健脾补肾方加味调节调节性T淋巴细胞/辅助性T淋巴细胞平衡的抗肝纤维化作用机制

Efficacy of Jiawei Jianpi Bushen prescription in treatment of liver fibrosis and its role in regulating Treg/Th17 balance

目的观察健脾补肾方加味对大鼠肝纤维化的治疗效果及对调节性T淋巴细胞(Treg)和辅助性T淋巴细胞(Th)17的影响。方法 32只雄性Wistar大鼠随机分为正常组、模型组、健脾补肾方组和健脾补肾方加味组,四氯化碳溶液皮下注射造模。用药组在第4周开始分别给予健脾补肾方和健脾补肾方加味,第8周末处死。测定大鼠血清肝功能,观察肝组织炎症及纤维化情况、Foxp3和白细胞介素(IL)17蛋白与基因表达及血清IL-4和IL-17表达情况。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验,等级资料比较采用Ridit检验。结果与正常组相比,模型组大鼠血清总胆汁酸(TBA)、ALP、TBil、ALT和AST均明显升高(P值均<0.05),肝脏胶原沉积明显、α平滑肌肌动蛋白(αSMA)表达增多(P值均<0.05)。与模型组相比,健脾补肾组血清ALP、TBA、TBil和ALT均明显降低(P值均<0.05);健脾补肾方加味组血清ALP、TBA、TBil、ALT和AST均明显降低(P值均<0.05);各用药组肝脏胶原沉积和αSMA表达明显减少(P值均<0.05)。与健脾补肾方组相比,健脾补肾方加味组TBA、ALT和AST均明显降低(P值均<0.05)。与正常组相比,模型组肝组织IL-17蛋白及基因表达显著增高且IL-17和Foxp3基因表达比值显著增高(P值均<0.05),血清IL-17水平明显增高且IL-4和IL-17水平比值明显降低(P值均<0.05)。与模型组相比,用药组肝组织IL-17蛋白及基因表达明显降低且IL-17和Foxp3基因表达比值明显降低(P值均<0.05),血清IL-17水平明显降低且IL-4和IL-17水平比值明显增高(P值均<0.05)。结论健脾补肾方加味可以提高抗肝纤维化的治疗效果,其作用机制可能与调节Treg/Th17失衡有关。

Objective To investigate the therapeutic effect of Jiawei Jianpi Bushen prescription in liver fibrosis in rats and its effect on regulatory T（ Treg） cells and T helper 17（ Th17） cells. Methods A total of 32 male Wistar rats were randomly divided into normal group,model group,Jianpi Bushen prescription group,and Jiawei Jianpi Bushen prescription group. The model was established by subcutaneous injection of CCl4. The administration groups were given Jianpi Bushen decoction and Jiawei Jianpi Bushen decoction since week 4 and sacrificed at the end of week 8. The serum markers of liver function were measured,the status of liver inflammation and fibrosis was observed,and the protein and mRNA expression of Fox3 and interleukin- 17（ IL- 17） and serum levels of interleukin- 4（ IL- 4） and IL- 17 were also observed. An analysis of variance was used for comparison of continueus data between multiple groups,the LSD t- test was used for comparison of countinuous data between two groups; the Ridit test was used for comparison of ranked data. Results Compared with the normal group,the model group showed significant increases in total bile acid（ TBA）,alkaline phosphatase（ ALP）,total bilirubin（ TBil）,alanine aminotransferase（ ALT）,and aspartate aminotransferase（ AST）（ all P〈0. 05）,as well as significant collagen deposition in the liver and significantly increased expression of α- smooth muscle actin（ α- SMA）（ both P〈0. 05）. Compared with the model group,the Jianpi Bushen decoction group showed significant reductions in serum ALP,TBA,TBil,and ALT（ all P〈0. 05）; the Jiawei Jianpi Bushen decoction group showed significant reductions in serum ALP,TBA,TBil,ALT,and AST（ all P〈0. 05）; the administration groups showed significant reductions in collagen deposition in the liver and expression of α- SMA（ all P〈0. 05）. Compared with the Jianpi Bushen decoction group,the Jiawei Jianpi Bushen decoction group had significantly reduced TBA,ALT,and AST（ all P〈0. 05）. Compared with the normal group,the model group showed significant increases in the protein and mRNA expression of IL- 17 in the liver tissue and a significant increase in the mRNA expression ratio of IL- 17 to Foxp3（ all P〈0. 05）,a significant increase in serum IL- 17 level,and significant reductions in serum levels of IL- 4 and IL- 17（ all P〈0. 05）. Compared with the model group,the administration groups showed significant reductions in the protein and mRNA expression of IL- 17 in the liver tissue and a significant reduction in the mRNA expression ratio of IL- 17 to Foxp3（ all P〈0. 05）,a significant reduction in serum IL- 17 level,and significant increases in serum levels of IL- 4 and IL- 17（ all P〈0. 05）. Conclusion Jiawei Jianpi Bushen prescription has improved efficacy in the treatment of liver fibrosis,and its mechanism of action may be associated with the regulation of Treg / Th17 imbalance.