丙戊酸钠通过AMPK/SIRT1轴抑制BMSCs铁死亡的机制研究  

Mechanism of sodium valproate in inhibiting ferroptosis of bone marrow mesenchymal stem cells via the adenosine monophosphate-activated protein kinase/Sirtuin 1 axis

作　　者：顾庆松 李剑桥 陈裕虎 汪林辉 李义恒 王子儒 王一聪 杨民 GU Qingsong;LI Jianqiao;CHEN Yuhu;WANG Linhui;LI Yiheng;WANG Ziru;WANG Yicong;YANG Min(Department of Traumatic Orthopedics,the First Affiliated Hospital of Wannan Medical College/Yijishan Hospital,Wuhu Anhui,241001,P.R.China)

机构地区：[1]皖南医学院第一附属医院/弋矶山医院创伤骨科,安徽芜湖241001

出　　处：《中国修复重建外科杂志》2025年第2期215-223,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：安徽省高校自然科学研究重大项目(2023AH040265);安徽省卫生健康委自然科学重大项目(AHWJ2023A10149);皖南医学院弋矶山医院人才引进项目(YR202226);皖南医学院弋矶山医院攀峰项目(PF2019005)。

摘　　要：目的探讨丙戊酸钠(sodium valproate,VPA)在抑制Erastin诱导的BMSCs铁死亡中的效果及其作用机制。方法从8周龄SD大鼠骨髓分离培养BMSCs并鉴定[流式细胞仪检测细胞表面抗原CD90、CD44、CD45表达,茜素红(alizarin red S,ARS)染色和油红O染色分别评估细胞的成骨和成脂分化能力]。取第3代细胞,使用Erastin诱导铁死亡模型,并通过不同浓度VPA进行干预,使用细胞计数试剂盒8法筛选最适药物浓度用于后续实验。实验分为4组,A组细胞在成骨诱导培养基中培养24 h,B组细胞在含最适浓度Erastin的成骨诱导培养基中培养24 h;C组细胞在含最适浓度Erastin和最适浓度VPA的成骨诱导培养基中培养24 h,D组细胞在含最适浓度Erastin、最适浓度VPA和8μmol/L EX527的成骨诱导培养基中培养24 h。评估细胞线粒体状态,包括丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)和活性氧(reactive oxygen species,ROS)的水平;同时评估成骨能力,包括ALP活性和ARS染色;使用Western blot检测成骨相关蛋白[Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨桥蛋白(osteopontin,OPN)]、铁死亡相关蛋白[谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、铁蛋白重链1(ferritin heavy chain 1,FTH1)、溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)],以及通路相关蛋白[腺苷单磷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)、去乙酰化酶1(Sirtuin 1,SIRT1)]的相对表达量。结果经鉴定所培养细胞为BMSCs。VPA能够抑制Erastin诱导的BMSCs铁死亡及成骨能力减弱,并通过激活AMPK/SIRT1通路发挥作用。其中,VPA显著降低了Erastin处理的BMSCs中ROS和MDA的水平,显著提高了GSH水平。同时,铁死亡相关蛋白(GPX4、FTH1和SLC7A11)的表达水平显著下降;VPA还上调了成骨相关蛋白(RUNX2和OPN)的表达,增强了矿化能力和成骨分化能力,并提高了通路相关蛋白(AMPK和SIRT1)的表达�Objective To investigate the effects of sodium valproate(VPA)in inhibiting Erastin-induced ferroptosis in bone marrow mesenchymal stem cells(BMSCs)and its underlying mechanisms.Methods BMSCs were isolated from bone marrow of 8-week-old Spragur Dawley rats and identified[cell surface antigens CD90,CD44,and CD45 were analyzed by flow cytometry,and osteogenic and adipogenic differentiation abilities were assessed by alizarin red S(ARS)and oil red O staining,respectively].Cells of passage 3 were used for the Erastin-induced ferroptosis model,with different concentrations of VPA for intervention.The optimal drug concentration was determined using the cell counting kit 8 assay.The experiment was divided into 4 groups:group A,cells were cultured in osteogenic induction medium for 24 hours;group B,cells were cultured in osteogenic induction medium containing optimal concentration Erastin for 24 hours;group C,cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA for 24 hours;group D,cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA,and 8μmol/L EX527 for 24 hours.The mitochondrial state of the cells was evaluated,including the levels of malondialdehyde(MDA),glutathione(GSH),and reactive oxygen species(ROS).Osteogenic capacity was assessed by alkaline phosphatase(ALP)activity and ARS staining.Western blot analysis was performed to detect the expressions of osteogenic-related proteins[Runt-related transcription factor 2(RUNX2)and osteopontin(OPN)],ferroptosis-related proteins[glutathione peroxidase 4(GPX4),ferritin heavy chain 1(FTH1),and solute carrier family 7 member 11(SLC7A11)],and pathway-related proteins[adenosine monophosphate-activated protein kinase(AMPK)and Sirtuin 1(SIRT1)].Results The cultured cells were identified as BMSCs.VPA inhibited Erastin-induced ferroptosis and the decline of osteogenic ability in BMSCs,acting through the activation of the AMPK/SIRT1 pathway.VPA significantly reduced the levels of ROS and MDA

关 键 词：丙戊酸钠 BMSCS 铁死亡 骨质疏松 腺苷单磷酸活化蛋白激酶 去乙酰化酶1 

分 类 号：R580[医药卫生—内分泌]