标本配穴对心肌缺血再灌注损伤糖尿病大鼠心肌细胞的影响及机制研究
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摘要
目的观察标本配穴预处理对糖尿病大鼠心肌缺血再灌注(I/R)损伤的钙稳态、心肌氧化应激和炎症细胞因子的影响。并从p38MAPK信号通路的角度分析其作用机制,为临床心血管疾病的防治方法提供新的理论探讨和实验基础。方法将50只大鼠随机分为5组:假手术组、模型组、电针预处理组、抑制剂预处理组和电针+抑制剂预处理组。假手术组大鼠开胸后暴露心脏不做其他处理,模型组大鼠行在体心肌缺血再灌注术,电针预处理组大鼠于造模前1周行电针内关穴、足三里穴治疗,抑制剂预处理组大鼠于造模前注射p38MAPK信号通路抑制剂SB203580,电针+抑制剂预处理组大鼠电针预处理1周后于造模前注射p38MAPK信号通路抑制剂SB203580。5组大鼠经相应处理后处死取材,测定大鼠心肌细胞相关钙稳态、心肌氧化应激和炎症细胞因子指标。结果模型组大鼠的Na~+-K~+-ATP酶、Mg~(2+)-ATP酶、Ca~(2+)-ATP酶、血清超氧化物歧化酶(SOD)、心肌丙二醛(MDA)和肿瘤坏死因子-α(TNF-α)与假手术组比较,有差异有统计学意义(P<0.05)。抑制剂预处理组、电针预处理组和电针+抑制剂预处理组的上述指标与模型组比较,差异有统计学意义(P<0.05)。结论标本配穴预处理能够维持心肌缺血再灌注损伤糖尿病大鼠心肌细胞的钙稳态,提高清除氧自由基的能力,抑制心肌炎症反应,进而明显减轻缺血再灌注对心功能的损害。
Objective To observe the influence of tip-root acupoint combination pretreatment on calcium homeostasis, myocardial oxidative stress and inflammatory cytokines in diabetic rats with myocardial ischemia-reperfusion(I/R) injury, analyze the mechanism from the aspect of p38 MAPK signaling pathway and provide a new theoretical discussion and experimental basis for prevention and treatment of cardiovascular diseases in clinic. Methods All rats(n=50) were randomly divided into 5 groups: sham-operation group, model group, electro-acupuncture retreatment group, inhibitor pretreatment group and electro-acupuncture+inhibitor pretreatment group(each n=10). The sham-operation group was not given other treatment after opening chest to expose heart, model group was given in vivo myocardial I/R operation, electro-acupuncture retreatment group was given needling in Neiguan(PC6) and Zusanli(ST36) one week before modeling, inhibitor pretreatment group was given injection of SB203580(inhibitor of p38 MAPK signaling pathway) before modeling, and electro-acupuncture+inhibitor pretreatment group was given injection of SB203580 one week after electro-acupuncture retreatment and before modeling. All rats in 5 groups were treated and executed for collecting samples. The indexes related to calcium homeostasis, myocardial oxidative stress and inflammatory cytokines were detected in all groups. Results The difference in Na~+-K~+-ATPase, Mg~(2+)-ATase, Ca~(2+)-ATPase, superoxide dismutase(SOD), malondialdehyde(MDA) and tumor necrosis factor-α(TNF-α) were significant between sham-operation group and model group(P<0.05). There difference in the above mentioned indexes had statistical significance compared among inhibitor pretreatment group and electro-acupuncture+inhibitor pretreatment group. Conclusion The pretreatment of tip-root acupoint combination aims at maintaining cardiomyocyte calcium homeostasis, improving ability of scavenging oxygen free radicals, inhibiting myocardial inflammation, and alleviating significantly damage of heart function due to I/R in diabetic rats with I/R injury.
Objective To observe the influence of tip-root acupoint combination pretreatment on calcium homeostasis, myocardial oxidative stress and inflammatory cytokines in diabetic rats with myocardial ischemia-reperfusion(I/R) injury, analyze the mechanism from the aspect of p38 MAPK signaling pathway and provide a new theoretical discussion and experimental basis for prevention and treatment of cardiovascular diseases in clinic. Methods All rats(n=50) were randomly divided into 5 groups: sham-operation group, model group, electro-acupuncture retreatment group, inhibitor pretreatment group and electro-acupuncture+inhibitor pretreatment group(each n=10). The sham-operation group was not given other treatment after opening chest to expose heart, model group was given in vivo myocardial I/R operation, electro-acupuncture retreatment group was given needling in Neiguan(PC6) and Zusanli(ST36) one week before modeling, inhibitor pretreatment group was given injection of SB203580(inhibitor of p38 MAPK signaling pathway) before modeling, and electro-acupuncture+inhibitor pretreatment group was given injection of SB203580 one week after electro-acupuncture retreatment and before modeling. All rats in 5 groups were treated and executed for collecting samples. The indexes related to calcium homeostasis, myocardial oxidative stress and inflammatory cytokines were detected in all groups. Results The difference in Na~+-K~+-ATPase, Mg~(2+)-ATase, Ca~(2+)-ATPase, superoxide dismutase(SOD), malondialdehyde(MDA) and tumor necrosis factor-α(TNF-α) were significant between sham-operation group and model group(P<0.05). There difference in the above mentioned indexes had statistical significance compared among inhibitor pretreatment group and electro-acupuncture+inhibitor pretreatment group. Conclusion The pretreatment of tip-root acupoint combination aims at maintaining cardiomyocyte calcium homeostasis, improving ability of scavenging oxygen free radicals, inhibiting myocardial inflammation, and alleviating significantly damage of heart function due to I/R in diabetic rats with I/R injury.
引文
[1] 简磊,高明杰,夏旋. 熊果酸减轻糖尿病大鼠心脏缺血再灌注损伤[J]. 天然产物研究与开发,2017,29(6):843-848. Jian L, Gao MJ, Xia X. Ursolic acid attenuates myocardial ischemia reperfusion injury in diabetic rats[J]. Nat Prod Res Dev, 2017, 29 (6): 843-848.
[2] 邱珍,夏中元,周斌,等. 细胞凋亡在糖尿病心肌缺血再灌注损伤的变化及作用机制[J]. 武汉大学学报(医学版),2017,38(1):1-5. Qiu Z, Xia ZY, Zhou B, et al. Changes and mechanisms of pyroptosis in myocardial ischemia-refperfusion in diabetic rats[J]. Medical Journal of Wuhan University, 2017, 38 (1): 1-5.
[3] Zhou GX, Yu L, Fang LL, et al. CD177(+) neutrophils as functionally activated neutrophils negatively regulate IBD[J]. Gut, 2017, 67(6): 1052-1063.
[4] Frangogiannis NG. The inflammatory response in myocardial injury, repair, and remodelling[J]. Nature Reviews Cardiology, 2014, 11(5): 255-265.
[5] Barancík M, Bohacova V, Kvackajova J, et al. SB203580,a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance[J]. European Journal of Pharmaceutical Sciences. 2001, 14(1): 29-36.
[6] Barancík M, Bohácová V, Kvackajová J, et al. SB203580,a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance[J]. European Journal of Pharmaceutical Sciences, 2001, 14(1): 29-36.
[7] 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.Hua XB, Zhou HL. Development of acupoint atlas of rats[J]. Experimental Animals and Animal Experiment, 1991(1): 1-5.
[8] Bhayana V, Henderson AR. Biochemical markers of myocardial damage[J]. Clinical Biochemistry, 1995, 28(1): 1-29.
[9] Fukushima Y, Yamada S,Nakao M. ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+, K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase[J]. J Biochem, 1984, 95(2): 359-368.
[10] 肖燕,顾一煌,陈昊. 针灸对心肌细胞钙超载保护机制的研究进展[J]. 针刺研究,2016,41(3):276-279.Xiao Y, Gu YH, Chen H. Progress of researches on protective effect of acupuncture and moxibustion in relieving intracellular calcium overload of cradiomyocytes[J]. Acupuncture study, 2016, 41(3): 276-279.
[11] Tao XG, Li K, Wang JQ, et al. Tetramethylpyrazine can ameliorate hepatocellular mitochondrial dysfunction by decreasing the inflammatory response and increasing AQP8 protein expression in septic rats[J]. European Journal of Inflammation, 2017, 15(3), 179-191.
[12] Michaelis ML, Kitos TE, Nunley EW, et al. Charateristic of Mg-dependent, ATP-activated Ca+ transport in synaptic and microsomal membranes and in permeabilized synaptosomes[J]. Journal of Biological Chemistry, 1987, 262(9): 4182-4189.
[13] 薛祚臣. 钙增敏剂对心肌钙稳态的影响[J]. 心血管病学进展,2014,35(6):653-656.Xue ZC. Effect of calcium sensitizer on calcium homeostasis in cardiomyocytes[J]. Adv Cardiovasc Dis, 2014, 35(6): 653-656.
[14] 荆娇,马海玲,张永忠,等. 辛伐他汀对大鼠肾缺血再灌注损伤后心肌氧自由基代谢的干预效应[J]. 大医生,2017,2(4):39-43.Jing J, Ma HL, Zhang YZ, et al. Intervention effect of simvastatin on myocardial oxygen free radical metabolism after renal ischemia-reperfusion injury in rats[J]. Doctor, 2017, 2(4):39-43.
[15] 杨冯睿,易汉,汪超,等. miR-208b-3p在右美托咪定减轻大鼠心肌缺血再灌注损伤中的作用研究[J]. 重庆医学,2018,47(2):153-155.Yang FR, Yi H, Wang C, et al. Study on role of miR-208b-3p in dexmedetomidine alleviating rat myocardial ischemia-reperfusion injury[J]. Chongqing Med, 2018, 47(2): 153-155.
[16] Shao ML. Protective effect of acupuncture preconditioning on oxidative stress injury induced by myocardial ischemia-reperfusion injury in rats[J]. China Medical Abstracts(Internal Medicine), 2017, 34(2): 9-10.
[17] 郭子江,高政,尹琳. 高血糖状态下大鼠脑缺血再灌注后不同时限点细胞间黏附分子1及肿瘤坏死因子α表达的差异[J]. 中国临床康复,2005,9(25):99-101.Guo ZJ, Gao Z, Yin L. Different expressions of intercellular adhesion molecule-1 and tumor necrosis factor-alpha under hyperglycemic state at different time limit in rats after cerebral ischemia reperfusion[J]. Chinese Journal of Clinical Rehabilitation, 2005, 9(25): 99-101.
[18] Paola RD, Fusco R, Gugliandolo E, et al. The antioxidant activity of pistachios reduces cardiac tissue injury of acute ischemia/reperfusion (I/R) in diabetic streptozotocin (STZ)-induced hyperglycaemic rats[J]. Frontiers in Pharmacology, 2018, 9(51): 1-17.
[19] 陈松,吴松,韩永丽,等.电针内关预处理对心肌缺血再灌注模型大鼠心功能、心肌损伤标志物和形态学的影响[J]. 时珍国医国药,2017,28(11):2801-2805.Chen S, Wu S, Han YL, et al. Effect of electroacupuncture preconditioning in Neiguan on cardiac function,markers of myocardial injury and morphology in rats with myocardial ischemia-reperfusion[J]. Lishizhen Medicine and Materia Medica Research, 2017, 28 (11): 2801-2805.
[20] 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.Chen S, Han YL, Wu S, et al. Electroacupuncture preconditioning at “Neiguan” prevents myocardial ischemia-reperfusion injury in rats by activating p38-MAPK pathway[J]. Acta Med Univ Sci Technol Huazhog(Medical Edition), 2017, 46(5): 526-530.
[21] 陈松,韩永丽,吴松,等. 电针内关穴预处理对大鼠心肌缺血再灌注后线粒体功能及超微结构的保护作用[J]. 中华中医药杂志,2017,32(10):4651-4654.Chen S, Han YL, Wu S, et al. Effects of electroacupuncture preconditioning at Neiguan (PC6) on the myocardial mitochondrial function in rats with myocardial ischemia-reperfusion injury[J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2017, 32(10): 4651-4654.
[22] 陈松. 电针“内关”预处理对心肌缺血再灌注大鼠p38MAPK信号通路的影响及机制研究[D]. 武汉:湖北中医药大学,2017.Chen S. Effect of electroacupuncture pretreatment at “neiguan” on the p38MAPK signaling pathway in rats with myocardial ischemia-reperfusion injury[D]. Wuhan: Hubei University of Chinese Medicine, 2017.
[23] 陈松,吴松,王华,等. P38信号转导通路与疾病谱的研究进展[J]. 湖北中医药大学学报,2017,19(1):110-113.Chen S, Wu S, Wang H, et al. Research progress of the relationship between P38MAPK signal transduction pathway and disease spectrure[J]. Journal of Hubei University of Chinese Medicine, 2017, 19(1): 110-113.
[24] 李强,徐芬,梁凤霞,等. “标本配穴”电针对胰岛素抵抗大鼠股四头肌线粒体超微结构和生物合成功能的影响[J]. 中国针灸,2014,34(6):578-582.Li Q, Xu F, Liang FX, et al. Effects of electroacupuncture with manifestation-root acupoint combination on ultrastructure and biosynthesis in mitochondrial of quadriceps femoris in rats with insulin resistance[J]. Chinese Acupuncture and Moxibustion, 2014, 34(6): 578-582.
[2] 邱珍,夏中元,周斌,等. 细胞凋亡在糖尿病心肌缺血再灌注损伤的变化及作用机制[J]. 武汉大学学报(医学版),2017,38(1):1-5. Qiu Z, Xia ZY, Zhou B, et al. Changes and mechanisms of pyroptosis in myocardial ischemia-refperfusion in diabetic rats[J]. Medical Journal of Wuhan University, 2017, 38 (1): 1-5.
[3] Zhou GX, Yu L, Fang LL, et al. CD177(+) neutrophils as functionally activated neutrophils negatively regulate IBD[J]. Gut, 2017, 67(6): 1052-1063.
[4] Frangogiannis NG. The inflammatory response in myocardial injury, repair, and remodelling[J]. Nature Reviews Cardiology, 2014, 11(5): 255-265.
[5] Barancík M, Bohacova V, Kvackajova J, et al. SB203580,a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance[J]. European Journal of Pharmaceutical Sciences. 2001, 14(1): 29-36.
[6] Barancík M, Bohácová V, Kvackajová J, et al. SB203580,a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance[J]. European Journal of Pharmaceutical Sciences, 2001, 14(1): 29-36.
[7] 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.Hua XB, Zhou HL. Development of acupoint atlas of rats[J]. Experimental Animals and Animal Experiment, 1991(1): 1-5.
[8] Bhayana V, Henderson AR. Biochemical markers of myocardial damage[J]. Clinical Biochemistry, 1995, 28(1): 1-29.
[9] Fukushima Y, Yamada S,Nakao M. ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+, K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase[J]. J Biochem, 1984, 95(2): 359-368.
[10] 肖燕,顾一煌,陈昊. 针灸对心肌细胞钙超载保护机制的研究进展[J]. 针刺研究,2016,41(3):276-279.Xiao Y, Gu YH, Chen H. Progress of researches on protective effect of acupuncture and moxibustion in relieving intracellular calcium overload of cradiomyocytes[J]. Acupuncture study, 2016, 41(3): 276-279.
[11] Tao XG, Li K, Wang JQ, et al. Tetramethylpyrazine can ameliorate hepatocellular mitochondrial dysfunction by decreasing the inflammatory response and increasing AQP8 protein expression in septic rats[J]. European Journal of Inflammation, 2017, 15(3), 179-191.
[12] Michaelis ML, Kitos TE, Nunley EW, et al. Charateristic of Mg-dependent, ATP-activated Ca+ transport in synaptic and microsomal membranes and in permeabilized synaptosomes[J]. Journal of Biological Chemistry, 1987, 262(9): 4182-4189.
[13] 薛祚臣. 钙增敏剂对心肌钙稳态的影响[J]. 心血管病学进展,2014,35(6):653-656.Xue ZC. Effect of calcium sensitizer on calcium homeostasis in cardiomyocytes[J]. Adv Cardiovasc Dis, 2014, 35(6): 653-656.
[14] 荆娇,马海玲,张永忠,等. 辛伐他汀对大鼠肾缺血再灌注损伤后心肌氧自由基代谢的干预效应[J]. 大医生,2017,2(4):39-43.Jing J, Ma HL, Zhang YZ, et al. Intervention effect of simvastatin on myocardial oxygen free radical metabolism after renal ischemia-reperfusion injury in rats[J]. Doctor, 2017, 2(4):39-43.
[15] 杨冯睿,易汉,汪超,等. miR-208b-3p在右美托咪定减轻大鼠心肌缺血再灌注损伤中的作用研究[J]. 重庆医学,2018,47(2):153-155.Yang FR, Yi H, Wang C, et al. Study on role of miR-208b-3p in dexmedetomidine alleviating rat myocardial ischemia-reperfusion injury[J]. Chongqing Med, 2018, 47(2): 153-155.
[16] Shao ML. Protective effect of acupuncture preconditioning on oxidative stress injury induced by myocardial ischemia-reperfusion injury in rats[J]. China Medical Abstracts(Internal Medicine), 2017, 34(2): 9-10.
[17] 郭子江,高政,尹琳. 高血糖状态下大鼠脑缺血再灌注后不同时限点细胞间黏附分子1及肿瘤坏死因子α表达的差异[J]. 中国临床康复,2005,9(25):99-101.Guo ZJ, Gao Z, Yin L. Different expressions of intercellular adhesion molecule-1 and tumor necrosis factor-alpha under hyperglycemic state at different time limit in rats after cerebral ischemia reperfusion[J]. Chinese Journal of Clinical Rehabilitation, 2005, 9(25): 99-101.
[18] Paola RD, Fusco R, Gugliandolo E, et al. The antioxidant activity of pistachios reduces cardiac tissue injury of acute ischemia/reperfusion (I/R) in diabetic streptozotocin (STZ)-induced hyperglycaemic rats[J]. Frontiers in Pharmacology, 2018, 9(51): 1-17.
[19] 陈松,吴松,韩永丽,等.电针内关预处理对心肌缺血再灌注模型大鼠心功能、心肌损伤标志物和形态学的影响[J]. 时珍国医国药,2017,28(11):2801-2805.Chen S, Wu S, Han YL, et al. Effect of electroacupuncture preconditioning in Neiguan on cardiac function,markers of myocardial injury and morphology in rats with myocardial ischemia-reperfusion[J]. Lishizhen Medicine and Materia Medica Research, 2017, 28 (11): 2801-2805.
[20] 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.Chen S, Han YL, Wu S, et al. Electroacupuncture preconditioning at “Neiguan” prevents myocardial ischemia-reperfusion injury in rats by activating p38-MAPK pathway[J]. Acta Med Univ Sci Technol Huazhog(Medical Edition), 2017, 46(5): 526-530.
[21] 陈松,韩永丽,吴松,等. 电针内关穴预处理对大鼠心肌缺血再灌注后线粒体功能及超微结构的保护作用[J]. 中华中医药杂志,2017,32(10):4651-4654.Chen S, Han YL, Wu S, et al. Effects of electroacupuncture preconditioning at Neiguan (PC6) on the myocardial mitochondrial function in rats with myocardial ischemia-reperfusion injury[J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2017, 32(10): 4651-4654.
[22] 陈松. 电针“内关”预处理对心肌缺血再灌注大鼠p38MAPK信号通路的影响及机制研究[D]. 武汉:湖北中医药大学,2017.Chen S. Effect of electroacupuncture pretreatment at “neiguan” on the p38MAPK signaling pathway in rats with myocardial ischemia-reperfusion injury[D]. Wuhan: Hubei University of Chinese Medicine, 2017.
[23] 陈松,吴松,王华,等. P38信号转导通路与疾病谱的研究进展[J]. 湖北中医药大学学报,2017,19(1):110-113.Chen S, Wu S, Wang H, et al. Research progress of the relationship between P38MAPK signal transduction pathway and disease spectrure[J]. Journal of Hubei University of Chinese Medicine, 2017, 19(1): 110-113.
[24] 李强,徐芬,梁凤霞,等. “标本配穴”电针对胰岛素抵抗大鼠股四头肌线粒体超微结构和生物合成功能的影响[J]. 中国针灸,2014,34(6):578-582.Li Q, Xu F, Liang FX, et al. Effects of electroacupuncture with manifestation-root acupoint combination on ultrastructure and biosynthesis in mitochondrial of quadriceps femoris in rats with insulin resistance[J]. Chinese Acupuncture and Moxibustion, 2014, 34(6): 578-582.
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