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目的：探讨电针“内关”对心肌缺血再灌注损伤（MIRI）大鼠不同时期（缺血期、再灌注期）线粒体自噬的调控作用，探讨电针“内关”的双向调控作用及可能机制。方法：将45只雄性SD大鼠按照随机数字表法分为6组，假手术组（9只）、模型A组（6只）、模型B组（9只）、电针A1组（6只）、电针B1组（6只）、电针B2组（9只）。除假手术组外，其余各组大鼠采用物理结扎加推管法复制MIRI模型，模型A组进行结扎后直接取材，模型B组进行结扎、再灌注后取材；电针A1组结扎的同时进行电针处理，之后直接取材；电针B1组结扎的同时进行电针处理，再灌注后取材；电针B2组结扎、开放冠状动脉左前降支的同时予电针处理，再灌注后取材。电针取双侧“内关”，采用疏密波，频率2 Hz/100 Hz，电流1 mA，时长为30 min。HE染色法观察大鼠心肌细胞形态，TUNEL法检测大鼠心肌细胞凋亡情况，转录组测序检测大鼠左心室差异表达基因，JC-1流式法检测大鼠心肌细胞线粒体膜电位（MMP）,Western blot法检测大鼠左心室磷酸酶和张力蛋白同源物诱导激酶1(Pink1)、Parkin和p62蛋白表达，ELISA法检测大鼠血清肌酸激酶同工酶（CK-MB）、心肌肌钙蛋白I(cTn-I)含量。结果：与假手术组比较，模型B组大鼠心肌细胞损伤严重，排列紊乱，边界不清，肌纤维断裂，细胞水肿、松散；电针B2组大鼠心肌细胞损伤轻微，细胞结构部分不清，细胞排列较规则，可见完整心肌细胞。与假手术组比较，模型B组大鼠心肌细胞凋亡加重（P<0.001）；与模型B组比较，电针B2组心肌细胞凋亡减轻（P<0.001）。电针B2组、假手术组和模型B组之间的差异表达基因与细胞自噬和线粒体自噬密切相关。与假手术组比较，模型B组大鼠心肌细胞MMP降低（P<0.001），左心室Pink1、Parkin、p62蛋白表达和血清CK-MB、cTn-I含量升高（P<0.001）；与模型A组比较，电针A1组心肌细胞MMP和血清CK-MB、cTn-I含量降低（P<0.001,P<0.05），左心室Pink1蛋白表达升高（P<0.01）；与模型B组比较，电针B1组和电针B2组心肌细胞MMP升高（P<0.001），左心室Pink1、Parkin、p62蛋白表达及血清CK-MB、cTn-I含量降低（P<0.001）；与电针A1组比较，电针B1组心肌细胞MMP升高（P<0.001），左心室Pink1、Parkin、p62蛋白表达及血清CK-MB、cTn-I含量降低（P<0.01）。结论：电针“内关”能改善大鼠MIRI，其机制可能通过Pink1/Parkin介导的线粒体自噬通路实现，在缺血期可以增强线粒体自噬来减轻心肌损伤，在再灌注期可以降低线粒体自噬来减轻再灌注损伤。
Abstract：Objective To investigate the regulatory effect of electroacupuncture(EA) at "Neiguan"(PC6) on mitochondrial autophagy in rats with myocardial ischemia-reperfusion injury(MIRI) at different phases(ischemia and reperfusion phases), and to explore the bidirectional regulatory effects of EA at "Neiguan"(PC6) and its potential mechanism.Methods Forty-five male SD rats were randomly divided into 6 groups according to the random number table method,namely, sham-operation group(n=9), model-A group(n=6), model-B group(n=9), EA-A1 group(n=6), EA-B1 group(n=6),and EA-B2 group(n=9). Except the rats in the sham-operation group, the MIRI model was established in the other groups with the physical ligation and tube pushing method. In the model-A group, the samples were collected directly after ligation,and in the model-B group, the samples were collected after ligation and reperfusion. In the EA-A1 group, EA was delivered while the ligation was performed, and afterwards, the samples were collected. In the EA-B1 group, while the ligation was performed, EA was operated at the same time, and after reperfusion, the samples were collected. In the EA-B2 group, during ligation and the opening of the left anterior descending branch of the coronary artery, EA was delivered, and after reperfusion,the samples were collected. EA was performed at bilateral "Neiguan"(PC6), with a disperse-dense wave, a frequency of 2 Hz/100 Hz, a current of 1 mA, and a duration of 30 min. HE staining was employed to observe the morphology of cardiomyocytes, TUNEL was adopted to detect the apoptosis of cardiomyocytes, transcriptome sequencing was to detect the differentially expressed genes in the left ventricle, JC-1 flow cytometry was to detect the mitochondrial membrane potential(MMP) of cardiomyocytes, Western blot was to detect the protein expression of phosphatase and tensin homolog-induced kinase 1(Pink1), Parkin and p62 in the left ventricle of rats, and ELISA was to detect the levels of serum creatine kinase isoenzyme(CK-MB) and cardiac troponin I(cTn-I) in the rats. Results Compared with the sham-operation group, the cardiomyocytes of rats in the model-B group were severely damaged, with disordered arrangement, unclear boundaries,broken muscle fibers, edema and loose distribution; and the cardiomyocytes in the EA-B2 group were slightly damaged, the cell structure was partially unclear, the cells were arranged more regularly, and the intact cardiomyocytes were visible.Compared with the sham-operation group, the apoptosis of cardiomyocytes increased in the model-B group(P<0.001); and when compared with the model-B group, the apoptosis alleviated in the EA-B2 group(P<0.001). The differentially expressed genes among the EA-B2 group, the sham-operation group and the model-B group were closely related to cell autophagy and mitochondrial autophagy. Compared with the sham-operation group, MMP of cardiomyocytes was reduced(P<0.001), the protein expression of Pink1, Parkin, and p62 of the left ventricle and the levels of serum CK-MB and cTn-I were elevated in the model B group(P<0.001). In comparison with model-A group, the MMP of cardiomyocytes and the levels of serum CK-MB and cTn-I were reduced(P<0.001, P<0.05), and the protein expression of Pink1 in the left ventricle rose in the EA-A1 group(P<0.01). Compared with the model-B group, MMP of cardiomyocytes increased(P<0.001), the protein expression of Pink1, Parkin, and p62 of the left ventricle, and the levels of serum CK-MB and cTn-I decreased(P<0.001) in the EA-B1 group and the EA-B2 group. When compared with the EA-A1 group, MMP of cardiomyocytes increased(P<0.001), and the protein expression of Pink1, Parkin, and p62 of the left ventricle, and the levels of serum CK-MB and cTn-I decreased in the EA-B1 group(P<0.01). Conclusion EA at "Neiguan"(PC6) can ameliorate MIRI in rats, which may be achieved through the Pink1/Parkin-mediated mitochondrial autophagy pathway. EA can alleviate myocardial injury by enhancing mitochondrial autophagy at the ischemia phase, and it can reduce reperfusion injury by weakening mitochondrial autophagy at the reperfusion phase.
[1] Matter MA, Paneni F, Libby P, et al. Inflammation in acute myocardial infarction:the good, the bad and the ugly[J]. Eur Heart J, 2024, 45(2):89-103.
[2] Michael Gibson C, Duffy D, Korjian S, et al. Apolipoprotein A1infusions and cardiovascular outcomes after acute myocardial infarction[J]. N Engl J Med, 2024, 390(17):1560-1571.
[3] Lindahl B, Mills NL. A new clinical classification of acute myocardial infarction[J]. Nat Med, 2023, 29(9):2200-2205.
[4] Lu YY, Li ZJ, Zhang SQ, et al. Cellular mitophagy:mechanism,roles in diseases and small molecule pharmacological regulation[J]. Theranostics, 2023, 13(2):736-766.
[5] Matsui Y, Takagi H, Qu XP, et al. Distinct roles of autophagy in the heart during ischemia and reperfusion:roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy[J]. Circ Res,2007, 100(6):914-922.
[6] Deng JX, Liu Q, Ye LX, et al. The Janus face of mitophagy in myocardial ischemia/reperfusion injury and recovery[J]. Biomed Pharmacother, 2024, 173:116337.
[7]陈松,韩永丽,吴松,等.针刺“内关”预处理对大鼠缺血再灌注心肌的保护作用及机制研究[J].华中科技大学学报(医学版),2017, 46(5):526-530.
[8] Sun QH, Cheng K, Dai XY, et al. Effect of electroacupuncture at Neiguan(PC6)at different time points on myocardial ischemia reperfusion arrhythmia in rats[J]. J Tradit Chin Med, 2024, 44(1):113-121.
[9]刘雅兰,文巧,肖清清,等.电针内关预处理干预大鼠心肌缺血再灌注损伤的文献分析[J].中国中医基础医学杂志, 2022,28(4):596-601.
[10]熊坚,黄小刚,赵惠芳,等.针灸治疗心肌缺血再灌注损伤研究现状与热点分析[J].世界科学技术–中医药现代化, 2025,27(1):179-194.
[11]黄婷,李绍旦,杨明会,等.大鼠心肌缺血再灌注实验技巧的经验总结[J].中国比较医药杂志, 2017, 27(9):80-82.
[12]华兴邦,周浩良.大鼠穴位图谱的研制[J].实验动物与动物实验, 1991(1):1-5.
[13] Moyzis AG, Sadoshima J, Gustafsson?B. Mending a broken heart:the role of mitophagy in cardioprotection[J]. Am J Physiol Heart Circ Physiol, 2015, 308(3):H183-H192.
[14] Rudokas MW, McKay M, Toksoy Z, et al. Mitochondrial network remodeling of the diabetic heart:implications to ischemia related cardiac dysfunction[J]. Cardiovasc Diabetol, 2024, 23(1):261.
[15] Baloglu E. Hypoxic stress-dependent regulation of Na, K-ATPase in ischemic heart disease[J]. Int J Mol Sci, 2023, 24(9):7855.
[16] Jimenez RE, Kubli DA, Gustafsson?B. Autophagy and mitophagy in the myocardium:therapeutic potential and concerns[J]. Br J Pharmacol, 2014, 171(8):1907-1916.
[17] Chen L, Mao LS, Xue JY, et al. Myocardial ischemia-reperfusion injury:the balance mechanism between mitophagy and NLRP3inflammasome[J]. Life Sci, 2024, 355:122998.
[18] Li YZ, Wu XD, Liu XH, et al. Mitophagy imbalance in cardiomyocyte ischaemia/reperfusion injury[J]. Acta Physiol(Oxf),2019, 225(4):e13228.
[19] Thrane PG, Olesen KKW, Thim T, et al. Mortality trends after primary percutaneous coronary intervention for ST-segment elevation myocardial infarction[J]. J Am Coll Cardiol, 2023,82(10):999-1010.
[20] Chacón-Diaz M, Custodio-Sánchez P, Rojas De la Cuba P, et al.Outcomes in ST-segment elevation myocardial infarction treated with primary percutaneous coronary intervention or pharmacoinvasive strategy in a Latin American country[J]. BMC Cardiovasc Disord, 2022, 22(1):296.
[21] Tanaka K. The PINK1-parkin axis:an overview[J]. Neurosci Res,2020, 159:9-15.
[22] Liu WH, Chen CG, Gu XJ, et al. AM1241 alleviates myocardial ischemia-reperfusion injury in rats by enhancing Pink1/Parkin-mediated autophagy[J]. Life Sci, 2021, 272:119228.
[23] Barazzuol L, Giamogante F, Brini M, et al. PINK1/parkin mediated mitophagy, Ca2+signalling, and ER-mitochondria contacts in Parkinson's disease[J]. Int J Mol Sci, 2020, 21(5):1772.
[24] Ma KY, Fokkens MR, Reggiori F, et al. Parkinson's disease-associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy[J]. Transl Neurodegener, 2021, 10(1):19.
[25] Wade Harper J, Ordureau A, Heo JM. Building and decoding ubiquitin chains for mitophagy[J]. Nat Rev Mol Cell Biol, 2018,19(2):93-108.
[26] Han R, Liu YT, Li SH, et al. PINK1-PRKN mediated mitophagy:differences between in vitro and in vivo models[J]. Autophagy,2023, 19(5):1396-1405.
[27] Geisler S, Holmstr?m KM, Skujat D, et al.PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1[J]. Nat Cell Biol, 2010, 12(2):119-131.
[28] Lee B, Kim YH, Lee W, et al. USP13 deubiquitinates p62/SQSTM1 to induce autophagy and Nrf2 release for activating antioxidant response genes[J]. Free Radic Biol Med, 2023, 208:820-832.
[29] Liu HH, Zheng Q, Yuan JX, et al. Modulating SQSTM1/p62-dependent selective autophagy of neurons by activating Nrf2 with multifunctional nanoparticles to eliminateα-synuclein aggregates and boost therapy of Parkinson's disease[J].Nano Today, 2023, 49:101770.
[30] Zhou YH, Hua SC, Song L. The versatile defender:exploring the multifaceted role of p62 in intracellular bacterial infection[J].Front Cell Infect Microbiol, 2023, 13:1180708.
[31] Guan SB, Xin YF, Ding YG, et al. Ginsenoside Rg1 protects against cardiac remodeling in heart failure via SIRT1/PINK1/parkin-mediated mitophagy[J]. Chem Biodivers,2023, 20(2):e202200730.
[32] Lazarou M, Sliter DA, Kane LA, et al. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy[J].Nature, 2015, 524(7565):309-314.
[33] Kataura T, Otten EG, Rabanal-Ruiz Y, et al. NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy[J]. EMBO J,2023, 42(5):e111372.
[34] Nguyen TN, Sawa-Makarska J, Khuu G, et al. Unconventional initiation of PINK1/parkin mitophagy by optineurin[J]. Mol Cell,2023, 83(10):1693-1709.e9.
[35] Narendra D, Kane LA, Hauser DN, et al. p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy;VDAC1 is dispensable for both[J]. Autophagy, 2010, 6(8):1090-1106.
[36]陈少宗.针灸双向良性调节作用规律的3种形式[J].针灸临床杂志, 2010, 26(12):62-65.
[37]陈少宗.针刺双向调节机制与机体的自组织原理[J].医学与哲学(A), 2017, 38(1):81-83.
[38] Zhuang Y, Xing JJ, Li J, et al. History of acupuncture research[J].Int Rev Neurobiol, 2013, 111:1-23.
基本信息:
DOI：10.13703/j.0255-2930.20241031-k0001
中图分类号:R245.97
引用信息:
[1]杨骐瑞,仇兴华,戴兴业,等.电针“内关”对心肌缺血再灌注损伤大鼠缺血期和再灌注期线粒体自噬的调控作用[J].中国针灸,2025,45(05):646-656.DOI:10.13703/j.0255-2930.20241031-k0001.
基金信息:
北京中医药大学揭榜挂帅资助项目：90011431210003
2025-02-27
2025-02-27
2025-02-27