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李雨晴, 何苗, 肖阳, 任卫琼, 徐菲, 傅音睿, 王志琪.乌头碱配伍甘草次酸前后对衰竭心肌细胞影响的比较研究[J].湖南中医药大学学报,2024,44(6):1001-1008[点击复制] |
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| 乌头碱配伍甘草次酸前后对衰竭心肌细胞影响的比较研究 |
| 李雨晴,何苗,肖阳,任卫琼,徐菲,傅音睿,王志琪 |
| (湖南中医药大学研究生院, 湖南 长沙 410208;四川大学华西药学院, 四川 成都 610041;湖南中医药大学附属第一医院, 湖南 长沙 410007;湖南中医药大学药学院, 湖南 长沙 410208;湖南省中药饮片标准化及功能工程技术研究中心, 湖南 长沙 410208) |
| 摘要: |
| 目的 比较乌头碱配伍甘草次酸前后对衰竭心肌细胞的影响。方法 将H9c2细胞随机分为9组，即空白组，模型组，甘草次酸组，30、120、480 μmol·L-1乌头碱组，30 μmol·L-1联用组（30 μmol·L-1乌头碱+30 μmol·L-1甘草次酸）、120 μmol·L-1联用组（120 μmol·L-1乌头碱+30 μmol·L-1甘草次酸）、480 μmol·L-1联用组（480 μmol·L-1乌头碱+30 μmol·L-1甘草次酸）。除空白组外，各组均以阿霉素法构建衰竭H9c2心肌细胞模型。造模成功后，空白组和模型组给予DMEM，其余各组给予相应药物干预24 h。显微镜观察细胞形态和线粒体微观结构，CCK-8法测定细胞存活率，ELISA检测乳酸脱氢酶（lactate dehydrogenase, LDH）、Na+-K+-ATP酶、Ca2+-ATP酶、Ca2+-Mg2+-ATP酶、超氧化物歧化酶（superoxide dismutase, SOD）、丙二醛（malondialdehyde, MDA）、过氧化氢酶（catalase, CAT）、谷胱甘肽（glutathione, GSH）、ATP，荧光探针法测定活性氧（reactive oxygen species, ROS）、膜电位、胞内及线粒体内Ca2+浓度。蛋白免疫印迹法（western blot, WB）检测AMPK通路和CaMKⅡ通路蛋白。结果 与模型组比较，120、480 μmol·L-1乌头碱降低细胞存活率，升高MDA含量（P<0.01）；480 μmol·L-1乌头碱破坏线粒体结构；30、120、480 μmol·L-1乌头碱升高ROS含量，降低SOD、CAT、GSH酶活性，降低线粒体膜电位和ATP生成，升高胞内及线粒体内钙离子浓度，降低Na+-K+-ATP酶、Ca2+-ATP酶、Ca2+-Mg2+-ATP酶活性（P<0.05或P<0.01）；480 μmol·L-1乌头碱抑制AMPK磷酸化，降低SERCA2a和PGC-1α蛋白表达，促进RyR2蛋白表达（P<0.05或P<0.01）。与单用乌头碱比较，配伍甘草次酸改善其线粒体结构损伤，降低ROS含量，升高SOD、CAT、Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性，降低胞内及线粒体内的钙离子浓度（P<0.05或P<0.01）；30、120 μmol·L-1联用组升高线粒体膜电位（P<0.05或P<0.01）；120、480 μmol·L-1联用组降低MDA含量，提高GSH、Ca2+-ATP酶活性，升高ATP含量（P<0.05或P<0.01）；480 μmol·L-1联用组可抑制CaMKⅡ磷酸化，升高PGC-1α蛋白表达（P<0.05或P<0.01）。结论 甘草次酸可拮抗乌头碱对衰竭心肌细胞的线粒体毒性，作用环节可能与缓解氧化应激和钙超载，增加线粒体产能有关。 |
| 关键词: 乌头碱 甘草次酸 衰竭心肌细胞 线粒体 钙超载 氧化应激 |
| DOI：10.3969/j.issn.1674-070X.2024.06.010 |
| 投稿时间：2024-01-05 |
| 基金项目:国家自然科学基金项目（81503492）；湖南省自然科学基金项目（2021JJ80059）；长沙市自然科学基金项目（kq2014093）；湖南省教育厅重点项目（23A0285）；湖南省大学生创新创业项目（S202310541091）；校级研究生创新课题（2022CX80）。 |
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| Comparison of the effects of aconitine on failing cardiomyocytes before and after combined with glycyrrhetinic acid |
| LI Yuqing, HE Miao, XIAO Yang, REN Weiqiong, XU Fei, FU Yinrui, WANG Zhiqi |
| (Graduate School, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Hunan Engineering Technology Research Center for Standardization and Function of Chinese Herbal Decoction Pieces, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To compare the different effects of aconitine on failing cardiomyocytes before and after combined with glycyrrhetinic acid. Methods H9c2 cells were randomized into nine groups, i.e., blank group, model group, glycyrrhetinic acid group, 30, 120, and 480 μmol·L-1 aconitine groups, 30 μmol·L-1 combination group (30 μmol·L-1 aconitine+30 μmol·L-1 glycyrrhetinic acid), 120 μmol·L-1 combination group (120 μmol·L-1 aconitine+30 μmol·L-1 glycyrrhetinic acid), and 480 μmol·L-1 combination group (480 μmol·L-1 aconitine+30 μmol·L-1 glycyrrhetinic acid). The model of failing H9c2 cardiomyocytes was established by an 24-hour intervention of 2 μmol·L-1 adriamycin in all groups except the blank group. After successful modeling, the blank and model groups were treated with DMEM, and the remaining groups with the corresponding drugs for 24 h. Cardiomyocyte morphology and mitochondrial microstructure were observed by microscopy, and cell viability was determined by CCK-8 assay. Enzyme immunoassay was used to examine the content of lactate dehydrogenase (LDH), Na+-K+-ATPase, Ca2+-ATPase, Ca2+-Mg2+-ATPase, superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione (GSH), and ATP. Fluorescent probe assay was employed to check the reactive oxygen species content (ROS), mitochondrial membrane potential, and intracellular and mitochondrial Ca2+ concentration. Proteins of AMPK pathway and CaMKII pathway were examined by Western blot. Results Compared with model group, 120 and 480 μmol·L-1 aconitine decreased cell viability, increased MDA content(P<0.05 or P<0.01), 480 μmol·L-1 aconitine disrupted the mitochondrial structure, 30, 120, and 480 μmol·L-1 aconitine increased ROS content, decreased SOD, CAT, and GSH enzyme activities, decreased mitochondrial membrane potential and ATP production, and increased intracellular and mitochondrial Ca2+ concentration, decreased Na+-K+-ATPase, Ca2+-ATPase, and Ca2+-Mg2+-ATPase activities (P<0.05 or P<0.01), and 480 μmol·L-1 aconitine inhibited AMPK phosphorylation, decreased SERCA2a and PGC-1α protein expression, and promoted RyR2 protein expression (P<0.05 or P<0.01). Compared with aconitine group, the combination of glycyrrhizic acid improved mitochondrial structural damage, reduced ROS content, elevated the activities of SOD, CAT, Na+-K+-ATPase and Ca2+-Mg2+-ATPase, and reduced the intracellular and mitochondrial calcium ion concentration (P<0.05 or P<0.01), and 30, 120 μmol·L-1 combination groups elevated the mitochondrial membrane potential (P<0.05 or P<0.01), the 120, 480 μmol·L-1 combination groups decreased MDA content, increased GSH and Ca2+-ATPase activities, and elevated ATP content (P<0.05 or P<0.01), and the 480 μmol·L-1 combination group inhibited CaMK Ⅱ phosphorylation and elevated PGC-1α protein expression (P<0.05 or P<0.01). Conclusion Glycyrrhetinic acid antagonizes the mitochondrial toxicity of aconitine on the failing cardiomyocytes, which may be related to alleviating oxidative stress and calcium overload, as well as increasing mitochondrial energy production. |
| Key words: aconitine glycyrrhetinic acid failing cardiomyocyte mitochondria calcium overload oxidative stress |
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