Protective Effect of Ganshuang Granules(肝爽颗粒)on Liver Cirrhosis by Suppressing Regulatory T Cells in Mouse Model

Objective: To investigate the potential antifibrotic mechanisms of Chinese medicine Ganshuang Granules(肝爽颗粒, GSG) and to provide clinical therapeutic evidence of its effects. Methods: A cirrhotic mouse model was established by intraperitoneally injecting a mixture of CCl_4(40%) and oil(60%) at 0.2 mL per 100 g of body weight twice a week for 12 weeks. After 12-week modeling, GSG was intragastric administrated to the mice for 2 weeks, and the mice were divided into low-, medium-and high-dose groups at doses of 1, 2 and 4 g/(kg·day), respectively. Liver morphology changes were observed using Masson's trichrome staining and B-ultrasound. The levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST) and hyaluronic acid(HA) in serum were detected using an automatic biochemistry analyzer. The expressions of desmin, smooth muscle actin(SMA) and Foxp3 in liver were detected by immunofluorescence. The regulatory T cell(Treg) frequency was determined through flow cytometry analysis. Collagen-Ⅰ, SMA, IL-6, tumor necrosis factor α(TNF-α), interleukin(IL)-1β and transforming growth factor β1(TGF-β1) expression levels were measured using quantitative polymerase chain reaction(qPCR). Results: Masson's staining result showed fewer pseudolobule structures and fibrous connective tissue in the GSG-treatment groups than in the spontaneous recovery group. Ultrasonography showed that GSG treatment reduced the number of punctate hyperechoic lesions in mice cirrhotic livers. The serum ALT, AST, HA levels were significantly ameliorated by GSG treatment(ALT: F=8.104, P=0.000; AST: F=7.078, P=0.002; and HA: F=7.621, P=0.001). The expression levels of collagen-Ⅰ and SMA in the cirrhotic livers were also attenuated by GSG treatment(collagen-Ⅰ: F=3.938, P=0.011; SMA: F=4.115, P=0.009). Tregs, which were elevated in the fibrotic livers, were suppressed by GSG treatment(F=8.268, P=0.001). The expressions of IL-6, TNF-α and IL-1β increased, and TGF-β levels decreased in the cirrhotic livers after GSG treatment(IL-6: F=5.457, P=0.004; TNF-α: F=6.023, P=0.002; IL-1β: F=6.658, P=0.001; and TGF-β1: F=11.239, P=0.000). Conclusions: GSG promoted the resolution/regression of cirrhosis and restored liver functions in part by suppressing Treg cell differentiation, which may be mediated by hepatic stellate cells.