灵仙新苷通过维持线粒体稳态保护骨髓间充质干细胞免受缺氧诱导的细胞凋亡  

Clematichinenoside AR protects bone marrow mesenchymal stem cells from hypoxia-induced apoptosis by maintaining mitochondrial homeostasis

作　　者：赵梓彤 涂鹏程 孙孝先 潘娅岚 郭杨[2,3] 王礼宁 马勇[1,2,3] ZHAO Zi-tong;TU Peng-cheng;SUN Xiao-xian;PAN Ya-lan;UO Yang;WANG Li-ning;MA Yong(School of Integrative Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China;Laboratory of New Techniques of Restoration and Reconstruction of Orthopedics and Traumatology,Nanjing University of Chinese Medicine,Nanjing 210023,China;Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;Wuxi Affiliated Hospital of Nanjing University of Chinese Medicine,Wuxi 214071,China;Institute of Integrative Medicine Nursing Research,Nanjing University of Chinese Medicine,Nanjing 210029,China)

机构地区：[1]南京中医药大学中西医结合学院,江苏南京210023 [2]南京中医药大学骨伤修复与重建新技术实验室,江苏南京210023 [3]南京中医药大学附属医院,江苏南京210029 [4]南京中医药大学无锡附属医院,江苏无锡214071 [5]南京中医药大学中西医结合护理研究所,江苏南京210029

出　　处：《中国中药杂志》2025年第5期1331-1339,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金青年科学基金项目(82205141,82405436);江苏省自然科学基金青年项目(BK20241155);江苏省中医院优秀青年博士项目(2024QB030);江苏省研究生科研与实践创新计划项目(KYCX24_2336)。

摘　　要：明确灵仙新苷保护骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)免受缺氧诱导的细胞凋亡的作用及机制。采用骨片法提取骨髓间充质干细胞,流式细胞技术鉴定BMSCs;在正常培养状态(37℃,5%CO_(2))和缺氧状态(37℃,90%N_(2),5%CO_(2))培养BMSCs,同时使用灵仙新苷进行干预,将BMSCs分为空白组(正常状态培养)、灵仙新苷组(正常状态培养+灵仙新苷)、缺氧24 h组(缺氧状态培养24 h)、灵仙新苷缺氧24 h组(缺氧状态培养24 h+灵仙新苷)、缺氧48 h组(缺氧状态培养48 h)、灵仙新苷缺氧48 h组(缺氧状态培养48 h+灵仙新苷)、缺氧72 h组(缺氧状态培养72 h)、灵仙新苷缺氧72 h组(缺氧状态培养72 h+灵仙新苷)8组,cell counting kit-8(CCK-8)检测各组细胞的增殖活性,TUNEL法检测细胞凋亡率,MitoTracker■Red CM-H2XRo线粒体染色检测线粒体总量,DCFH-DA荧光探针染色检测细胞内活性氧水平,JC-1染色检测细胞线粒体膜电位,Western blot检测细胞内B淋巴细胞瘤-2相关X蛋白(B-cell lymphoma-2 associated X protein,BAX)、半胱天冬蛋白酶-3(caspase-3)和视神经萎缩蛋白1(optic atrophy 1,OPA1)蛋白的表达变化。结果显示,灵仙新苷可以显著提高细胞的增殖活性;与空白组相比,缺氧组的BMSCs凋亡率显著增加,线粒体数量显著减少,ROS水平显著升高,线粒体膜电位显著下降,BAX和caspase-3蛋白表达显著升高,OPA1蛋白表达显著下降;与相同缺氧时间下的缺氧组相比,灵仙新苷干预后细胞凋亡率显著下降,线粒体数量显著增多,ROS水平显著下降,线粒体膜电位显著升高,BAX和caspase-3蛋白表达显著下降,OPA1蛋白表达显著升高。由此得出,灵仙新苷可能通过调控OPA1来维持BMSCs线粒体稳态,从而发挥对缺氧环境下的BMSCs的抗凋亡作用。This study aims to elucidate the role and mechanism of clematichinenoside AR(CAR)in protecting bone marrow mesenchymal stem cells(BMSCs)from hypoxia-induced apoptosis.BMSCs were isolated by the bone fragment method and identified by flow cytometry.Cells were cultured under normal conditions(37℃,5%CO_(2))and hypoxic conditions(37℃,90%N_(2),5%CO_(2))and treated with CAR.The BMSCs were classified into eight groups:control(normal conditions),CAR(normal conditions+CAR),hypoxia 24 h,hypoxia 24 h+CAR,hypoxia 48 h,hypoxia 48 h+CAR,hypoxia 72 h,and hypoxia 72 h+CAR.The cell counting kit-8(CCK-8)assay and terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)were employed to measure cell proliferation and apoptosis,respectively.The number of mitochondria and mitochondrial membrane potential were measured by MitoTracker■Red CM-H2XRo staining and JC-1 staining,respectively.The level of reactive oxygen species(ROS)was measured with the DCFH-DA fluorescence probe.The protein levels of B-cell lymphoma-2 associated X protein(BAX),caspase-3,and optic atrophy 1(OPA1)were determined by Western blot.The results demonstrated that CAR significantly increased cell proliferation.Compared with the control group,the hypoxia groups showed increased apoptosis rates,reduced mitochondria,elevated ROS levels,decreased mitochondrial membrane potential,upregulated expression of BAX and caspase-3,and downregulated expression of OPA1.In comparison to the corresponding hypoxia groups,CAR intervention significantly decreased the apoptosis rate,increased mitochondria,reduced ROS levels,elevated mitochondrial membrane potential,downregulated the expression of BAX and caspase-3,and upregulated the expression of OPA1.Therefore,it can be concluded that CAR may exert an anti-apoptotic effect on BMSCs under hypoxic conditions by regulating OPA1 to maintain mitochondrial homeostasis.

关 键 词：灵仙新苷 骨髓间充质干细胞 细胞凋亡 线粒体稳态 OPA1 

分 类 号：R285[医药卫生—中药学]