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目的研究六味五灵片(Liuweiwuling Tables,LWWL)对刀豆蛋白A(concanavalin A,Con A)诱导的小鼠急性免疫性肝损伤的保护作用及机制。方法将小鼠随机分成正常组对照组、模型组、双环醇组(Bicyclol,39 mg·kg~(-1))、六味五灵片低剂量组(8 g·kg~(-1))、六味五灵片高剂量组(16 g·kg~(-1)),各给药组每天给药1次,连续7 d,末次给药1 h后,除正常对照组外,其他各组尾静脉注射Con A(15 mg·kg~(-1))制备小鼠急性免疫性肝损伤模型。HE染色观察小鼠肝组织病理变化;比色法检测小鼠血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBIL);实时定量RT-q PCR测定法检测肝组织中白介素-12(IL-12)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、白介素-4(IL-4)、白介素-10(IL-10)mRNA的表达;流式细胞术(FCM)观察脾脏Th1(IFN-γ)/Th2(IL-4)细胞的变化;免疫印迹(Western blot)法检测肝组织中Th1/Th2转录因子T-bet/GATA-3的表达。结果与正常对照组相比,模型组小鼠血清中ALT、AST、TBIL明显升高,小鼠肝组织病理损伤严重,肝细胞大量坏死、凋亡,表明造模成功。与模型组比较,六味五灵片低、高剂量组小鼠血清中ALT、AST、TBIL的水平明显降低;脾脏中Th1细胞减少,Th2细胞增多。肝组织中IL-12、IFN-γ、TNF-α的mRNA表达下降,IL-4、IL-10的mRNA表达上升,GATA-3蛋白表达上调,T-bet蛋白表达无明显变化。结论六味五灵片通过调节Th1/Th2的平衡,保护Con A诱导的急性免疫性肝损伤。
Objective To study the protective effect and mechanism of Liuweiwuling Tables (LWWL) on acute immunity-induced liver injury induced by concanavalin A (Con A) in mice. Methods The mice were randomly divided into normal control group, model group, Bicyclol group (39 mg · kg -1), low dose of Liuwei Wuling tablets group (8 g · kg -1) Liuwei Wuling tablets high-dose group (16 g · kg -1), each administration group administered once daily for 7 days, the last administration of 1 h, except for the normal control group, the other groups tail Con A (15 mg · kg -1) was injected intravenously to establish acute immunological liver injury model in mice. HE staining was used to observe the pathological changes of liver tissue in mice. The levels of ALT, AST and TBIL were detected by colorimetric assay. The real-time quantitative RT-q PCR (IL-12), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL- 10) mRNA was detected by flow cytometry. The changes of Th1 (IFN-γ) / Th2 (IL-4) cells in spleen were observed by flow cytometry (FCM). The expressions of Th1 / Th2 transcription factor T- bet / GATA-3 expression. Results Compared with the normal control group, the levels of ALT, AST and TBIL in the model group were significantly increased, and the pathological damage of the liver tissue in the model group was severe. A large number of necrosis and apoptosis of the hepatocytes were observed, indicating successful modeling. Compared with the model group, the levels of ALT, AST and TBIL in the serum of low and high dose Liuwei Wuling tablets were significantly decreased; the number of Th1 cells and Th2 cells in the spleen were decreased. The mRNA expression of IL-12, IFN-γ and TNF-α in liver tissue decreased, the mRNA expressions of IL-4 and IL-10 increased, the expression of GATA-3 protein increased, and the expression of T-bet protein did not change significantly. Conclusion Liuwei Wuling tablet can protect Con A-induced acute immune liver injury by regulating the balance of Th1 / Th2.