穿心莲内酯通过激活NRF2/HO-1信号通路减轻膝骨关节炎大鼠软骨损伤的研究

Andrographolide reduces cartilage damage in rats with knee osteoarthritis by activating the NRF2/HO-1 signaling pathway

关键词:  穿心莲内酯  膝骨关节炎  软骨损伤  NRF2/HO-1信号通路  氧化应激  炎症  凋亡

Key words:  Andrographolide  knee osteoarthritis  cartilage injury  NRF2/HO-1 signaling pathway  oxidative stress  inflammation  apoptosis

摘要:

[目的] 研究穿心莲内酯对膝骨关节炎（KOA）大鼠软骨损伤及核因子E2相关因子2/血红素加氧酶-1（NRF2/HO-1）信号通路的影响。[方法] 采用膝关节腔内注射（2次，间隔3 d）木瓜蛋白酶和L-半胱氨酸混合液的方法制备KOA大鼠模型，设正常组、模型组、氨基葡萄糖组（130 mg/kg）、穿心莲内酯低剂量组（30 mg/kg）、穿心莲内酯高剂量组（60 mg/kg）和穿心莲内酯高剂量+NRF2抑制剂鸦胆子苦醇组（60 mg/kg+2 mg/kg），每组8只。每天1次给药治疗4周后，通过Lequesne MG评分评估大鼠行为学表现；Von Frey实验、热板实验测定机械痛阈值（PWMT）和热痛阈值（PWTL）；酶联免疫吸附法（ELISA）检测膝关节液中抗氧化酶[总超氧化物歧化酶（T-SOD）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化物酶（CAT）]活性和丙二醛（MDA）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、单核细胞趋化蛋白1（MCP-1）含量；苏木精-伊红（HE）、番红O-固绿染色法考察软骨组织病理学改变，进行Mankin’s评分评估软骨退变程度；原位末端标记法（TUNEL）观察细胞凋亡；蛋白免疫印迹法（Western blot）检测软骨组织中NRF2/HO-1信号通路相关蛋白表达。[结果] 与正常组相比，模型组大鼠Lequesne MG评分升高，PWMT和PWTL降低（P＜0.05）；膝关节液中T-SOD、GSH-Px、CAT活性降低，MDA、TNF-α、IL-1β、MCP-1含量升高（P＜0.05）；软骨组织出现厚度变薄、分裂、表层缺损、细胞数量减少且排列失序、炎性细胞浸润等病理学改变，软骨细胞凋亡数量明显增多，Mankin’s评分和细胞凋亡指数（AI）升高（P＜0.05）；软骨组织中NRF2、HO-1、B淋巴细胞瘤-2基因（Bcl-2）蛋白表达水平降低，核因子-κB p65（NF-κB p65）、Bcl-2相关X蛋白（Bax）、活化型半胱天冬氨酸蛋白酶-3（Cleaved Caspase-3）表达水平及Bax/Bcl-2升高（P＜0.05）。与模型组相比，氨基葡萄糖组和穿心莲内酯低、高剂量组Lequesne MG评分降低，PWMT和PWTL升高（P＜0.05）；膝关节液中T-SOD、GSH-Px、CAT活性升高，MDA、TNF-α、IL-1β、MCP-1含量降低（P＜0.05）；软骨组织病理学改变和细胞凋亡情况不同程度改善，Mankin’s评分和AI降低（P＜0.05）；软骨组织中NRF2、HO-1、Bcl-2蛋白表达水平升高，NF-κB p65、Bax、Cleaved Caspase-3表达水平及Bax/Bcl-2降低（P＜0.05）。穿心莲内酯高剂量组对各项指标的调节作用优于穿心莲内酯低剂量组和氨基葡萄糖组，穿心莲内酯高剂量+鸦胆子苦醇组对各指标的调节作用弱于穿心莲内酯高剂量组（P＜0.05）。[结论] 穿心莲内酯可能通过激活NRF2/HO-1信号通路减轻氧化应激、炎症反应和细胞凋亡，对KOA大鼠软骨损伤起到保护作用。

Abstract:

[Objective] To investigate the effect of Andrographolide on cartilage injury and nuclear factor E2 related factor 2/heme oxygenase-1（NRF2/HO-1） signaling pathway in rats with knee osteoarthritis（KOA）. [Methods] The KOA rat models were established by injecting a mixture of papain and L-cysteine into the knee joint cavity twice with a 3 days interval. The rats were randomly assigned to the following groups：normal，model，glucosamine（130 mg/kg），Andrographolide low dose group（30 mg/kg），Andrographolide high dose group（60 mg/kg） and Andrographolide high dose+NRF2 inhibitor Brusatol group（60 mg/kg + 2 mg/kg），with 8 rats in each group. After 4 weeks of treatment once a day，the behavioral performance of rats was evaluated by Lequesne MG score. The paw withdrawal mechanical threshold（PWMT） and paw withdraw thermal latency（PWTL） were determined by Von Frey experiment or hot plate experiment. The activity of antioxidant enzyme [total superoxide dismutase（T-SOD），glutathione peroxidase（GSH-Px），catalase（CAT）] and the content of malondialdehyde（MDA），tumor necrosis factor-α（TNF-α），interleukin-1β（IL-1β），monocyte chemoattractant protein-1（MCP-1） in knee joint fluid were detected by enzyme-linked immunosorbent assay（ELISA）. The pathological changes of cartilage tissue were examined by hematoxylin-eosin（HE） staining and safranin O-green staining，the Mankin’s score was used to evaluate the degree of cartilage degeneration；cell apoptosis was investigated by TUNEL staining. The expression of NRF2/HO-1 signaling pathway related proteins in cartilage tissue were detected by Western blot. [Results] Compared with the normal group，the Lequesne MG score of the rats in model group was increased，while the PWMT and PWTL were decreased（P＜0.05）. The activities of T-SOD，GSH-Px，and CAT in knee joint fluid were decreased，while the level of MDA，TNF-α，IL-1β，MCP-1 were increased（P＜0.05）. The cartilage tissue showed pathological changes such as thinning，division，surface defects，cell number decreased and disordered，inflammatory cell infiltration；the number of apoptotic chondrocytes was significantly increased；the Mankin’s score and apoptosis index（AI） were increased（P＜0.05）. The expression of NRF2，HO-1，B-cell lymphoma-2（Bcl-2） proteins were decreased，the expression of nuclear factor-κB p65（NF-κB p65），Bcl-2 related X protein（Bax），Cleaved Caspase-3 proteins and the Bax/Bcl-2 were increased（P＜0.05）. Compared with the model group，the Lequesne MG score of the rats in Glucosamine group and Andrographolide low，high dose groups was decreased，the PWMT and PWTL were increased（P＜0.05）. The activity of T-SOD，GSH-Px，CAT were increased，and the level of MDA，TNF-α，IL-1β，MCP-1 were decreased（P＜0.05）. The pathological changes of cartilage tissue and the apoptosis of chondrocytes were improved to varying degrees，the Mankin's score and AI were decreased（P＜0.05）. The expression of NRF2，HO-1，Bcl-2 proteins were increased，the expression of NF-κB p65，Bax，Cleaved Caspase-3 proteins and the Bax/Bcl-2 were decreased（P＜0.05）. The regulating effect of Andrographolide high dose group on various indexes were better than those of Andrographolide low dose group and Glucosamine group，the regulating effect of Andrographolide high dose+Brusatol group on various indexes were weaker than those of Andrographolide high dose group（P＜0.05）. [Conclusion] Andrographolide may alleviate oxidative stress，inflammatory response and cell apoptosis by activating the NRF2/HO-1 signaling pathway，and thereby protect against cartilage damage in rats with KOA.