摘要
目的探讨ω-3多不饱和脂肪酸(以下简称“ω-3”)对MC3T3-E1细胞抗氧化应激及促进成骨分化的作用机制,并揭示ω-3与MC3T3-E1细胞中核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)/醌氧化还原酶1[NAD(P)H quinone oxidoreductase 1,NQO1]抗氧化应激关键通路之间的关系。方法通过细胞计数试剂盒8法筛选H_(2)O_(2)最佳浓度(用于建立MC3T3-E1细胞体外氧化应激模型)和ω-3最佳干预浓度。将MC3T3-E1细胞分为空白对照组、氧化应激组(H_(2)O_(2))、低剂量ω-3组(H_(2)O_(2)+低剂量ω-3)和高剂量ω-3组(H_(2)O_(2)+高剂量ω-3)。各组细胞经成骨诱导分化7 d或14 d后进行以下检测:采用细胞荧光染色和流式细胞术测定细胞内活性氧自由基(reactive oxygen species,ROS)含量;生物透射电镜观察线粒体形态变化;Western blot检测Nrf2/HQO1通路关键蛋白Nrf2、NQO1、血红素氧合酶1(heme oxygenase 1,HO-1)及线粒体形态相关蛋白线粒体融合蛋白1(Mitofusin 1,Mfn1)、Mfn2表达水平,以评估细胞抗氧化应激能力;细胞免疫荧光染色和Western blot检测成骨蛋白Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨钙素(osteocalcin,OCN)表达水平;采用ALP染色和茜素红染色评估MC3T3-E1细胞成骨能力。结果与氧化应激组相比,低剂量和高剂量ω-3组ROS含量显著降低,Nrf2、NQO1和HO-1蛋白表达量明显升高,差异均有统计学意义(P<0.05);同时,MC3T3-E1细胞的线粒体形态得到改善,线粒体形态相关蛋白Mfn1和Mfn2表达量显著增加,差异有统计学意义(P<0.05);ALP染色和茜素红染色示,低剂量和高剂量ω-3组表现出更强成骨能力,且成骨相关蛋白RUNX2、OCN表达量显著增加,差异有统计学意义(P<0.05)。并且上述结果在两个ω-3处理组表现出剂量依赖性(P<0.05)。结论ω-3能够增强氧化应激条件下MC3T3-E1细胞的抗氧化能力并上调其成骨活性,其机制可能与Nrf2/NQO1信号通路有关。
Objective To explore the mechanism by whichω-3 polyunsaturated fatty acids(hereinafter referred to as“ω-3”)exert antioxidant stress protection and promote osteogenic differentiation in MC3T3-E1 cells,and to reveal the relationship betweenω-3 and the key antioxidant stress pathway involving nuclear factor E2-related factor 2(Nrf2)and NAD(P)H quinone oxidoreductase 1(NQO1)in MC3T3-E1 cells.Methods The optimal concentration of H_(2)O_(2)(used to establish the oxidative stress model of MC3T3-E1 cells in vitro)and the optimal intervention concentrations ofω-3 were screened by cell counting kit 8.MC3T3-E1 cells were divided into blank control group,oxidative stress group(H_(2)O_(2)),low-doseω-3 group(H_(2)O_(2)+low-doseω-3),and high-doseω-3 group(H_(2)O_(2)+high-doseω-3).After osteoblastic differentiation for 7 or 14 days,the intracellular reactive oxygen species(ROS)level was measured by fluorescence staining and flow cytometry,and the mitochondrial morphological changes were observed by biological transmission electron microscope;the expression levels of Nrf2,NQO1,heme oxygenase 1(HO-1),Mitofusin 1(Mfn1),and Mfn2 were detected by Western blot to evaluate the cells’antioxidant stress capacity;the expression levels of Runt-related transcription factor 2(RUNX2)and osteocalcin(OCN)were detected by immunofluorescence staining and Western blot;osteogenic potential of MC3T3-E1 cells was evaluated by alkaline phosphatase(ALP)staining and alizarin red staining.Results Compared with the oxidative stress group,the content of ROS in the low and high doseω-3 groups significantly decreased,and the protein expressions of Nrf2,NQO1,and HO-1 significantly increased(P<0.05).At the same time,the mitochondrial morphology of MC3T3-E1 cells improved,and the expressions of mitochondrial morphology-related proteins Mfn1 and Mfn2 significantly increased(P<0.05).ALP staining and alizarin red staining showed that the low-dose and high-doseω-3 groups showed stronger osteogenic ability,and the expressions of osteogenesis-related proteins RUNX2 and OCN significantly increased(P<0.05).And the above results showed a dose-dependence in the twoω-3 treatment groups(P<0.05).Conclusionω-3 can enhance the antioxidant capacity of MC3T3-E1 cells under oxidative stress conditions and upregulate their osteogenic activity,possibly through the Nrf2/NQO1 signaling pathway.
作者
黄家辉
陈龙
徐陈
于浩杰
周士帅
官建中
HUANG Jiahui;CHEN Long;XU Chen;YU Haojie;ZHOU Shishuai;GUAN Jianzhong(Department of Orthopaedics,the First Affiliated Hospital of Bengbu Medical College,Bengbu Anhui,233000,P.R.China;Key Laboratory of Anhui Province for Tissue Transplantation,Bengbu Anhui,233000,P.R.China)
出处
《中国修复重建外科杂志》
北大核心
2025年第11期1459-1467,共9页
Chinese Journal of Reparative and Reconstructive Surgery
基金
安徽省自然科学基金项目(2408085MH235)
安徽省优秀科研创新团队(2024AH010020)
安徽省厅级重点实验室开放课题重点项目(AHTT2024A002)
安徽省卫生健康委青年项目(AHWJ2024Aa30092)。
