二甲双胍激活核因子E2及相关通路保护脂多糖诱导的软骨细胞损伤  

Protective effect of metformin against lipopolysaccharide-induced chondrocyte injury by activating nuclear factor E2 and its relevant signaling pathway

作　　者：王旭东 韩俊柱[1] 王文锐 夏启鑫 郭成 Wang Xudong;Han Junzhu;Wang Wenrui;Xia Qixin;Guo Cheng(Anhui Key Laboratory of Tissue Transplantation,Department of Orthopedics,Second Affiliated Hospital of Bengbu Medical University,Bengbu 233000,Anhui Province,China;Anhui Provincial Key Laboratory of Translational Cancer Research,School of Life Science,Bengbu Medical University,Bengbu 233030,Anhui Province China)

机构地区：[1]蚌埠医学院第二附属医院骨科,组织移植安徽省重点实验室,安徽省蚌埠市233000 [2]蚌埠医学院生命科学院,癌症转化医学安徽省重点实验室,安徽省蚌埠市233000

出　　处：《中国组织工程研究》2023年第26期4147-4153,共7页Chinese Journal of Tissue Engineering Research

基　　金：安徽省蚌埠医学院自然科学重点项目(2020byzd186),项目负责人:韩俊柱。

摘　　要：背景:既往研究表明,核因子E2相关性因子2/血红素加氧酶1信号通路在骨关节炎发生发展中至关重要,二甲双胍对软骨细胞具有一定的保护作用。目的:探讨二甲双胍对脂多糖诱导的软骨细胞损伤的保护作用及机制研究。方法:取SD大鼠软骨细胞进行体外分离培养和鉴定。选取第3代软骨细胞,分别用不同质量浓度的脂多糖(0,10,50,100,500,1000和5000μg/L)和二甲双胍(0,50,100,500,1000,5000和10000μmol/L)处理细胞24 h,采用CCK8法检测细胞活性,筛选最适脂多糖和二甲双胍质量浓度。按照处理因素不同将软骨细胞分为空白组、脂多糖诱导组(5000μg/L)和脂多糖(5000μg/L)+二甲双胍组(1000μmol/L)。流式细胞术检测各组软骨细胞凋亡情况;通过活性氧、丙二醛和超氧化物歧化酶试剂盒检测细胞氧化应激;RT-PCR和Western Blot检测软骨细胞白细胞介素1β、环氧化酶2、Ⅱ型胶原、核因子E2相关性因子2和血红素加氧酶1的mRNA和蛋白表达。结果与结论:①脂多糖降低了软骨细胞活力,诱导软骨细胞凋亡,而二甲双胍干预后细胞活力上升,凋亡率下降;②脂多糖诱导软骨细胞活性氧和丙二醛表达增加,超氧化物歧化酶活性下降,而二甲双胍干预后活性氧和丙二醛表达降低,超氧化物歧化酶活性增加;③脂多糖诱导软骨细胞白细胞介素1β、环氧化酶2表达增加,Ⅱ型胶原、核因子E2相关性因子2和血红素加氧酶1表达降低,而二甲双胍干预后白细胞介素1β和环氧化酶2表达降低,Ⅱ型胶原、核因子E2相关性因子2和血红素加氧酶1表达增加;④结果说明,二甲双胍能够抑制脂多糖诱导的软骨细胞损伤,发挥对软骨细胞的保护作用,其作用可能通过激活核因子E2相关性因子2/血红素加氧酶1信号通路实现的。BACKGROUND:Previous studies have shown that the nuclear factor E2-related factor 2/heme oxygenase 1 signaling pathway is crucial for the development of osteoarthritis,and metformin has a certain protective effect on chondrocytes.OBJECTIVE:To investigate the protective effect and mechanism of metformin on chondrocyte injury induced by lipopolysaccharide.METHODS:Sprague-Dawley rat chondrocytes were isolated,cultured,and identified in vitro.Passage 3 chondrocytes were selected and treated with different concentrations of lipopolysaccharide(0,10,50,100,500,1000,and 5000μg/L)and metformin(0,50,100,500,1000,5000,and 10000μmol/L)for 24 hours.Cell viability was then detected by cell counting kit-8 method,and the optimal mass concentrations of lipopolysaccharide and metformin were screened.According to different treatment factors,the chondrocytes were divided into blank group,lipopolysaccharide(5000μg/L)induced group,and 5000μg/L lipopolysaccharide+1000μmol/L group.Cell apoptosis was detected by flow cytometry.Oxidative stress was detected by reactive oxygen species,malondialdehyde,and superoxide dismutase kits.The mRNA and protein expressions of interleukin-1β,cyclooxygenase 2,type Ⅱ collagen,nuclear factor E2-related factor 2,and heme oxygenase were detected by RT-PCR and western blot,respectively.RESULTS AND CONCLUSION:Lipopolysaccharide reduced the activity of chondrocytes and induced apoptosis of chondrocytes,while metformin increased the activity of chondrocytes and decreased the apoptosis rate.The levels of reactive oxygen species and malondialdehyde were increased and the activity of superoxide dismutase was decreased in lipopolysaccharide-induced chondrocytes,while treatment with metformin decreased the levels of reactive oxygen species and malondialdehyde and increased the activity of superoxide dismutase in lipopolysaccharide-induced chondrocytes.Lipopolysaccharide increased the expressions of interleukin-1βand cyclooxygenase-2 in chondrocytes,and decreased the expressions of type Ⅱ collagen,nuclear

关 键 词：二甲双胍 关节炎 氧化损伤 Nrf2/HO-1通路 

分 类 号：R496[医药卫生—康复医学] R318[医药卫生—临床医学] R684.3