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.
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。.
Keywords: Nrf2-Keap1 signaling pathway; cyclophosphamide (CTX); liver injury; oxidative stress; wheat-grain moxibustion.