Liver fibrosisHuman liver stellate cell line LX-2Shugan Jianpi Huoxue RecipeCUGBP1IFN-γSmad7
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