姜黄素对H_(2)O_(2)诱导软骨细胞自噬的调控  

Regulatory role of curcumin in hydrogen peroxide-induced autophagy in chondrocytes

作　　者：曹舒兴[1] 宋永周[1] 李明[1] 张洪亮 赵立辉 马维[1] Cao Shuxing;Song Yongzhou;Li Ming;Zhang Hongliang;Zhao Lihui;Ma Wei(Department of Orthopedics,Second Hospital of Hebei Medical University,Shijiazhuang 050000,Hebei Province,China;Department of Radiology,Shenze County Hospital,Shenze 052560,Hebei Province,China)

机构地区：[1]河北医科大学第二医院骨科,河北省石家庄市050000 [2]河北省深泽县医院放射科,河北省深泽县052560

出　　处：《中国组织工程研究》2022年第14期2161-2166,共6页Chinese Journal of Tissue Engineering Research

基　　金：河北省卫生厅科研基金(20190068),项目负责人:曹舒兴;河北省财政厅老年病课题(361004),项目负责人:李明。

摘　　要：背景:姜黄素对骨关节炎具有良好的治疗作用,但具体机制还不清楚;研究表明自噬的激活可降低骨关节炎的严重程度。目的:探讨姜黄素对H_(2)O_(2)诱导软骨细胞自噬的调控作用。方法:体外培养C57小鼠关节软骨细胞,分为对照组、模型组以及姜黄素不同剂量组(10,20,40,80μmol/L)。模型组给予H_(2)O_(2)(1 mmol/L)处理,模拟骨关节炎体内氧化应激状态;姜黄素组中不同浓度姜黄素与软骨细胞培养48 h后,加入H_(2)O_(2),24 h后收集细胞进行检测。CCK-8法检测各组细胞活力;酶联免疫吸附法检测炎症因子白细胞介素6和肿瘤坏死因子α水平;TUNEL染色检测凋亡状况;流式细胞法检测各组细胞凋亡率;Western-blot法检测凋亡相关蛋白(活化半胱天冬酶3、Bax/Bcl-2)、细胞自噬相关蛋白(Beclin-1、LC3-Ⅱ/Ⅰ蛋白)及自噬相关信号通路蛋白(蛋白激酶B、哺乳动物雷帕霉素靶蛋白)的表达情况;实时荧光定量PCR检测软骨基质降解相关基因(基质金属蛋白酶13、血小板结合蛋白基序的解聚蛋白样金属蛋白酶5、Ⅱ型胶原蛋白α1、软骨蛋白聚糖)的表达水平。结果与结论:①与模型组相比,姜黄素干预后软骨细胞的活力明显升高(P<0.05);炎症因子表达明显下降(P<0.05),凋亡率明显下降(P<0.05),凋亡相关标志物活化半胱天冬酶3以及Bax/Bcl-2比值显著下降(P<0.05),自噬相关标志物LC3-Ⅱ/Ⅰ和Beclin-1表达水平显著升高(P<0.05),蛋白激酶B、哺乳动物雷帕霉素靶蛋白的表达水平显著降低(P<0.05);②实时荧光定量PCR显示,基质金属蛋白酶13和血小板结合蛋白基序的解聚蛋白样金属蛋白酶5 mRNA表达降低,Ⅱ型胶原蛋白α1和软骨蛋白聚糖mRNA表达升高(P<0.05);③提示姜黄素通过促进自噬降低H_(2)O_(2)诱导的软骨细胞凋亡,这种作用可能与蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路有关。BACKGROUND:Curcumin has a good therapeutic effect on osteoarthritis,but the specific mechanism is still unclear.Activation of autophagy can reduce the severity of osteoarthritis OBJECTIVE:To investigate the regulatory effect of curcumin on hydrogen peroxide(H_(2)O_(2))-induced autophagy in chondrocytes.METHODS:C57 mouse articular chondrocytes were cultured in vitro and randomly divided into control group,model group(treated with H_(2)O_(2))and curcumin low-,medium-and high-dose groups(10,20,40,80μmol/L).Model group was treated with H_(2)O_(2)(1 mmol/L)to simulate oxidative stress in osteoarthritis.In the different curcumin groups,chondrocytes were treated with different concentrations of curcumin for 48 hours,followed by addition of H_(2)O_(2).Twenty-four hours later,the cells were collected for detection.Cell counting kit-8 assay was used to detect cell viability in each group.Interleukin-6 and tumor necrosis factor-αlevels were detected by ELISA.TUNEL staining was used to detect cell apoptosis.The apoptosis rate of each group was detected by flow cytometry.The expression levels of cleaved caspase-3,Bax/Bcl2,Beclin1,LC3-Ⅱ/Ⅰ,protein kinase B(Akt)and mammalian target of rapamycin(mTOR)were detected by western blot.The expression of cartilage matrix degradation related genes,including matrix metalloproteinase 13,ADAM metallopeptidase with thrombospondin type 1 motif 5,type Ⅱ collagenα1,and Aggrecan,were detected by qRT-PCR.RESULTS AND CONCLUSION:Compared with the model group,the activity of chondrocytes was significantly increased after curcumin intervention(P<0.05).The expression of inflammatory cytokines was significantly decreased(P<0.05).Apoptosis was significantly decreased(P<0.05).Apoptosis-related markers,cleaved caspase-3 and Bax/Bcl2 ratio,were significantly decreased(P<0.05).The expression of autophagy-related markers,LC3-Ⅰ/Ⅱ and Beclin1,was significantly increased(P<0.05).The expression levels of Akt and mTOR were significantly decreased(P<0.05).qRT-PCR showed that the mRNA expressions of ma

关 键 词：姜黄素 骨关节炎 软骨细胞 自噬 H_(2)O_(2) 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]