摘要
目的:探讨人参-三七对缺氧/复氧HL-1心肌细胞线粒体功能的影响。方法:根据大鼠等效剂量,每日取2.50 g人参粉与1.25 g三七粉用44 mL温开水冲调后给22只大鼠各灌胃2 mL,为给药组;再给10只大鼠灌胃2 mL生理盐水,为正常组。根据课题组前期提取大鼠含药血清经验以及预实验结果,两组皆灌胃持续7 d,断粮1 d但不断水后将大鼠麻醉取血,离心收集血清。给药组大鼠收集的血清为人参-三七高剂量含药血清,正常组大鼠收集的血清为正常血清。人参-三七高剂量含药血清与筛选出用于后续实验的最佳浓度比例正常血清分别按照2∶3、1∶3的比例稀释得到人参-三七中剂量含药血清与人参-三七低剂量含药血清。然后,将HL-1心肌细胞设为正常组、模型组以及人参-三七低剂量组、中剂量组、高剂量组。其中,正常组细胞不予特殊方法干预;模型组细胞先缺氧6 h,再复氧2 h,以此建立缺氧/复氧模型;低、中、高剂量组细胞先加入相应的低、中、高含药血清预培养细胞12 h,再缺氧6 h,复氧2 h进行造模。以上5组细胞采用流式细胞分析仪检测HL-1心肌细胞凋亡率;比色法检测超氧化物歧化酶(SOD)、丙二醛(MDA)以及血清心肌梗死标志物乳酸脱氢酶(LDH);免疫荧光技术检测线粒体膜电位与线粒体膜通透性转换孔(MPTP)的开放程度;蛋白质印迹法(Wersternblotting)检测线粒体复合物相关蛋白表达。结果:与正常组比较,模型组细胞活性与SOD活性降低,MDA活性、LDH释放率与细胞凋亡率升高,线粒体膜电位水平降低,MPTP的开放程度升高。泛醌氧化还原酶核心亚基A9(NDUFA9)、泛醌氧化还原酶核心亚基S3(NDUFS3)、琥珀酸脱氢酶复合铁硫亚基B(SDHB)、腺嘌呤核苷三磷酸合成酶δ亚基(ATP5D)、泛醌细胞色素C还原酶核心蛋白2(UQCRC2)等蛋白表达水平均降低,S100钙结合蛋白A8/A9复合物(S100A8/A9)表达水平升高(均P<0.01)。与模型组比较,经人参-三七预处理后,细胞活性与SOD活性明显升高,MDA活性、LDH释放率与细胞凋亡率显著降低,线粒体膜电位水平升高,MPTP的开放程度降低,NDUFS3、NDUFA9、SDHB、ATP5D与UQCRC2等蛋白表达水平升高,S100A8/A9蛋白表达水平下降(均P<0.01)。结论:人参-三七可维持线粒体稳态,改善缺氧/复氧后HL-1心肌细胞损伤,其机制可能与S100A8/A9介导的线粒体功能相关。
Objective:To investigate the effects of Renshen(Ginseng Radix et Rhizoma)and Sanq(i Notoginseng Radix et Rhizoma)on mitochondrial function in hypoxia/reoxygenation-induced HL-1 cardiomyocyte.Methods:According to the equivalent dose for rats,2.50 g of Renshen powder and 1.25 g of Sanqi powder were mixed with 44 mL of warm boiled water daily.Then,2 mL of the mixture was intragastrically administered to each of the 22 rats in the drug-administration group.Another 10 rats were intragastrically administered with 2 mL of normal saline as the normal group.Based on the previous experience of extracting drugcontaining serum from rats in our research group and the results of preliminary experiments,intragastric administration was continued for 7 days in both groups.After fasting for 1 day(with free access to water),the rats were anesthetized and blood was collected.Serum was obtained by centrifugation.The serum collected from the drug-administration group was the high-dose drug-containing serum of Renshen and Sanqi,and the serum collected from the normal group was the normal serum.The high-dose drug-containing serum of Renshen and Sanqi and the normal serum with the optimal concentration ratio selected for subsequent experiments were diluted at the ratios of 2∶3 and 1∶3 respectively to obtain the medium-dose drug-containing serum of Renshen and Sanqi and the low-dose drug-containing serum of Renshen and Sanqi.Then,HL-1 cardiomyocytes were divided into a normal group,a model group,and low-dose,mediumdose,and high-dose groups of Renshen and Sanqi.In the normal group,cells were not given special intervention.In the model group,cells were exposed to hypoxia for 6 h and then reoxygenated for 2 h to establish the hypoxia/reoxygenation model.In the low-dose,medium-dose and high-dose groups,cells were pre-cultured with the corresponding low-dose,medium-dose and high-dose drug-containing sera for 12 h,and then exposed to hypoxia for 6 h and reoxygenation for 2 h to establish the model.The apoptosis rate of HL-1 cardiomyocytes in the above 5 groups was detected by flow cytometry.Colorimetry was used to measure the activities of superoxide dismutase(SOD),malondialdehyde(MDA),and the release rate of lactate dehydrogenase(LDH),a serum marker of myocardial infarction.Immunofluorescence technology was used to detect the opening degree of mitochondrial membrane potential and mitochondrial membrane permeability transition pore(MPTP).The expression of mitochondrial complex-associated proteins was detected by Western blotting(P<0.01).Results:Compared with the normal group,cell activity and SOD activity in the model group decreased,while the activity of MDA,the release rate of LDH,and the apoptosis rate of cells increased(P<0.01).The level of mitochondrial membrane potential decreased,and the opening degree of MPTP increased(P<0.01).The expression levels of proteins such as ubiquinone oxidoreductase core subunit A9(NDUFA9),ubiquinone oxidoreductase core subunit S3(NDUFS3),succinate dehydrogenase complex iron-sulfur subunit B(SDHB),Adenine nucleoside triphosphate synthaseδsubunit(ATP5D),ubiquinone cytochrome C reductase core protein 2(UQCRC2)decreased(P<0.01).The expression level of S100 calcium-binding protein A8/A9 complex(S100A8/A9)increased(P<0.01).Compared with the model group,after pretreatment with Renshen and Sanqi,cell activity and SOD activity increased significantly,while the activity of MDA,the release rate of LDH,and the apoptosis rate of cells decreased significantly(P<0.01).The level of mitochondrial membrane potential increased,and the opening degree of MPTP decreased(P<0.01).The expression levels of NDUFS3,NDUFA9,SDHB,ATP5D,and UQCRC2 increased,and the expression level of S100A8/A9 decreased(P<0.01).Conclusion:Renshen and Sanqi can maintain mitochondrial homeostasis and improve the hypoxia/reoxygenation-induced HL-1 cardiomyocyte injury.The mechanism may be related to the mitochondrial function mediated by S100A8/A9.
作者
高薇涵
孙煜
邓金兰
李洁
GAO Weihan;SUN Yu;DENG Jinlan;LI Jie(Shandong University of Traditional Chinese Medi-cine,Jinan 250355,China)
出处
《山东中医药大学学报》
2025年第4期481-492,共12页
Journal of Shandong University of Traditional Chinese Medicine
基金
中国博士后基金特别资助项目(编号:2022T150393)
国家中医药管理局科技司共建科技项目(编号:GZY-KJS-2023-036)。
关键词
人参
三七
缺氧/复氧损伤
线粒体
能量代谢
HL-1心肌细胞
Renshen(Ginseng Radix et Rhizoma)
Sanqi(Notoginseng Radix et Rhizoma)
hypoxia/reoxygenation-induced injury
mitochondria
energy metabolism
HL-1 cardiomyocytes
