高迁移率族蛋白1通过PINK1/Parkin介导的线粒体自噬促进骨髓干细胞的趋化及成骨分化  

Effect of HMGB1 on Chemotaxis and Osteogenic Differentiation of Bone Marrow Stem Cells through PINK1/Parkin⁃mediated Mitophagy

作　　者：陈施羊 周爱国[2] 闫文龙[2] 张健[2] CHEN Shiyang;ZHOU Aiguo;YAN Wenlong;ZHANG Jian(Department of Orthopaedics,Chongqing General Hospital,Chongqing 401147,China;Department of Orthopaedics,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400042,China)

机构地区：[1]重庆市人民医院骨科,重庆401147 [2]重庆医科大学附属第一医院骨科,重庆400042

出　　处：《骨科》2023年第5期445-452,共8页ORTHOPAEDICS

基　　金：重庆市卫生计生委医学科研项目(2017ZDXM006);重庆市科学技术局技术创新与应用发展专项项目(cstc2019jscx-msxmX0245)。

摘　　要：目的探讨高迁移率族蛋白1(HMGB1)通过PTEN诱导假定激酶1(PINK1)/帕金蛋白(Parkin)介导的线粒体自噬对骨髓干细胞的趋化及成骨分化的影响。方法将人骨髓间充质干细胞(hBMSCs)分为7组:control组、siRNA-NC组、siRNA-HMGB1组、pcDNA-NC组、pcDNA-HMGB1组、pcDNAHMGB1+siRNA-NC组、pcDNA-HMGB1+siRNA-PINK1组。采用Transwell检测细胞迁移能力;茜素红染色检测各组细胞中钙结节数目;ELISA检测骨桥蛋白(OPN)、碱性磷酸酶(ALP)的含量;RT-qPCR检测各组细胞中成骨细胞特异性转录因子(Osterix)、HMGB1及Runt相关转录因子2(RUNX2)、转录因子CCAAT/增强子结合蛋白(C/EBPα)、过氧化物酶体增殖物激活受体γ(PPARγ)水平;透射电镜检测线粒体自噬小体数目;Western Blot检测hBMSCs中Parkin及微管相关蛋白1A/1B-轻链3(LC3Ⅱ/Ⅰ)、选择性自噬接头蛋白62(P62)和自噬关键分子酵母Atg6同系物(Beclin1)等线粒体自噬相关蛋白的表达。结果HMGB1通过PINK1/Parkin介导的线粒体自噬对hBMSCs的趋化作用研究表明:与pcDNA-NC组相比,pcDNAHMGB1组HMGB1表达、细胞迁移率、线粒体自噬数目及自噬相关蛋白Beclin-1、LC3B-Ⅱ/Ⅰ的表达增加,Parkin、P62等蛋白的表达降低(P<0.05);与siRNA-NC组相比,siRNA-HMGB1组HMGB1表达、细胞迁移率、线粒体自噬数目及Beclin-1、LC3B-Ⅱ/Ⅰ等表达降低,Parkin、P62表达增高(P<0.01)。HMGB1通过PINK1/Parkin介导的线粒体自噬对hBMSCs的成骨分化作用研究结果显示:与pcDNA-NC组相比,pcDNAHMGB1组钙结节数量、Osterix及RUNX2含量、ALP及OPN的表达升高(P<0.01),PPARγ、C/EBPα的表达降低(P<0.05);与pcDNA-HMGB1+siRNA-NC组相比,pcDNA HMGB1+siRNA-PINK1组钙结节数量、Osterix及RUNX2含量、ALP及OPN的表达降低(P<0.05),PPARγ、C/EBPα的表达升高(P<0.05)。结论HMGB1高表达能通过PINK1/Parkin介导的线粒体自噬促进hBMSCs的趋化、成骨细胞分化及抑制成脂细胞分化,可能是骨质疏松症的潜在治疗靶点。Objective To investigate the effect of mitophagy mediated by high mobility group box 1(HMGB1)through PTEN induced putative kinase 1(PINK1)/parkin on chemotaxis and osteogenic differentiation of bone marrow stem cells.Methods Human bone marrow mesenchymal stem cells(hBMSCs)were divided into 7 groups:control group,siRNA⁃NC group,siRNA⁃HMGB1 group,pcDNA⁃NC group,pcDNA⁃HMGB1 group,pcDNA⁃HMGB1+siRNA⁃NC group,and pcDNA⁃HMGB1+siRNA⁃PINK1 group.Cell migration was detected by Transwell assay.The number of calcium nodules in each group was detected by alizarin red staining.The contents of osteopontin(OPN)and alkaline phosphatase(ALP)were detected by ELISA.The levels of osteoblast⁃specific transcription factor(Osterix),HMGB1 and Runt⁃related transcription factor 2(RUNX2),transcription factor CCAAT/enhancer binding protein(C/EBPα),and peroxisome proliferator⁃activated receptorγ(PPARγ)in each group were detected by RT⁃qPCR.The number of mitophagosomes was detected by transmission electron microscopy.The expression of mitophagy⁃related proteins such as Parkin and microtubule⁃associated protein 1A/1B⁃light chain 3(LC3Ⅱ/Ⅰ),selective autophagic adaptor protein 62(P62),and yeast Atg6 homologue(Beclin1)in hBMSCs was detected by Western blotting.Results The chemotactic effect of HMGB1 on hBMSCs mediated by PINK1/Parkin mitophagy showed that HMGB1 expression,cell migration rate,number of mitophagy and expression of autophagy⁃related proteins(Beclin⁃1 and LC3B⁃Ⅱ/Ⅰ)were increased,and the expression of Parkin,P62 and other proteins was decreased in the pcDNA⁃HMGB1 group as compared with the pcDNA⁃NC group(P<0.05).HMGB1 expression,cell migration rate,number of mitophagy and expression of Beclin⁃1 and LC3B⁃Ⅱ/Ⅰwere decreased,Parkin and P62 expression was increased in the siRNA⁃HMGB1 group as compared with the siRNA⁃NC group(P<0.01).The effect of HMGB1 on osteogenic differentiation of hBMSCs through PINK1/Parkin⁃mediated mitophagy results showed that as compared with the pcDNA

关 键 词：人骨髓间充质干细胞 PINK1/Parkin 线粒体自噬 高迁移率族蛋白1 成骨分化 骨质疏松 

分 类 号：R580[医药卫生—内分泌]