Methyl helicterte ameliorates liver fibrosis by regulating miR-21-mediated ERK and TGF-β1/Smads pathways

Methyl helicterate (MH) has been reported to have protective effects against CCl4-induced hepatic injury and fibrosis in rats, but its protective mechanism, especially on hepatic stallete cells (HSCs), remains unclear. Recently, our pilot experiment showed that MH could inhibit miR-21 expression in HSC-T6 cells, suggesting that miR-21 may be one of the targets of MH to intervene liver fibrosis. To verify the hypothesis, the present study would focus on the regulatory effect of MH on the miR-21-mediated ERK and TGF-beta 1/Smads pathways. Briefly, rats were intraperitoneally injected with 0.5 ml porcine serum (PS) twice a week for 24 weeks to induce liver fibrosis, and meanwhile, the rats were treated with MH from weeks 16 to 24. In vitro experiment, miR-21 expression in HSC-T6 cells was up- or down-regulated using lentiviral transfection assay. Collagen accumulation, inflammatory cytokines, cell apoptosis, miR-21 expression, and activation of the ERK and TGF-beta 1/smad2/3 pathways were then assessed. The results showed that MH treatment markedly alleviated PS-induced liver injury, as evidenced by the attenuation of histopathological changes and the decrease in serum alanine and aspartate aminotransferases activity. MH significantly decreased the content of inflammatory cytokines and recruited the anti-oxidative defense system. Moreover, MH treatment significantly decreased miR-21 expression and inhibited the activation of the ERK and TGF-beta 1/smad2/3 pathways in liver tissues. In vitro experiments showed that MH strongly inhibited HSC-T6 cell activation and reduced collagen accumulation. Interestingly, miR-21 overexpression significantly promoted HSC-T6 cell proliferation, reduced HSC apoptosis, and increased collagenation, while these abnormal changes induced by miR-21overexpression were significantly reversed by MH treatment. Furthermore, miR-21 overexpression notably activated the ERK and TGF-beta 1/Smads pathways via repressing SPRY2 and Smad7 expression respectively, however, these effects were largely abolished by MH treatment. In conclusion, our study demonstrates that MH significantly alleviates PS-induced liver injury and fibrosis by inhibiting miR-21-mediated ERK and TGF-beta 1/Smads pathways.