药学学报  2020, Vol. 55 Issue (10): 2398-2404     DOI: 10.16438/j.0513-4870.2020-1015   PDF    
黄芪甲苷通过调节线粒体稳态减轻大鼠心肌细胞缺氧复氧损伤
刘啊敏1, 牟幼灵2, 徐紫薇2, 刘骞2     
1. 浙江大学医学院附属第二医院临床研究中心, 浙江 杭州 310009;
2. 浙江中医药大学药学院, 浙江 杭州 311402
摘要: 探讨黄芪甲苷对缺氧复氧损伤大鼠心肌细胞及线粒体形态和功能的影响及机制。大鼠心肌细胞H9c2分为正常对照组、缺氧复氧组和黄芪甲苷组。CCK-8(cell counting kit-8)法检测细胞活力;酶标法检测细胞培养液上清乳酸脱氢酶(lactate dehydrogenase,LDH)活性、超氧化物歧化酶(superoxide dismutase,SOD)活性、谷胱甘肽(glutathione,GSH)含量和丙二醛(malondialdehyde,MDA)含量;DHE(dihydroethidium)和MitoSOX荧光探针法检测细胞和线粒体活性氧(reactive oxygen species,ROS)含量;JC-1荧光探针法检测线粒体膜电位;calcein-AM(acetoxymethyl ester)荧光探针法检测线粒体通透性转换孔开放性;TUNEL(terminal-deoxynucleoitidyl transferase mediated nick end labeling)法检测细胞凋亡率;Western blot法检测线粒体分裂/融合蛋白Drp1(dynamin-related protein 1)、Mfn1(mitofusin1)和Mfn2,以及细胞凋亡蛋白(B-cell lymphoma-2,Bcl-2)、Bax和cleaved caspase(cysteine-aspartic protease)-3的表达量。与正常对照组相比,缺氧复氧损伤造成H9c2细胞活力、SOD活性和GSH含量显著降低,LDH漏出量和细胞MDA含量明显增加;细胞和线粒体ROS含量显著增加;线粒体膜电位去极化,伴随线粒体通透性转换孔显著开放;线粒体分裂蛋白表达量上调,融合蛋白表达量下调;细胞凋亡蛋白量及凋亡率显著增加。黄芪甲苷(100 μmol·L-1)预处理可显著改善缺氧复氧造成的H9c2细胞损伤、线粒体形态改变和功能障碍。此外,在大鼠原代心肌细胞上也验证了黄芪甲苷抗缺氧复氧损伤的作用。动物福利和实验过程均遵循浙江中医药大学动物伦理委员会的规定。结果表明,黄芪甲苷可能通过调节线粒体形态动态稳定维持线粒体正常功能,抑制ROS过度合成,改善氧化应激内环境并减轻细胞凋亡,从而发挥抗心肌细胞缺氧复氧损伤作用。
关键词: 黄芪甲苷    心肌细胞    缺氧复氧损伤    线粒体稳态    细胞凋亡    
Astragaloside IV ameliorates hypoxia/reoxygenation injury via regulating mitochondrial homeostasis in rat cardiomyocytes
LIU A-min1, MOU You-ling2, XU Zi-wei2, LIU Qian2     
1. Clinical Research Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China;
2. College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 311402, China
Abstract: This study was designed to investigate the effect and mechanism of astragaloside IV (ASIV) on mitochondrial morphology and function of rat cardiomyocytes under hypoxia/reoxygenation injury. H9c2 cells were divided into control group, hypoxia/reoxygenation (H/R) group, and H/R + ASIV group. Cell viability and lactate dehydrogenase (LDH) leakage were measured by cell counting kit-8 (CCK-8) and LDH assay kit, respectively. Oxidative stress levels, such as superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA), were analyzed by commercial kits. Intracellular and mitochondrial reactive oxygen species (ROS) levels were detected by dihydroethidium (DHE) and MitoSOX. Changes of the mitochondrial membrane potential were detected using the fluorescent probe JC-1. Opening of mitochondrial permeability transition pore was examined via calcein acetoxymethyl ester (calcein-AM). Apoptosis was assessed using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay kit. To detect protein expression of dynamin-related protein 1 (Drp1), mitofusin1 (Mfn1), Mfn2, Bax, B-cell lymphoma-2 (Bcl-2), and cleaved cysteine-aspartic protease (caspase)-3, Western blot analysis was carried out. Compared with the control group, ASIV (100 μmol·L-1) significantly improved H/R induced cell injury, LDH leakage, decrease of SOD activity, and GSH content, increase of MDA content and ROS content, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, ROS production activation, mitochondrial fission/fusion imbalance, and cell apoptosis. In addition, the effect of ASIV against H/R injury was also verified on primary rat cardiomyocytes. The animal welfare and experimental process follow the rules of Animal Ethics Committee of Zhejiang Chinese Medical University. In conclusion, ASIV may play a protective role in mitochondria by regulating morphological dynamic stability and mitochondrial function, inhibiting excessive synthesis of ROS, improving the internal environment of oxidative stress, reducing cell apoptosis, and thereby protecting against cardiomyocytes' hypoxia/reoxygenation injury.
Key words: astragaloside IV    cardiomyocyte    hypoxia/reoxygenation injury    mitochondrial homeostasis    apoptosis    

急性心肌梗死(acute myocardial infarction, AMI)的全球发病率和致死率一直居高不下, 目前临床治疗方案是迅速恢复心肌梗死区血流供应, 通过介入性心脏治疗如经皮冠脉成形术等能够快速重建缺氧区氧供应, 有效减少心肌不可逆坏死程度[1]。然而, 即使在及时恢复血液供应的情况下, 心梗患者1年内心衰发生率仍高达25%, 其主要原因之一是心肌恢复血液供应时发生了缺血再灌注损伤(ischemia-reperfusion injury, IRI)[2]。心肌缺血再灌注损伤是指当缺血心肌恢复血流供应时, 受损心肌细胞合成并释放大量活性氧(reactive oxygen species, ROS), 诱发多种病理因素的级联反应, 如氧化应激、钙超载、炎症和线粒体功能障碍等, 最终导致更加严重的心肌损伤[3]。因此, 如何有效干预心肌缺血再灌注损伤并维持心肌细胞存活是心梗治疗面对的重点问题, 并且由于心肌组织高能量需求的特殊性, 对其线粒体功能的保护更加重要。

传统中药材黄芪是临床常用的补气要药, 具有补气固表和利尿消肿等功效[4], 同时兼有活血之功, 能够延缓衰老[5], 用于治疗恶性肿瘤[6]、缺铁性贫血[7]、脑缺血[8]、认知障碍[9]、糖尿病[10]、糖尿病心肌病[11]、心肌纤维化[12]和心肌缺血[13]等疾病。黄芪主要活性成分之一黄芪甲苷(astragaloside IV, ASIV)具有抗炎[14]、抗氧化[15]、抗脑缺血再灌注损伤[16]、抗动脉粥样硬化[17]、抗心肌纤维化[18]及抗心肌缺血再灌注损伤[19]等活性。此外, 研究发现ASIV能够清除自由基和抗氧化应激损伤, 维持细胞氧化还原系统稳态[20], 对线粒体功能调节具有重要作用[21], 但其是否通过调节线粒体稳态提升心肌存活以实现抗心肌缺血再灌注损伤的作用机制仍不清楚。本研究应用大鼠心肌细胞H9c2缺氧复氧(hypoxia/reoxygenation, H/R)损伤模型研究ASIV对心肌细胞线粒体稳态的调控作用, 以进一步阐明其对缺血再灌注心肌细胞氧化损伤的保护作用及机制。

材料与方法

药物与细胞   H9c2细胞购自中国科学院细胞库; ASIV (质量分数≥98%, 批号140913), 购自四川省维克奇生物科技有限公司。

试剂  二甲基亚砜(dimethylsulfoxide, DMSO)和JC-1荧光探针(美国Sigma公司); DMEM (Dulbecco's modified eagle medium)培养基和胎牛血清(美国Gibco公司); CCK-8 (cell counting kit-8)试剂盒、BCA (bicinchoninic acid)蛋白检测试剂盒、DAPI (4', 6-diamidino-2-phenylindole)荧光探针和β-actin抗体(上海碧云天生物技术有限公司); 乳酸脱氢酶(lactate dehydrogenase, LDH)、超氧化物歧化酶(superoxide dismutase, SOD)、丙二醛(malondialdehyde, MDA)和谷胱甘肽(glutathione, GSH)检测试剂盒(南京建成生物工程研究所); DHE (dihydroethidium)荧光探针(美国Invitrogen公司); MitoSOX荧光探针和calcein-AM (acetoxymethyl ester)荧光探针(美国Life Technologies公司); TUNEL (terminal-deoxynucleoitidyl transferase mediated nick end labeling)试剂盒(美国Roche公司); Bax、Bcl-2 (B-cell lymphoma-2)和cleaved caspase (cysteine-aspartic protease)-3抗体(美国Cell Signaling Technology公司)。

仪器   iMark酶标仪和ChemiDoc凝胶成像系统(美国Bio-Rad公司); FACSAriaII流式细胞仪(美国BD公司); IX70荧光显微镜(日本Olympus公司)。

细胞培养与实验分组 H9c2细胞培养于高糖DMEM培养基(含10%胎牛血清)中, 培养条件为5% CO2、37 ℃恒温。ASIV用DMEM配制成终浓度为10 mmol·L-1的储备液, 临用前使用相应培养液稀释到对应浓度, 确保DMSO终浓度不高于0.1%。H9c2细胞随机分成4组: ①正常对照组(control):置于正常培养液和培养箱中培养; ②缺氧复氧组(H/R):置于无糖DMEM培养液中缺氧[混合气N2:O2:CO2 (94:1:5)]培养16 h后, 更换高糖DMEM培养液并常氧条件[空气:CO2 (95:5)]培养2 h; ③ ASIV组(H/R + ASIV):培养液中加入ASIV (1、10和100 μmol·L-1)预处理1 h, 再进行缺氧复氧处理, 整个过程不同条件的培养液中均含有相应浓度的黄芪甲苷。

大鼠原代心肌细胞分离与培养操作如下: SD (Sprague-Dawley)乳大鼠6只, 酒精浸泡30 s左右后取出心脏, 加入冰磷酸缓冲盐溶液(phosphate buffer saline, PBS)冲洗。用眼科剪剪成1 mm3组织块后加入0.1%胰酶消化吹打, 并于37 ℃、100 r·min-1的摇床中进行消化, 每次消化结束后反复吹打消化液10次, 每次10 min, 使组织块自然沉淀, 并将上清转移至10%胎牛血清(fetal bovine serum, FBS)的中和液中, 结束消化后, 于室温1 500 r·min-1离心10 min, 去除上清, 用含10% FBS的DMEM培养液重悬沉淀并种板。根据差速贴壁法, 于90 min后将上清转移至新的培养皿中, 用含10% FBS的DMEM培养液培养细胞, 并加入5-溴脱氧尿嘧啶核苷(5-bromodeoxyuridine, Brdu)抑制非心肌细胞的生长。缺氧复氧损伤及ASIV预处理操作同H9c2细胞。动物福利和实验过程均遵循浙江中医药大学动物伦理委员会的规定。

细胞活力检测   H9c2细胞以每孔5×103个接种于96孔板, 分组给药后, 用CCK-8法检测各组细胞活力:每孔加入10 μL CCK-8试剂, 继续孵育2 h后, 用酶标仪检测各孔在450 nm波长处的吸光度值(A450)。细胞活力(%) = [(A450处理组-A450溶剂对照孔)/(A450正常对照组-A450溶剂对照孔)]×100%。

培养液上清LDH活性检测   H9c2细胞以每孔1×105个接种于6孔板, 分组给药后, 收集培养液上清, 根据LDH检测试剂盒的方法检测培养液上清中LDH活性。

细胞SOD活力、MDA和GSH含量检测  分别收集培养液上清和细胞总蛋白, 根据SOD、MDA及GSH检测试剂盒和BCA蛋白含量检测试剂盒的方法分别检测细胞SOD活力、MDA和GSH含量及细胞总蛋白量, 计算结果以单位总蛋白含量(mg)均一化处理。

细胞ROS含量和线粒体ROS含量检测  使用DHE荧光探针(10 μmol·L-1)避光孵育细胞30 min, PBS润洗2次后, 于荧光显微镜下拍照, 每组随机拍摄6个视野, Image J软件分析荧光强度。H9c2细胞经MitoSOX荧光探针(5 μmol·L-1)避光标记30 min后, 用Hank's平衡盐溶液(Hank's balanced salt solution, HBSS) (含Ca2+/Mg2+离子)润洗2次后, 消化并计数, 调整细胞数为每管(5~8)×106个, 流式细胞仪检测荧光强度(发射波长为580 nm)。

线粒体膜电位检测  使用JC-1荧光探针(2 μmol·L-1)避光孵育细胞20 min, PBS润洗2次后, 于荧光显微镜下拍照, 每组随机拍摄6个视野, Image J软件分析荧光强度。

线粒体通透性转换孔检测  使用calcein-AM (1 μmol·L-1)和CoCl2 (2 mmol·L-1)避光孵育细胞20 min, PBS润洗2次后, 于荧光显微镜下拍照, 每组随机拍摄6个视野, Image J软件分析荧光强度。

细胞凋亡检测  按照TUNEL检测试剂盒提供的方法结合DAPI荧光探针标记凋亡细胞和总细胞, 在原代心肌细胞凋亡染色的同时共染troponin I。荧光显微镜每组随机拍摄6个视野, 细胞凋亡率(%) =凋亡细胞数/总细胞数×100%。

Western blot检测目的蛋白表达   H9c2细胞经预冷PBS润洗3次后, 使用RIPA (radio immunoprecipitation assay)裂解液制备总蛋白提取液, 用于检测Drp1 (dynamin-related protein 1, 1:1 000)、Mfn1 (mitofusin 1, 1:1 000)、Mfn2 (1:1 000)、Bax (1:1 000)、Bcl-2 (1:1 000)、cleaved caspase-3 (1:1 000)和β-actin (1:4 000)蛋白含量。使用化学发光成像仪收集目的蛋白条带图像并通过Image Lab软件分析计算相对蛋白表达量。

统计分析  使用Minitab 14软件统计分析实验结果, 以x ± s表示实验结果, 使用ANOVA分析组间差异, 其中P < 0.05认为差异具有统计学意义。

结果 1 ASIV对H/R诱导的H9c2细胞损伤的影响

ASIV对H/R诱导的H9c2细胞损伤的影响结果见图 1, 与正常对照组比较, H/R处理显著降低H9c2细胞活力(P < 0.01), 同时增加H9c2细胞LDH泄露(P < 0.01); ASIV (10和100 μmol·L-1)预处理能够显著恢复细胞活力(P < 0.01), 且呈剂量依赖性; ASIV (100 μmol·L-1)预处理能够明显降低培养液上清中LDH活性(P < 0.05)。以上实验结果发现, ASIV (100 μmol·L-1)具有较好的抗细胞缺氧复氧损伤活性, 后续实验均采用100 μmol·L-1浓度ASIV开展实验。

Figure 1 Effects of astragaloside IV (ASIV) on hypoxia/reoxygenation (H/R) induced H9c2 cells injury. A: The cell viability was determined by cell counting kit-8 (CCK-8) assay; B: The lactate dehydrogenase (LDH) release was determined by LDH assay kit. n = 6, x± s. ##P < 0.01 vs control; *P < 0.05, **P < 0.01 vs model (H/R)
2 ASIV对H/R诱导的H9c2细胞氧化应激的影响

图 2结果显示, 与正常对照组比较, H/R处理显著降低细胞SOD活力(P < 0.01), 减少GSH含量(P < 0.01)并增加MDA含量(P < 0.01); ASIV (100 μmol·L-1)预处理能够显著增加SOD活力(P < 0.05)和GSH含量(P < 0.05), 并降低MDA含量(P < 0.01)。

Figure 2 Effects of ASIV on H/R induced H9c2 cells oxidative stress injury. A: Superoxide dismutase (SOD) activity was determined by SOD assay kit; B: Glutathione (GSH) content was estimated by GSH assay kit; C: Malondialdehyde (MDA) content was estimated by MDA assay kit. n = 6, x± s. ##P < 0.01 vs control; *P < 0.05, **P < 0.01 vs model (H/R)
3 ASIV对ROS含量的影响

与正常对照组比较(图 3), H/R处理显著增加细胞ROS含量和线粒体ROS含量(P < 0.01); ASIV (100 μmol·L-1)预处理能够显著降低细胞ROS含量和线粒体ROS含量(P < 0.01)。

Figure 3 Effects of ASIV on reactive oxygen species (ROS) content in H/R induced H9c2 cells. A: Representative fluorescence images of dihydroethidium (DHE) fluorescence (×200). Scale bar: 100 μm; B: Quantification of DHE fluorescence intensity; C: Quantification of MitoSOX fluorescence intensity. n = 6, x± s. ##P < 0.01 vs control; **P < 0.01 vs model (H/R)
4 ASIV对线粒体膜电位的影响

JC-1是一种常用于检测线粒体膜电位的荧光探针, 当线粒体膜电位较高时, JC-1在线粒体内形成聚合物, 产生红色荧光; 当线粒体膜电位较低时, JC-1在线粒体内处于离散状态, 产生绿色荧光。图 4结果显示, 与正常对照组比较, H/R处理显著增强绿色荧光(P < 0.01); ASIV (100 μmol·L-1)预处理能够显著降低绿色荧光(P < 0.01), 维持线粒体膜电位。

Figure 4 Effects of ASIV on the collapse of mitochondrial transmembrane potential in H/R induced H9c2 cells. A: Representative fluorescence images of JC-1 fluorescence (×200). Scale bar: 100 μm; B: Quantification of JC-1 fluorescence. n = 6, x± s. ##P < 0.01 vs control; **P < 0.01 vs model (H/R)
5 ASIV对线粒体通透性转换孔开放的影响

Calcein-AM荧光探针进入细胞线粒体后被切割成calcein而无法正常离开线粒体, CoCl2能够进入细胞淬灭calcein的绿色荧光但无法进入线粒体, 因此线粒体通透性转换孔的开放程度与绿色荧光强度成负相关。图 5结果显示, 与正常对照组比较, H/R处理显著降低绿色荧光(P < 0.01); ASIV (100 μmol·L-1)预处理能够显著增强绿色荧光(P < 0.01), 降低线粒体通透性转换孔开放程度。

Figure 5 Effects of ASIV on opening of the mitochondrial permeability transition pore in H/R induced H9c2 cells. A: Representative fluorescence images of calcein fluorescence (×200). Scale bar: 100 μm; B: Quantification of calcein fluorescence. n = 6, x± s. ##P < 0.01 vs control; **P < 0.01 vs model (H/R)
6 ASIV对线粒体分裂/融合平衡的影响

与正常对照组比较, H/R处理显著上调线粒体分裂蛋白Drp1表达量(P < 0.001), 下调线粒体融合蛋白Mfn2表达量(P < 0.01); ASIV (100 μmol·L-1)预处理显著改善Drp1蛋白的过表达(P < 0.01)和Mfn2蛋白低表达(P < 0.05), 结果见图 6

Figure 6 Effect of ASIV on modulation of mitochondrial fission/fusion proteins in H/R induced H9c2 cells. Relative expressions of dynamin-related protein 1 (Drp1), mitofusin1 (Mfn1), and Mfn2 were determined by Western blot. A: Representative blots of Drp1, Mfn1, and Mfn2; B: Quantified expression of Drp1 protein; C: Quantified expression of Mfn1 protein; D: Quantified expression of Mfn2 protein. n = 4, x± s. ##P < 0.01, ###P < 0.001 vs control; *P < 0.05, **P < 0.01 vs model (H/R)
7 ASIV对细胞凋亡的影响

图 7结果显示, 与正常对照组比较, H/R处理显著增加细胞凋亡率(P < 0.01)和凋亡蛋白cleaved caspase-3表达量(P < 0.01), 降低Bcl-2/Bax比值(P < 0.001); ASIV (100 μmol·L-1)预处理能够显著降低细胞凋亡率(P < 0.01)和cleaved caspase-3表达量(P < 0.05), 增加Bcl-2/Bax比值(P < 0.01)。

Figure 7 Effects of ASIV on apoptosis of H/R induced H9c2 cells. A: Representative fluorescence images of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL, red) and 4', 6-diamidino-2-phenylindole (DAPI, blue) fluorescence (×200). Scale bar: 100 μm; B: Quantification of TUNEL positive cells (n = 6); C: Representative blots of B-cell lymphoma-2 (Bcl-2), Bax, and cleaved caspase-3; D: Quantified expression of Bcl-2/Bax ratio (n = 4); E: Quantified expression of cleaved caspase-3 protein (n = 4). Results were represented as x± s. ##P < 0.01, ###P < 0.001 vs control; *P < 0.05, **P < 0.01 vs model (H/R)
8 大鼠原代心肌细胞验证ASIV抗H/R损伤的作用

ASIV对H/R诱导的大鼠原代心肌细胞损伤的影响结果见图 8, 与正常对照组比较, H/R处理显著降低大鼠原代心肌细胞活力(P < 0.001), 降低线粒体膜电位(P < 0.001), 增加细胞凋亡率(P < 0.001); ASIV (100 μmol·L-1)预处理能够显著恢复细胞活力(P < 0.01), 维持线粒体膜电位(P < 0.01), 降低细胞凋亡率(P < 0.05)。

Figure 8 Effects of ASIV on apoptosis of H/R induced primary neonatal Sprague-Dawley (SD) rat cardiomyocytes. A: The cell viability was determined by CCK-8 assay; B: Quantification of JC-1 fluorescence; C: Representative fluorescence images of TUNEL (red), DAPI (blue), and troponin (green) fluorescence (×200). Scale bar: 100 μm; D: Quantification of TUNEL positive cells. n = 6, x± s. ###P < 0.001 vs control; *P < 0.05, **P < 0.01 vs model (H/R)
讨论

对于包括急性心肌缺血在内的各类组织缺血, 临床常规治疗手段都是优先恢复血流再灌注, 此时会面对包括血管、内皮及线粒体功能障碍, 水肿和炎症等一系列并发症, 最终导致细胞不可逆坏死等严重组织损伤[22]。因此, 能够减轻心肌缺血再灌注过程中各类损伤的辅助治疗都有助于改善急性心肌梗死患者的预后。黄芪作为传统补气药材, 能够提高机体免疫力和抗氧化能力[23], 治疗心肌缺血在内的多种疾病, 实验研究发现其主要活性成分之一ASIV具有抗心肌肥大[24]、抗心肌缺氧损伤[25]、抗心肌细胞氧化损伤[26]等心肌保护活性, 但其具体作用机制仍不十分明确。本实验结果显示, ASIV能够显著改善缺氧复氧导致的H9c2细胞活力下降和LDH泄露, 表明ASIV对心肌细胞氧化应激损伤具有一定的保护作用。

缺血心肌再灌注时, 心肌细胞会在恢复血氧供应时合成过量ROS, 直接氧化细胞内脂质、蛋白质和DNA等生物大分子, 对细胞造成不可逆损伤[27], 因此提高缺血心肌组织的抗氧化能力对防止再灌注损伤具有重要作用。本实验发现ASIV显著提升细胞SOD活性, 增加GSH含量, 降低了MDA含量, 显示出良好的抗氧化能力。为了检测更直接的抗氧化指标, 本研究通过DHE和MitoSOX荧光探针分别检测了H9c2细胞ROS含量和线粒体中ROS含量, 发现ASIV能够显著降低细胞ROS含量和线粒体中ROS含量。

心肌细胞中ROS主要合成于线粒体, 而线粒体功能障碍会导致ROS爆发性合成并大量释放到细胞质中, 造成严重的氧化损伤[28]。本实验通过JC-1和calcein荧光探针检测线粒体膜电位和通透性转换孔开放程度, 发现ASIV能够显著恢复线粒体膜电位去极化并抑制通透性转换孔过度开放, 提示ASIV能够有效维持线粒体形态和功能正常。此外, 线粒体形态的动态稳定对保障线粒体功能正常也具有重要意义[29], 线粒体过度分裂造成的碎片化会使线粒体失去正常功能, 同时诱发细胞凋亡。本实验发现ASIV能够显著恢复线粒体分裂蛋白Drp1和线粒体融合蛋白Mfn2的表达失衡, 抑制线粒体过度分裂, 促进线粒体融合, 表明ASIV对调节心肌细胞线粒体形态动态稳定具有重要作用。

综上, 本研究结果表明ASIV可能通过稳定心肌细胞线粒体形态动态稳定, 维持线粒体功能, 抑制ROS过度合成, 从而减轻缺氧复氧诱导的氧化应激损伤。

作者贡献:刘啊敏和牟幼灵负责实验设计和具体实验; 徐紫薇负责实验数据分析; 刘骞负责实验指导和论文修改

利益冲突:所有作者声明不存在利益冲突

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