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

Abstract

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.

Keywords: Alcoholic liver fibrosis; Epithelial-to-mesenchymal transition; QGHXR; Snail; Transforming growth factor-β1/Smad.

Figure 1

Qinggan Huoxue recipe ameliorates liver injury associated with alcoholic liver fibrosis with respect to alanine transaminase, aspartate transaminase, laminin and hyaluronidase levels. ^aP < 0.05, ^bP < 0.01 vs Normal healthy control group; ^cP < 0.05, ^dP < 0.01 vs Model ALF group. N: Normal healthy control group; M: Model ALF group; Q: Qinggan Recipe group; H: Huoxue Recipe group; QH: Qinggan Huoxue Recipe group.

Figure 2

Changes in liver histopathology detected by hematoxylin and eosin staining and light microscopy (× 200 magnification). A: Normal healthy control group; B: Model ALF group; C: Qinggan Recipe group; D: Huoxue Recipe group; E: Qinggan Huoxue Recipe group. ALF: Alcoholic liver fibrosis.

Figure 3

Changes in liver histopathology observed by Sirius Red staining and light microscopy (× 200 magnification). A: Normal healthy control group; B: Model ALF group; C: Qinggan Recipe group; D: Huoxue Recipe group; E: Qinggan Huoxue Recipe group.ALF: Alcoholic liver fibrosis.

Figure 4

Qinggan Huoxue Recipe-induced expression of epithelial-to-mesenchymal transition-associated factors. A: E-cadherin, vimentin and fibronectin mRNA expression evaluated by RT-PCR; B: E-cadherin, vimentin and fibronectin protein expression evaluated by Western blot. ^aP < 0.05, ^bP < 0.01 vs Normal healthy control group; ^cP < 0.05, ^dP < 0.01 vs Model ALF group. N: Normal healthy control group; M: Model ALF group; Q: Qinggan Recipe group; H: Huoxue Recipe group; QH: Qinggan Huoxue Recipe group.

Figure 5

Qinggan Huoxue Recipe suppresses epithelial-to-mesenchymal transition by inhibiting the Smad signaling pathway. A: TGF-β1, Smad3 and Snail mRNA expression evaluated by RT-PCR; B: TGF-β1, Smad3 and Snail protein expression evaluated by Western blot. ^aP < 0.05 and ^bP < 0.01 vs Normal healthy control group; ^cP < 0.05 and ^dP < 0.01 vs Model ALF group. N: Normal healthy control group; M: Model ALF group; Q: Qinggan Recipe group; H: Huoxue Recipe group; QH: Qinggan Huoxue Recipe group.

Figure 6

Potential mechanism underlying Qinggan Huoxue Recipe-mediated inhibition of epithelial-to-mesenchymal transition in alcoholic liver fibrosis rats. TGF-β1 stimulates responsive cells through binding and activating the transmembrane receptors TGF-β type I (TGF-βR-I) and type II (TGF-βR-II). Receptor ternary complexes phosphorylate and activate Smad2/3. Once activated, Smad2/3 forms heterocomplexes with Smad4, and these translocate to the nucleus and activate TGF-β1 signaling. Snail, a gene associated with the TGF-β/Smad signaling pathway, inhibits E-cadherin expression, increases vimentin and fibronectin levels, promotes EMT and decreases the levels of occludin, ZO-1 and claudin. QGR, HXR and QGHXR suppressed the effects of ALF-induced modulation of the TGF-β/Smad signaling pathway and ameliorated EMT-induced alcoholic fibrosis. QGR, HXR and QGHXR affected molecules associated with the TGF-β/Smad signaling pathway in the same manner. However, QGR and HXR exerted no significant changes in vimentin mRNA expression, QGR exerted no significant changes in fibronectin mRNA expression, and HXR exerted no significant changes in Snail and TGF-β1 mRNA expression.