摘要
目的探讨羟基酪醇(HT)对氧化应激诱导的小鼠软骨细胞损伤的改善作用及其机制。方法将HT 0~400μmol·L^(-1)与小鼠软骨细胞共同孵育24 h,CCK-8法检测细胞存活率。用H_(2)O_(2)200μmol·L^(-1)制备氧化应激软骨细胞模型,设细胞对照组、H_(2)O_(2)组及H_(2)O_(2)+HT 10、50和250μmol·L^(-1)组,共培养24 h后用实时荧光定量PCR检测小鼠软骨细胞中白细胞介素6(IL-6)、环氧合酶2(COX-2)、前列腺素E_(2)(PGE_(2))、诱导型一氧化氮合酶(iNOS)、基质金属蛋白酶3(MMP-3)、MMP-13、含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶4(ADAMTS-4)、ADAMTS-5、S RY-box转录因子9(SOX-9)和聚集蛋白聚糖(ACAN)mRNA表达水平,2,7-二氯荧光素二乙酸酯(DCFH-DA)染色检测小鼠软骨细胞内活性氧(ROS)水平,JC-1染色检测线粒体膜电位;或共培养48 h后用Western印迹法检测小鼠软骨细胞中iNOS、COX-2、MMP-13和Ⅱ型胶原(Col-2)蛋白表达水平。结果HT浓度≤350μmol·L^(-1)时对小鼠软骨细胞存活率无明显影响。与细胞对照组相比,处理24 h后,H_(2)O_(2)组小鼠软骨细胞中IL-6、COX-2、PGE_(2)、iNOS、MMP-3、MMP-13、ADAMTS-4和ADAMTS-5 mRNA水平显著升高,SOX-9和ACAN mRNA水平显著降低,ROS水平显著升高,线粒体膜电位显著降低。与H_(2)O_(2)组相比,H_(2)O_(2)+HT 10、50和250μmol·L^(-1)组IL-6、COX-2、PGE_(2)、iNOS、MMP-3、MMP-13、ADAMTS-4和ADAMTS-5 mRNA水平显著降低,SOX-9和ACAN mRNA水平显著升高,ROS水平显著降低,H_(2)O_(2)+HT 50和250μmol·L^(-1)组线粒体膜电位显著升高。处理48 h后,与细胞对照组相比,H_(2)O_(2)组小鼠软骨细胞中iNOS、COX-2和MMP-13蛋白表达升高,Col-2蛋白表达降低;与H_(2)O_(2)组相比,H_(2)O_(2)+HT 10、50和250μmol·L^(-1)组小鼠软骨细胞中iNOS、COX-2和MMP-13蛋白表达降低,H_(2)O_(2)+HT 50和250μmol·L^(-1)组Col-2蛋白表达升高。结论HT可改善H_(2)O_(2)诱导的软骨细胞损伤,其机制可能与降低软骨细胞内ROS水平、缓解线粒体膜电位降低、抑制炎症细胞因子表达并抑制促分解代谢、促进促合成代谢有关。
OBJECTIVE To investigate the effect and underlying mechanism of hydroxytyrosol(HT)on mouse chondrocyte injury induced by oxidative stress.METHODS Mouse chondrocytes were incubated with varying concentrations of HT 0-400μmol·L^(-1)for 24 h,and the viability of the mouse chondrocytes was assessed using CCK-8 kit.An oxidative stress model of chondrocytes was estab-lished by the addition of H_(2)O_(2)200μmol·L^(-1).The experimental groups included the cell control group,H_(2)O_(2)group,and H_(2)O_(2)+HT 10,50 and 250μmol·L^(-1)groups.After 24 h,the mRNA expression levels of interleukin-6(IL-6),cyclooxygenase-2(COX-2),prostaglandin E_(2)(PGE_(2)),inducible nitric oxide synthase(iNOS),matrix metalloproteinase-3(MMP-3),MMP-13,a disintegrin and metalloproteinase with throm-bospondin motifs-4(ADAMTS-4),ADAMTS-5,SRY-box transcription factor-9(SOX-9)and aggrecan(ACAN)in mouse chondrocytes were detected by real-time quantitative PCR,the intracellular reactive oxygen species(ROS)level in chondrocytes was measured with 2,7-dichlorodihydrofluorescein diace-tate(DCFH-DA)staining,while the mitochondrial membrane potential was evaluated using JC-1 staining.After 48 h,the protein expression levels of iNOS,COX-2,MMP-13,and typeⅡcollagen(Col-2)in mouse chondrocytes were detected using Western blotting.RESULTS HT at concentrations≤350μmol·L^(-1)had no significant effect on the survival of mouse chondrocytes.Compared with the cell control group,after 24 h,the mRNA expression levels of IL-6,COX-2,PGE_(2),iNOS,MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5 in the chondrocytes of mice in the H_(2)O_(2)group were increased,while the mRNA expression levels of SOX-9 and ACAN were decreased.Additionally,there was an elevation in the ROS level and a significant loss of mitochondrial membrane potential in the chondrocytes of mice.Compared with the H_(2)O_(2)group,after treatment with HT 10,50 and 250μmol·L^(-1),there were significant decreases in mRNA expression levels of IL-6,COX-2,PGE_(2),iNOS,MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5,the mRNA expressions of SOX-9 and ACAN were increased,the ROS level was lowered.After treatment with HT 50 and 250μmol·L^(-1),the loss of mitochondrial membrane potential was ameliorated.Compared to the cell control group,the protein expressions of iNOS,COX-2 and MMP-13 were upregulated in the H_(2)O_(2)group,while the protein expression of Col-2 was downregulated after 48 h.Compared to the H_(2)O_(2)group,treatment with HT at concentrations of 10,50 and 250μmol·L^(-1)resulted in decreased protein expressions of iNOS,COX-2 and MMP-13 in mouse chondrocytes,but the protein expression of Col-2 increased following treatment with HT 50 and 250μmol·L^(-1).CONCLUSION HT can ameliorate H_(2)O_(2)-induced chondrocyte injury by reducing intracellular ROS levels and alleviating the loss of mitochondrial membrane potential,suppressing the release of inflammatory cytokines,inhibiting catabolic processes,and promoting anabolic activities.
作者
于海超
王文超
段君昭
王华
张雪松
YU Haichao;WANG Wenchao;DUAN Junzhao;WANG Hua;ZHANG Xuesong(School of Medicine,Nankai University,Tianjin 300071,China;Department of Orthopaedics,the Fourth Medical Center of PLA General Hospital,Beijing 100048,China;Academy of Military Medical Sciences,Beijing 100850,China)
出处
《中国药理学与毒理学杂志》
北大核心
2025年第3期183-190,共8页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(82372466)。
关键词
羟基酪醇
骨关节炎
软骨细胞
氧化应激
炎症因子
hydroxytyrosol
osteoarthritis
chondrocytes
oxidative stress
inflammatory factor
