摘要
目的探讨白藜芦醇(resveratrol,Res)对H2O2处理的佐剂性关节炎(adjuvant arthritis,AA)大鼠软骨细胞增生的影响及其与自噬的关系。方法采取弗氏完全佐剂注射SD大鼠左侧足趾皮下制作AA动物模型。第15天时,动物处死,取股动脉血,检测AA大鼠氧化应激状态。酶消化法体外培养和鉴定AA大鼠原代软骨细胞,给予低浓度H2O2(5μmol/L)处理原代软骨细胞,模拟体内氧化应激状态。给予不同浓度Res处理,MTT法检测软骨细胞增生活性,激光共聚焦检测软骨细胞中自噬相关基因5(Atg5)的表达,Western blot法检测软骨细胞中p62、Beclin-1自噬相关蛋白的表达。结果组织染色结果显示:AA大鼠踝关节滑膜组织增生,滑膜中炎细胞明显浸润,软骨膜的连续性被破坏,排列紊乱。AA模型中血清丙二醛含量(11.65±1.06)μmol/L增加,谷胱甘肽过氧化物酶含量(140.18±8.65)μmol/L、超氧化物歧化酶活性(133.72±8.36)U/mL均显著下降,差异有统计学意义(P<0.05)。细胞组织化学染色示原代软骨细胞胞质Ⅱ型胶原表达阳性。不同浓度Res(1、3、5、10、20、40、80、160 mg/L)可抑制H2O2处理的软骨细胞增生,抑制率分别为(2.2±0.4)%、(5.3±0.6)%、(12.1±1.3)%、(26.6±2.4)%、(40.3±3.1)%、(52.7±4.2)%、(69.2±4.5)%、(72.5±5.1)%,呈剂量依赖性(P<0.05);半数致死量值为37.2 mg/L。软骨细胞质中Atg5绿色荧光呈剂量依赖性增加;软骨细胞中p62蛋白表达呈剂量依赖性降低,Beclin-1蛋白表达呈剂量依赖性升高,差异有统计学意义(P<0.05)。结论Res可抑制氧化应激状态下AA大鼠软骨细胞增生,其作用机制可能与Res诱导的软骨细胞自噬改变有关。
Objective To investigate the effects of resveratrol(Res)on chondrocytes of rat adjuvant arthritis(AA)treated with H2O2 and the expression of autophagy related protein p62 and beclin-1.Methods In the experimental animal model of AA,Fresno's adjuvant was subcutaneously injected into the left toe of SD rats,and the SD rats in the control group were injected with the same amount of normal saline.Enzyme digestion cultivation and identification of AA rat chondrocytes in vitro,different concentrations Res(final concentration:0,1,3,10,20,40,80,160 mg/L)determined to detect of low concentration of H2O2(≤5μmol/L)cartilage cell proliferation inhibition effects of AA rats by MTT method.Laser confocal was used to detect Res(final concentration 0,10,40 mg/L)deal with low concentration of H2O2(including 5μmol/L)AA rat chondrocytes autophagy related gene 5(Atg5),Western blot was used to detect p62 and beclin-1 autophagy related proteins in AA rat chondrocytes.Results HE staining showed synovial hyperplasia of ankle in adjuvant arthritis rat chondrocytes.The inflammatory cells in synovium were infiltrated,and the continuity of cartilage membrane disorderly arranged even destroyed.Compared with the control group,the serum oxidative stress level of AA model rats was increased,the content of malondialdehyde(11.65±1.06)μmol/L increased,the content of glutathione peroxidase(140.18±8.65)μmol/L and the activity of superoxide dismutase(133.72±8.36)U/mL decreased significantly.Cytochemical staining confirmed the success of chondrocyte culture in AA rats,Res showed a concentration-dependent inhibition of chondrocyte proliferation treated with low H2O2 concentration(5μmol/L).The inhibition rate respectively were(2.2±0.4)%,(5.3±0.6)%,(12.1±1.3)%,(26.6±2.4)%,(40.3±3.1)%,(52.7±4.2)%,(69.2±4.5)%,(72.5±5.1)%,and the half of the lethal dose was 37.2 mg/L,increased green fluorescence of Atg5 in chondrocytoplasm,decreased the protein expression of p62,and increased the protein expression of beclin-1 in chondrocyte by Western blot.Conclusion Res can reduce the oxidative stress in AA rats and inhibit the proliferation of chondrocytes in AA rats,which may be related to the mechanism of Res induced chondrocyte autophagy.
作者
季丹
黄大可
周瑾
潘献柱
祝捷
JI Dan;HUANG Da-ke;ZHOU Jin;PAN Xian-zhu;ZHU Jie(Department of Foundation Courses, Anhui Medical College, Hefei 230601, China;College of Basic Medicine,Anhui Medical University, Hefei 230032, China;Department of Endocrinology, The First Affiliated Hospital of USTC,Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230001, China)
出处
《中华骨质疏松和骨矿盐疾病杂志》
CSCD
北大核心
2020年第3期236-242,共7页
Chinese Journal Of Osteoporosis And Bone Mineral Research
基金
安徽省教育厅2019年度高校优秀青年骨干人才国内访学研修项目(gxgnfx2019102)。
关键词
白藜芦醇
佐剂性关节炎
软骨细胞
氧化应激
自噬
resveratrol
adjuvant arthritis rat
chondrocytes
oxidative stress
autophagy
