Transcriptional Repression of CYP3A4 by Increased miR-200a-3p and miR-150-5p Promotes Steatosis in vitro.

Hepatic cytochrome P450 enzyme activities correlate with non-alcoholic fatty liver disease (NAFLD) and hepatic steatosis. The decreased activity of CYP3A4, an important drug-metabolizing enzyme, is associated with the progression of NAFLD. CYP3A4 is predicted as a target gene of miR-200a-3p and miR-150-5p by MicroInspector and TargetScan algorithms analyses. Here, we found decreased CYP3A4 and increased miR-200a-3p and miR-150-5p in LO2 cells with free fatty acid (FFA)-induced steatosis. Dual-luciferase assay confirmed that both miR-200a-3p and miR-150-5p targeted the 3'-untranslated region (3'-UTR) of CYP3A4 and that such interaction was abolished by miRNA binding site mutations in 3'-UTR of CYP3A4. Using miR-200a-3p and miR-150-5p mimics and inhibitors, we further confirmed that endogenous CYP3A4 was regulated posttranscriptionally by miR-200a-3p or miR-150-5p. Moreover, miR-200a-3p and miR-150-5p inhibitors attenuated FFA-induced steatosis in LO2 cells, and such effect was dependent on CYP3Y4 expression. These results suggest that miR-200a-3p and miR-150-5p, through directly targeting 3'-UTR of CYP3A4, contribute to the development of FFA-induced steatosis.

[Effects of three Wenyang Jianpi Tang on cell proliferation and apoptosis of nonalcoholic fatty liver cells].

To investigate the effects of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang on the cell proliferation and apoptosis of nonalcoholic fatty liver cells through the nonalcoholic fatty liver cell model established by inducing L02 cells with oleic acid. Different concentrations of oleic acid were added into L02 cells to induce the nonalcoholic fatty liver cell model. Oil red O staining was used to observe fatty droplets of fatty liver cells. Automatic biochemical analyzer was used to detect the levels of aspartic transaminase(AST), alanine aminotransferase(ALT), total cholesterol(TC), and triglyceride(TG) in the cell supernatants. There were five groups, namely normal group, model group, model and Sijunzi Tang group, model and Lizhong Tang group, and model and Fuzi Lizhong Tang group. The cell proliferation and apoptosis of the five groups were detected by MTT colorimetry test and flow cytometer. The expressions of PCNA, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax and Bcl-2 proteins of the five groups were detected by Western blot. The oil red O staining results showed that the optimum concentration of oleic acid that was used to induce nonalcoholic fatty liver cell models was 80 mg•L-1. The levels of AST, ALT, TC and TG in the nonalcoholic fatty liver cell supernatants were higher than that in normal liver cell supernatants(P<0.01). MTT colorimetry test and flow cytometer results showed that all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(P<0.01). And Fuzi Lizhong Tang showed the best effect. Western blot results showed that Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could down-regulate the expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax proteins, and up-regulate the expressions of PCNA and Bcl-2 proteins of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. In conclusion, all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. The pharmacodynamic mechanism may be related to the expressions of key factors in pathways related with proliferation and apoptosis mediated by the three decoctions.

Effects of three Wenyang Jianpi Tang on cell proliferation and apoptosis of nonalcoholic fatty liver cells / 中国中药杂志

To investigate the effects of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang on the cell proliferation and apoptosis of nonalcoholic fatty liver cells through the nonalcoholic fatty liver cell model established by inducing L02 cells with oleic acid. Different concentrations of oleic acid were added into L02 cells to induce the nonalcoholic fatty liver cell model. Oil red O staining was used to observe fatty droplets of fatty liver cells. Automatic biochemical analyzer was used to detect the levels of aspartic transaminase(AST), alanine aminotransferase(ALT), total cholesterol(TC), and triglyceride(TG) in the cell supernatants. There were five groups, namely normal group, model group, model and Sijunzi Tang group, model and Lizhong Tang group, and model and Fuzi Lizhong Tang group. The cell proliferation and apoptosis of the five groups were detected by MTT colorimetry test and flow cytometer. The expressions of PCNA, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax and Bcl-2 proteins of the five groups were detected by Western blot. The oil red O staining results showed that the optimum concentration of oleic acid that was used to induce nonalcoholic fatty liver cell models was 80 mg•L-1. The levels of AST, ALT, TC and TG in the nonalcoholic fatty liver cell supernatants were higher than that in normal liver cell supernatants(P<0.01). MTT colorimetry test and flow cytometer results showed that all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(P<0.01). And Fuzi Lizhong Tang showed the best effect. Western blot results showed that Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could down-regulate the expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax proteins, and up-regulate the expressions of PCNA and Bcl-2 proteins of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. In conclusion, all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. The pharmacodynamic mechanism may be related to the expressions of key factors in pathways related with proliferation and apoptosis mediated by the three decoctions.