基于TLR2/MyD88/NF-κB和NALP3信号通路探讨藏族药短穗兔耳草提取物抗慢性酒精性肝损伤大鼠的作用机制

目的:通过酒精灌胃8周建立大鼠慢性酒精性肝损伤模型,研究藏族药短穗兔耳草提取物对Toll样受体(TLR2/髓样分化因子88(MyD88)/核转录因子-κB(NF-κB)和NOD样受体蛋白3(NALP3)信号通路的影响,探讨其提取物抗慢性酒精性肝损伤的作用机制。方法:60只SD雄性大鼠随机分成正常组、模型组、联苯双酯组(0.1 g·kg^-1)及短穗兔耳草低、中、高剂量组(0.5,1,2 g·kg^-1),每日上午各给药组按10 m L·kg^-1给予相应的药物,下午梯度乙醇灌胃法给予56度白酒灌胃,连续8周后,取血,测定各组大鼠血清中天门冬氨酸氨基转移酶(AST),丙氨酸氨基转移酶(ALT),甘油三酯(TC),总胆固醇(TG)及肝脏谷胱甘肽(GSH)水平;酶联免疫吸附测定(ELISA)检测大鼠血清中白细胞介素-1β(IL-1β)水平;蛋白免疫印迹法(Western blot)检测肝脏中TLR2,MyD88,NF-κB和NALP3蛋白的表达;苏木素-伊红(HE)染色观察肝脏病理学形态改变。结果:与正常组比较,模型组血清中AST,ALT,TC,TG,IL-1β水平均明显升高(P<0.05,P<0.01);与模型组比较,藏族药短穗兔耳草各剂量组能降低血清AST,ALT,TC,TG,IL-1β水平,其中高剂量组的效果尤为显著(P<0.05,P<0.01)。与正常组比较,模型组肝匀浆中GSH水平下降;模型组肝组织中TLR2,MyD88,NF-κB和NALP3蛋白水平明显升高(P<0.05,P<0.01);与模型组比较,藏族药短穗兔耳草各剂量组能降低肝脏中GSH水平以及TLR2,MyD88,NF-κB和NALP3的蛋白表达(P<0.05,P<0.01);肝脏病理切片表明,藏族药短穗兔耳草能改善大鼠肝组织病理变化。结论:藏族药短穗兔耳草对酒精诱导的慢性酒精性肝损伤大鼠具有一定的保护作用,其机制可能与TLR/MyD88/NF-κB和NALP3信号通路有关。

Mechanism of Anti-chronic Alcoholic Liver Injury in Rats of Tibetan Medicine Lagotis brachystachys Extracts by TLR2/MyD88/NF-κB and NALP3 Signaling Pathway

Objective: To established the model of chronic alcoholic liver injury in rats by long-term(8 weeks) alcoholic gavage,to study the effects of Tibetan medicine Lagotis brachystachys extracts on Toll-like receptor(TLR) 2/myeloid differentiation factor 88(MyD88)/nuclear factor kappa B(NF-κB) and NOD like receptor protein 3(NALP3) signaling pathways and study preliminary the mechanism of action of chronic alcoholic liver injury.Method: Sixty male Sprague-Dawley rats were randomly divided into normal group,model group,bifendate positive drug group(0.1 g·kg^-1) and L.brachystachys low,medium and high-dose groups(0.5,1,2 g·kg^-1),the corresponding drugs were given at 10 m L·kg^-1 in each morning,and the 56 degree Liquor was administered by the afternoon gradient alcoholic gavage method.After 8 weeks,the levels of serum aspartate transaminase(AST),serum alanineaminotransfease(ALT),serum total cholesterol(TC),triglyceride(TG),interleukin-1β(IL-1β),and the liver levels of L-glutathione(GSH) were measured.The expression of TLR2,MyD88,NF-κB and NALP3 protein in liver were detected by Western blot.Hematoxylin-eosin(HE) staining was used to observe the pathological changes of liver tissue.Result: Compared with normal group,the serum levels of AST,ALT,TC,TG and IL-1β in model group were significantly increased(P<0.05,P<0.01).Compared with model group,the serum AST,ALT,TC,TG and IL-1β levels were decreased in the various doses of L.brachystachys,and the high dose group was particularly effective(P<0.05,P<0.01).Compared with normal group,the GSH level in the liver homogenate of model group decreased significantly,and the difference was not statistically significant.The levels of TLR2,My D88,NF-κB and NALP3 in the liver tissue of model group were significantly increased(P<0.05,P<0.01).The GSH levels in the liver and the protein expression of TLR2,MyD88,NF-κB and NALP3 were decreased in L.brachystachys group(P<0.05, P<0.01).The liver pathological section showed that L.brachystachys can improve the pathological changes of rat liver tissue.Conclusion: L.brachystachys can protect liver from alcohol-induced chronic liver injury in rats.The mechanism was related to TLR2/MyD88/NF-κB and NALP3 signaling pathway.