摘要
目的探讨橘络黄酮急性缺氧肠平滑肌细胞的保护作用及其线粒体机制。方法人肠道平滑肌细胞(human intestinal smooth muscle cells,HISMCs)分为对照组、缺氧1组(1%O2,5%CO2,94%N2)。缺氧2组(3%O2,5%CO2,92%N2)和缺氧3组(5%O2,5%CO2,90%N2)。在研究不同缺氧时间对细胞影响过程中,HISMCs分为对照组、3 h缺氧组(1%O2,3 h)、6 h缺氧组(1%O2,6 h)和9 h缺氧组(1%O2,9 h);给予缺氧HISMCs(1%O2预处理)不同浓度的橘络黄酮(300、600μg/mL)干预6 h;在研究TR3功能实验中,HISMCs分为正常对照组、缺氧组(1%O2)、缺氧细胞橘络酮干预组(1%O2,600μg/mL橘络酮)、缺氧细胞橘络酮干预TR3高表达组(1%O2,600μg/mL橘络酮,TR3高表达)。流式细胞仪检测肠平滑肌细胞线粒体膜电位和凋亡率;分别采用Ac-DEVD-pNA和Ac-IETD-pNA显色法检测caspase-3和caspase-8酶活性;Western-blot技术检测缺氧细胞中TR3的总含量和线粒体中TR3含量。结果急性缺氧导致肠平滑肌细胞线粒体膜电位显著下降、caspase-3和caspase-8酶活性均显著升高、细胞凋亡率显著升高(P<0.05)。橘络黄酮干预后可显著提高缺氧肠平滑肌细胞线粒体膜电位水平并有效抑制线粒体凋亡通路的活化(P<0.05)。橘络黄酮抑制细胞内TR3向线粒体移位,TR3过表达可逆转橘络黄酮对缺氧细胞的保护作用(P<0.05)。结论橘络黄酮可通过调控缺氧细胞内TR3的线粒体移位,继而保护肠平滑肌细胞免受缺氧损伤。
Objective To investigate the protective effect of citrus flavone on cultured human intestinal smooth muscle cells(HISMCs)against acute hypoxia and explore the mitochondrial mechanism.Methods Cultured HISMCs were exposed to acute hypoxia with 1%,3%,or 5%O2(5%CO2,94%N2)for 6 h or with 1%O2 for 3,6,and 9 h.The cells cultured in 1%O2(5%CO2,94%N2)were treated with citrus flavonoids(300 or 600μg/mL)for 6 h.Mitochondrial membrane potential and the cell apoptotic rate were determined with flow cytometry,and the activities of caspase-3 and caspase-8 were detected using Ac-DEVD-pNA and AC-IETD-pNA colorimetry,respectively.The total content of TR3 nuclear orphan receptor(TR3)in the hypoxic cells and its content in the mitochondria were determined with Western blotting.We also tested the effect of TR3 overexpression on the protective effect of 600μg/mL citrus flavonoids in the HISMCs exposed to 1%O2.Results Acute hypoxia resulted in significantly decreased mitochondrial membrane potential,enhanced caspase-3 and caspase-8 activities,and increased apoptosis in cultured HISMCs(P<0.05).Citrus flavonoids effectively enhanced mitochondrial membrane potential level,suppressed the mitochondrial apoptotic pathway(P<0.05),and inhibited the translocation of TR3 to the mitochondria in the hypoxic cells.Overexpression of TR3 significantly attenuated the protective effect of citrus flavonoids on the hypoxic cells(P<0.05).Conclusion Citrus flavonoids protect intestinal smooth muscle cells against hypoxic injury by regulating mitochondrial translocation of TR3.
作者
刘景君
王晓明
沈成凤
邓炳楠
陈德美
蒲玲玲
刘伟丽
陈照立
王新兴
LIU Jingjun;WANG Xiaoming;SHEN Chengfeng;DENG Bingnan;CHEN Demei;PU Lingling;LIU Weili;CHEN Zhaoli;WANG Xinxing(Institute of Environmental and Operational Medicine,Military Medical Research Institute,Academy of Military Sciences,Tianjin,300050;Tianjin Center for Disease Control and Prevention,Tianjin,300011,First Outpatient Department;No.983 Hospital of PLA,Tianjin,300220,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2020年第6期594-600,共7页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(31971106,81372124),BWS(18CXZ044,16J010)。
关键词
橘络黄酮
缺氧
肠平滑肌细胞
线粒体
凋亡
citrus flavonoids
hypoxia
intestinal smooth muscle cells
mitochondria
apoptosis
