姜黄素联合二至丸抑制刀豆蛋白A诱导小鼠免疫性肝炎机制的研究

Study on the Mechanism of Curcumin Combined with Erzhi Pill Inhibiting Concanavalin A Inducing Immune Hepatitis in Mice

目的对姜黄素联合二至丸对刀豆蛋白A诱导的小鼠免疫性肝炎的肝组织中炎性细胞因子(IL-6、IFN-γ)水平变化以及对NF-κB信号通路的影响。方法建立刀豆蛋白A(Con A)诱导小鼠免疫性肝炎模型;小鼠随机分为:正常组、Con A模型对照组、二至丸组、姜黄素组、二至丸+姜黄素组。原位末端凋亡法(TUNEL)观察肝内细胞凋亡情况;酶联免疫吸附法(ELISA)测定肝组织中细胞因子(IL-6、IFN-γ)水平变化;实时荧光定量PCR法(Realtime PCR)检测肝内IL-6、IFN-γ、IL-10 mRNA的表达情况;蛋白印迹法(Western Blot)检测肝内NF-κB信号通路相关蛋白表达变化。结果ELISA结果显示二至丸联合姜黄素组小鼠血清中炎性细胞因子水平(IL-6、INF-γ)较Con A模型组均有显著下降(P<0.05);Real-time PCR结果显示,与Con A模型对照组相比,二至丸联合姜黄素组小鼠肝脏内IL-6和IFN-γ上调以及IL-10下调的现象被明显抑制,差异有统计学意义(P<0.05);Western Blot结果显示,给予Con A后,NF-κB信号通路被激活,二至丸+姜黄素组小鼠明显抑制了NF-κB的磷酸化,从而起到抗炎、抗氧化作用;同时还减少Caspase-3的活化,降低肝细胞凋亡率。结论在Con A诱导的小鼠自身免疫性肝炎模型中,二至丸和姜黄素联用可通过减少血清和肝脏中炎性细胞因子的释放,减少肝细胞坏死,而这种保护肝脏的免疫调节作用是与阻碍NF-κB信号通路的激活相关。

Objective To investigate the changes of inflammatory cytokines(IL-6,IFN-γ)levels in liver tissues of mice with immunological hepatitis induced by curcumin combined with Erzhi Pills and concanavalin A,and the impact to NF-κB signal pathway.Methods A mouse model of immune hepatitis induced by concanavalin A(Con A)was established.The mice were randomly divided into normal group,Con A model control group,Erzhi pill group,curcumin group and Erzhi pill+curcumin group.In situ apoptosis method(TUNEL)was used to observe the apoptosis of liver cells;enzyme-linked immunosorbent assay(ELISA)was used to measure the changes of cytokines(IL-6,IFN-γ)in liver tissue;Real-time PCR method(Real-time PCR).The expression of IL-6,IFN-γand IL-10 mRNA in liver was detected.The expression of NF-κB signaling pathway-related proteins in liver was detected by Western Blot.Results The results of ELISA showed that the levels of inflammatory cytokines(IL-6,INF-γ)in the serum of Erzhi Pill combined with curcumin group were significantly lower than those in Con A model group(P<0.05).RealtimePCR results showed that with Con Compared with the control group of A model,the up-regulation of IL-6 and IFN-γand the down-regulation of IL-10 in the liver of Erzhi Pill combined with curcumin group were significantly inhibited(P<0.05);Western Blot The results showed that NF-κB signaling pathways were simultaneously activated after Con A administration,and the phosphorylation of NF-κB were significantly inhibited in the mice treated by erzhi pill+curcumin group.It has anti-inflammatory and anti-oxidative effects;it also reduces the activation of caspase-3 and reduces the apoptosis rate of hepatocytes.Conclusion In Con A-induced autoimmune hepatitis in mice,the combination of Erzhi Pill and curcumin can reduce hepatocyte necrosis by reducing the release of inflammatory cytokines in serum and liver,and this protects the liver from immune regulation.It is associated with blocking the activation of NF-κB signaling pathways.