羽扇豆醇通过SIRT3/mTOR通路调节自噬以改善骨关节炎软骨细胞衰老  

Lupeol Alleviates Chondrocytes Senescence in Osteoarthritis by Regulating Autophagy via the Sirtuin 3/Mechanistic Target of Rapamycin Kinase Pathway

作　　者：马运锋[1] 曹玉净[2] 韩小飞[3] MA Yunfeng;CAO Yujing;HAN Xiaofei(Department of Osteopathics,The Second Affiliated Hospital of Henan University of Traditional Chinese Medicine/Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China;Department of Traumatic Orthopedics,The Second Affiliated Hospital of Henan University of Traditional Chinese Medicine/Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China;Department of Rheumatology,The Second Affiliated Hospital of Henan University of Traditional Chinese Medicine/Henan Province Hospital of Traditional Chinese Medicine,Zhengzhou 450002,China)

机构地区：[1]河南中医药大学第二附属医院/河南省中医院骨病一科,郑州450002 [2]河南中医药大学第二附属医院/河南省中医院骨伤科,郑州450002 [3]河南中医药大学第二附属医院/河南省中医院风湿病科,郑州450002

出　　处：《四川大学学报(医学版)》2025年第1期83-93,共11页Journal of Sichuan University(Medical Sciences)

基　　金：2020年度河南省中医药科学研究专项课题(No.20-21ZY3013);2019年河南省中医药科学研究专项课题(No.2019ZY2149)资助。

摘　　要：目的探讨羽扇豆醇(Lupeol)通过去乙酰化酶3(sirtuin 3,SIRT3)/雷帕霉素靶蛋白(mechanistic target of rapamycin kinase,mTOR)通路调控自噬在骨关节炎(osteoarthritis,OA)软骨细胞衰老中的作用机制。方法分离原代小鼠膝关节软骨细胞,分为对照组,Lupeol(2.5、5、10、20、40μmol/L)组,50μmol/L叔丁基过氧化氢(tert-butyl hydroperoxide,TBHP)组,TBHP+Lupeol组,TBHP+Lupeol+氯喹(chloroquine,CQ;自噬抑制剂,20μmol/L)组,TBHP+Lupeol+si-NC组,TBHP+Lupeol+si-SIRT3组。利用CCK-8、DCFH-DA探针、流式细胞术检测细胞增殖、活性氧(reactive oxygen species,ROS)水平、细胞凋亡;利用β-gal染色评估细胞衰老;利用Western blot检测SIRT3、mTOR、衰老标志蛋白(p21和p16)、细胞外基质(extracellular matrix,ECM)降解相关蛋白(aggrecan、collagenⅡ、ADAMTS5、MMP13)以及自噬相关蛋白(LC3BⅠ、LC3BⅡ、P62)的表达;利用RT-qPCR检测衰老相关分泌表型(senescence-associated secretory phenotype,SASP;包括IL-6、Cxcl10、MCP1、MMP3)的mRNA水平;利用免疫荧光检测LC3斑点;利用透射电镜观察自噬小体。将30只C57BL/6雄性野生型小鼠分为(n=10):Sham组、OA组、OA+Lupeol〔50 mg/(kg·d),灌胃给药〕组,利用番红O-固绿染色评估软骨损伤程度。结果根据细胞活力测定结果,Lupeol的最佳处理浓度和时间选择为20μmol/L和24 h。与TBHP组相比,TBHP+Lupeol组细胞活力升高(P<0.05);ROS产生、β-gal阳性细胞比例、p21和p16蛋白表达水平以及SASP mRNA水平降低(P<0.05);aggrecan和collagenⅡ蛋白水平升高、ADAMTS5和MMP13蛋白水平降低(P<0.05);细胞凋亡减少(P<0.05);P62蛋白水平降低、LC3BⅡ/LC3BⅠ的比值升高、LC3B荧光斑点强度以及自噬小体数量增加(P<0.05);SIRT3表达水平升高、mTOR磷酸化水平降低(P<0.05)。CQ处理有效废除Lupeol对细胞活力和自噬的促进作用,对ROS水平、细胞衰老、ECM降解、细胞凋亡的抑制作用(P<0.05)。沉默SIRT3逆转Lupeol对mTOR磷酸化水平的抑制�Objective To investigate the role of lupeol in mitigating chondrocyte senescence in osteoarthritis(OA)by regulating autophagy through the sirtuin 3(SIRT3)/mechanistic target of rapamycin kinase(mTOR)pathway.Methods Knee articular chondrocytes from primary-generation mice were isolated and divided into different groups,including a control group,a lupeol group(given 2.5,5,10,20,and 40μmol/L lupeol),a tert-butyl hydrogen peroxide(TBHP)group(receiving 50μmol/L TBHP),TBHP+lupeol group,TBHP+lupeol+chloroquine(CQ)group(receiving 20μmol/L CQ,an autophagy inhibitor),TBHP+lupeol+si-NC group,and TBHP+lupeol+si-SIRT3 group.Cell proliferation,reactive oxygen species(ROS)levels,and apoptosis were determined by CCK-8,DCFH-DA probe,and flow cytometry.Cell senescence was evaluated byβ-gal staining.Western blot was used to determine the expressions of SIRT3,mTOR,senescence marker proteins(p21 and p16),extracellular matrix(ECM)degradation-related proteins(aggrecan,collagenⅡ,ADAMTS5,and MMP13),and autophagy-related proteins(LC3BⅠ,LC3BⅡ,and P62).RT-qPCR was used to determine the mRNA levels of senescence-associated secretory phenotypes(SASP),including IL-6,Cxcl10,MCP1,and MMP3.The expression of LC3 was detected by immunofluorescence.Autophagosomes were observed by transmission electron microscopy.A total of 30 male wild-type C57BL/6 mice were divided into different groups(n=10),including a Sham group,an OA group,and an OA+lupeol group receiving 50 mg/(kg·d)lupeol via gastric gavage.Cartilage damage was evaluated by safranin O-fast green staining.Results Based on the results of cell viability assay,20μmol/L lupeol treatment for 24 h was identified as the optimal intervention concentration and duration.Compared with that in the TBHP group,cell viability was elevated in the TBHP+lupeol group(P<0.05);ROS production,the proportion ofβ-galpositive cells,the protein expression levels of p21 and p16,and the mRNA levels of SASP were decreased(P<0.05);the protein levels of aggrecan and collagenⅡwere elevated and the protein leve

关 键 词：羽扇豆醇 SIRT3/mTOR通路 自噬 衰老 软骨细胞 

分 类 号：R73[医药卫生—肿瘤]