ABSTRACT
Objective To investigate the therapeutic effect of Taohong Siwu decoction on a mouse model of carbon tetrachloride (CCl 4 )-induced liver fibrosis and its mechanism of action. Methods A total of 24 male C57BL/6 mice were randomly divided into normal group, model group, and Taohong Siwu decoction group, with 8 mice in each group. The mice in the model group and the Taohong Siwu decoction group were given intraperitoneal injection of 10% CCl 4 , and Taohong Siwu decoction was given by gavage since week 3 for 4 consecutive weeks. Liver function [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] was measured, and liver pathomorphology was observed. Real-time PCR was used to measure the mRNA expression of α-smooth muscle actin (α-SMA), hyaluronic acid synthase-2 (HAS-2), and collagen type Ⅰ(Col1), and Western blotting was used to measure the protein expression of α-SMA, Col1, and HAS-2. Primary hepatic stellate cells (HSCs) were isolated, and HAS-2 was silenced by siRNA to observe its influence on HSC activation. The t -test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the SNK or least significant difference t -test was used for further comparison between two groups. Results Compared with the normal group, the model group had significant increases in serum liver function parameters (ALT, AST) and the Taohong Siwu decoction group had significant reductions in the serum levels of ALT and AST (all P < 0.01). Pathological staining showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers, and the Taohong Siwu decoction group had loose fibrous septa and alleviated inflammatory cell infiltration. Compared with the model group, the Taohong Siwu decoction group had significant reductions in the mRNA and protein expression of α-SMA and Col1(all P < 0.001). Compared with the normal group, the model group had a significant increase in the mRNA expression level of HAS-2 in liver tissue ( t =6.14, P < 0.05), and compared with the model group, the Taohong Siwu decoction group had a significant reduction in the protein expression level of HAS-2 (0.29±0.10 vs 1.00±0.12, t =70.73, P < 0.001). After HAS-2 was silenced by siRNA, the Si HAS-2+transforming growth factor β (TGFβ) group (treated with TGFβ) had significant reductions in the mRNA expression levels of α-SMA and Col-Ⅰ compared with the NC+TGFβ group ( P < 0.01). Conclusion Taohong Siwu decoction exerts a marked therapeutic effect on CCl 4 -induced liver fibrosis in mice by inhibiting HAS-2.
ABSTRACT
ObjectiveTo investigate whether Xiayuxue decoction exerts an anti-liver fibrosis effect by inhibiting glial cell line-derived neurotrophic factor (GDNF). MethodsA total of 24 C57BL/6 mice were randomly divided into control group, model group, and Xiayuxue decoction group. The mice in the model group and the Xiayuxue decoction group were given intraperitoneal injection of 10% CCl4, and those in the Xiayuxue decoction group were given 0.4678 g/kg Xiayuxue decoction by gavage since week 4. The liver function parameters alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and liver histopathology was observed. Immunohistochemistry was used to measure the protein expression of alpha-smooth muscle actin (α-SMA) and GDNF. GFP-Col-HSC and human primary hepatic stellate cells (HSCs) were treated with GDNF (10 ng/ml), and HSC activation was measured. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the control group, the model group had significant increases in the levels of ALT and AST, and compared with the model group, the Xiayuxue decoction group had significant reductions in the levels of ALT and AST (all P＜0.01). Liver histopathology showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers, and the Xiayuxue decoction group had loose fibrous septa and alleviated inflammatory cell infiltration. Immunohistochemistry showed that compared with the control group, the model group had significant increases in the expression of α-SMA and GDNF (both P＜0.01), which were observed in fibrous septa, and compared with the model group, the Xiayuxue decoction group had significant reductions in the expression of α-SMA and GDNF (both P＜0.05). Western blotting showed that the control group had relatively low expression of GDNF in liver tissue, the formation of liver fibrosis at week 6 of CCl4 modeling, and an around 10-fold increase in the expression of GDNF, and the Xiayuxue decoction group had significantly inhibited protein expression of GDNF (P＜0.01); there were significant increases in the expression of α-SMA and collagen type I α1 (Col1) in mice with liver fibrosis, with significant reductions in α-SMA and Col1 after treatment with Xiayuxue decoction (all P＜0.01). The in vitro experiment showed that GDNF induced the significant increases in the protein expression of α-SMA and Col1 in HSCs, which was significantly inhibited by Xiayuxue decoction (all P＜0.01). ConclusionThe expression of GDNF is significantly upregulated in the formation of liver fibrosis. GDNF can induce HSC activation, and Xiayuxue decoction can exert an anti-liver fibrosis effect by inhibiting GDNF.