基于Nrf2-Keap1信号通路探讨麦粒灸对环磷酰胺致肝损伤小鼠的抗氧化作用机制

Antioxidative effect of wheat-grain moxibustion on cyclophosphamide-induced liver injury in mice based on Nrf2-Keap1 signaling pathway

目的:观察麦粒灸对环磷酰胺(CTX)引起的小鼠肝损伤的保护作用,并基于核因子E2相关因子2(Nrf2)-Kelch样ECH相关蛋白1(Keap1)信号通路探讨其作用机制。方法:将24只雄性CD-1(ICR)小鼠随机分为空白组、模型组、麦粒灸组,每组8只。模型组、麦粒灸组小鼠采用腹腔注射CTX(80 mg/kg)制备肝损伤模型。麦粒灸组予麦粒灸“关元”及双侧“足三里”“三阴交”,每穴3壮,每壮时间约30 s,每日1次,连续7 d。干预后,观察小鼠一般情况;测量小鼠肝脏质量并计算肝脏指数;HE染色法观察小鼠肝脏形态,并评定肝脏组织病理评分;ELISA法检测小鼠血清天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、谷氨酸脱氢酶(GLDH)及肝脏丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)含量;Western blot法和实时荧光定量PCR法检测小鼠肝脏Nrf2、Keap1、醌氧化还原酶1(NQO1)蛋白及mRNA表达。结果:与空白组比较,模型组小鼠反应迟钝、步态不稳、体质量下降;肝脏指数升高(P<0.01);肝细胞排列较为疏松,少部分细胞肿胀、气球样变,肝脏组织病理评分升高(P<0.05);血清AST、ALT、GLDH及肝脏MDA含量均增加(P<0.05),肝脏SOD、GSH-Px含量降低(P<0.05);肝脏Nrf2、NQO1蛋白及mRNA表达均下降(P<0.01),Keap1蛋白及mRNA表达升高(P<0.01)。与模型组比较,麦粒灸组小鼠一般情况好转;肝脏指数降低(P<0.01);肝细胞结构相对完整清晰,排列较为整齐,肝脏组织病理评分降低(P<0.05);血清AST、ALT、GLDH及肝脏MDA含量均减少(P<0.05),肝脏SOD、GSH-Px含量增加(P<0.05,P<0.01);肝脏Nrf2、NQO1蛋白及mRNA表达均升高(P<0.05),Keap1蛋白及mRNA表达降低(P<0.05)。结论:麦粒灸可能通过激活Nrf2-Keap1信号通路,增强机体的抗氧化酶系统表达,减轻CTX引起的肝损伤。

Objective To observe the protective effect of wheat-grain moxibustion on cyclophosphamide(CTX)-induced liver injury in mice,and explore its mechanism based on the nuclear factor E2-related factor 2(Nrf2)-Kelch-like ECH-associated protein 1(Keap1)signaling pathway.Methods Twenty-four male CD-1(ICR)mice were randomly divided into a blank group,a model group,and a moxibustion group,with 8 mice in each group.The mice in the model group and the moxibustion group were intraperitoneally injected with CTX(80 mg/kg)to induce liver injury.The mice in the moxibustion group were treated with wheat-grain moxibustion at"Guanyuan"(CV 4)and bilateral"Zusanli"(ST 36)and"Sanyinjiao"(SP 6),with each acupoint being treated by 3 cones,approximately 30 seconds per cone,once daily for 7 days.After intervention,the general condition of the mice was observed;the liver mass was measured and the liver index was calculated;HE staining was used to observe the morphology of the liver,and the liver tissue pathological score was assessed;ELISA was used to detect the serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),glutamate dehydrogenase(GLDH)and the levels of malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)in the liver;Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of Nrf2,Keap1,and quinione acceptor oxidoreductase 1(NQO1)in the liver.Results Compared with the blank group,the mice in the model group showed sluggishness,unsteady gait,and decreased body weight;liver index was increased(P<0.01);liver cells were loosely arranged,with a small number of cell swollen and exhibiting balloon-like changes;liver tissue pathological score was increased(P<0.05);the serum levels of AST,ALT,GLDH,and level of MDA in the liver were increased(P<0.05),and the levels of SOD and GSH-Px in the liver were decreased(P<0.05);protein and mRNA expression of Nrf2 and NQO1 in the liver was decreased(P<0.01),protein and mRNA expression of Keap1 in the liver was increased(P<0.01).Compared with the model group,the mice in the moxibustion group showed improvement in general condition;liver index was decreased(P<0.01);liver cell structure was relatively intact and clear,and liver tissue pathological score was decreased(P<0.05);the serum levels of AST,ALT,GLDH,and level of MDA in the liver were decreased(P<0.05),and the levels of SOD and GSH-Px in the liver were increased(P<0.05,P<0.01);protein and mRNA expression of Nrf2 and NQO1 in the liver was increased(P<0.05),protein and mRNA expression of Keap1 in the liver was decreased(P<0.05).Conclusion The wheat-grain moxibustion may alleviate CTX-induced liver injury by activating the Nrf2-Keap1 signaling pathway and enhancing the expression of antioxidative enzyme system in the body.