Qinggan Huoxue Recipe suppresses epithelial-to-mesenchymal transition in alcoholic liver fibrosis through TGF-β1/Smad signaling pathway.

AIM: To investigate the mechanism by which Qinggan Huoxue Recipe (QGHXR) inhibits epithelial-to-mesenchymal transition (EMT) in rats with alcoholic liver fibrosis (ALF).

METHODS: A total of 75 male SD rats were used to induce ALF. Serum biochemical indicators, including alanine aminotransferase, aspartate aminotransferase, laminin and hyaluronidase, were measured. Liver histopathological changes were evaluated using hematoxylin-eosin and Sirius red staining. EMT was examined by analyzing the expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and fibronectin using RT-PCR and Western blot. The inhibitory effect of QGHXR on EMT markers, as well as its effect on molecules associated with the transforming growth factor (TGF)-β1/Smad signaling pathway, including TGF-β1, Smad3, snail, occludin, ZO-1 and claudin, was also examined.

RESULTS: Compared with normal control rats, ALF rats exhibited a decrease in E-cadherin levels (mRNA: ALF 0.16 ± 0.05 vs control 1.00 ± 0.08; protein: ALF 0.09 ± 0.05 vs control 0.70 ± 0.17, P < 0.01) and an increase in vimentin and fibronectin levels (mRNA: 11.43 ± 0.39 vs 1.00 ± 0.19 and 9.91 ± 0.34 vs 1.00 ± 0.44, respectively, P < 0.01; protein: 1.13 ± 0.42 vs 0.09 ± 0.03 and 1.16 ± 0.43 vs 0.09 ± 0.00, respectively, P < 0.01). This indicates that EMT occurred in ALF rats. In addition, the TGF-β1/Smad signaling pathway was activated in ALF rats, as evidenced by the increase in TGF-β1 and snail levels (mRNA: 1.76 ± 0.12 vs 1.00 ± 0.05 and 6.98 ± 0.41 vs 1.00 ± 0.10, respectively, P < 0.01; protein: 1.43 ± 0.05 vs 0.12 ± 0.03 and 1.07 ± 0.29 vs 0.07 ± 0.02, respectively, P < 0.01) and the decrease in Smad3 levels (mRNA: 0.05 ± 0.01 vs 1.00 ± 0.12, P < 0.01; protein: 0.06 ± 0.05 vs 0.89 ± 0.12, P < 0.01). Furthermore, levels of the tight junction markers occludin, ZO-1 and claudin decreased in ALF rats compared with healthy control rats (mRNA: 0.60 ± 0.09 vs 1.00 ± 0.12, 0.11 ± 0.00 vs 1.00 ± 0.12 and 0.60 ± 0.01 vs 1.00 ± 0.08, respectively, P < 0.01; protein: 0.05 ± 0.01 vs 0.87 ± 0.40, 0.09 ± 0.05 vs 0.89 ± 0.18 and 0.04 ± 0.03 vs 0.95 ± 0.21, respectively, P < 0.01). In ALF rats treated with QGHXR, E-cadherin levels increased (mRNA: QGHXR 0.67 ± 0.04 vs ALF model 0.16 ± 0.05, P < 0.01; protein: QGHXR 0.66 ± 0.21 vs ALF model 0.09 ± 0.05, P < 0.01), and vimentin and fibronectin levels decreased (mRNA: 6.57 ± 1.05 vs 11.43 ± 0.39 and 1.45 ± 1.51 vs 9.91 ± 0.34, respectively, P < 0.01; protein: 0.09 ± 0.03 vs 1.13 ± 0.42 and 0.10 ± 0.01 vs 1.16 ± 0.43, respectively, P < 0.01). In addition, QGHXR inhibited the expression of TGF-β1 and increased the expression of Smad3 (mRNA: 1.03 ± 0.11 vs 1.76 ± 0.12, 0.70 ± 0.10 vs 0.05 ± 0.01, respectively, P < 0.05 and P < 0.01; protein: 0.12 ± 0.03 vs 1.43 ± 0.05 and 0.88 ± 0.20 vs 0.06 ± 0.05, respectively, P < 0.01). QGHXR treatment also reduced the levels of the EMT-inducing transcription factor snail (mRNA: 2.28 ± 0.33 vs 6.98 ± 0.41, P < 0.01; protein: 0.08 ± 0.02 vs 1.07 ± 0.29, P < 0.01) and increased the occludin, ZO-1 and claudin levels (mRNA: 0.73 ± 0.05 vs 0.60 ± 0.09, 0.57 ± 0.04 vs 0.11 ± 0.00 and 0.68 ± 0.03 vs 0.60 ± 0.01, respectively, P < 0.01, P < 0.01 and P < 0.05; protein: 0.92 ± 0.50 vs 0.05 ± 0.01, 0.94 ± 0.22 vs 0.09 ± 0.05 and 0.94 ± 0.29 vs 0.04 ± 0.03, respectively, P < 0.01). The effects of QGR and HXR on the TGF-β1/Smad signaling pathway were similar to that of QGHXR; however, the QGR- and HXR-induced changes in vimentin mRNA levels, the QGR-induced changes in fibronectin mRNA levels and the HXR-induced changes in snail and TGF-β1 mRNA levels were not significant.

CONCLUSION: Qinggan Huoxue Recipe inhibits EMT in ALF rats by modulating the TGF-β1/Smad signaling pathway, suggesting that the mechanism underlying the amelioration of ALF induced by QGHXR is associated with this pathway.