艾灸对慢性心力衰竭大鼠心肌细胞自噬功能的影响
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摘要
目的:观察艾灸对慢性心力衰竭(CHF)大鼠心功能及自噬相关蛋白LC3、p62表达的影响,探讨艾灸防治CHF的可能机制。方法:SD大鼠随机分为正常组12只与造模组48只,采用腹腔注射盐酸多柔比星复制CHF模型,将建模成功的大鼠随机均分为模型组、艾灸组、自噬抑制剂3-甲基腺嘌呤(3-MA)组、自噬激动剂雷帕霉素(RAPA)组。艾灸组用艾条温和灸双侧"肺俞""心俞"穴,每日1次,每次20min,治疗3周。3-MA组按15 mg/kg给予3-MA混悬液腹腔注射,每日1次,共3周。RAPA组按2mg/kg给予RAPA混悬液灌胃,每日1次,共3周。观察大鼠的一般情况及行为体征;心功能测量仪检测大鼠心率(HR)、心输出量(CO)、左心室收缩压(LVSP)、左心室舒张末压(LVEDP)、左室内压最大上升或下降速率(±dp/dtmax);HE染色法观察心肌病理变化;Western blot法检测心肌细胞自噬相关蛋白LC3及p62表达。结果:与正常组比较,模型组大鼠心肌细胞出现肿胀、空泡,肌纤维断裂、溶解,排列紊乱;与模型组比较,艾灸组和3-MA组大鼠心肌细胞的数量正常,心肌纤维排列相对整齐,RAPA组心肌细胞出现肿胀、空泡,心肌纤维断裂、溶解,排列紊乱等状况。与正常组比较,模型组HR和LVEDP明显升高(P<0.01),CO、LVSP及±dp/dtmax明显下降(P<0.01),LC3-Ⅱ表达、LC3-Ⅱ/Ⅰ明显升高(P<0.01),而p62表达明显下降(P<0.01)。与模型组比较,艾灸组和3-MA组HR和LVEDP明显下降(P<0.05),CO、LVSP及±dp/dtmax明显升高(P<0.05),LC3-Ⅱ表达、LC3-Ⅱ/Ⅰ明显降低(P<0.01),p62表达明显升高(P<0.01),而RAPA组LC3-Ⅱ/Ⅰ明显升高(P<0.01),p62表达明显降低(P<0.01)。结论:艾灸可以改善CHF大鼠心肌损伤,其作用可能与降低心肌细胞LC3-Ⅱ/Ⅰ、升高p62蛋白表达水平,发挥类似于3-MA的自噬抑制作用,抑制心肌细胞过度自噬、减轻心肌损伤有关。
Objective To observe the effect of moxibustion on cardiac function and the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3(LC3)and selective autophagy receptor signaling adaptor sequestosome 1(SQSTM1/p62)in rats with chronic heart failure(CHF),so as to explore its underlying mechanisms in preventing and treating CHF.Methods Sixty male SD rats were randomly divided into normal,model,moxibustion,autophagy inhibitor 3-methyladenine(3-MA)and autophagy agonist rapamycin(RAPA)groups(n=12 rats/group).The CHF model was established by intraperitoneal injection of adriamycin(ADR,2 mg/kg,once every week for 12 weeks).Mild moxibustion was applied to bilateral"Feishu"(BL13)and"Xinshu"(BL15)for 20 min every time.Rats of the 3-MA group were treated by intraperitoneal injection of 3-MA suspension(15 mg/kg),and those of the RAPA group treated by gavage of RAPA suspension(2 mg/kg).All the treatments were given once a day for 3 weeks.The heart rate(HR),cardiac output(CO),left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure(LVEDP)and maximum rising and lowering rates of left ventricular pressure(±dp/dtmax)were measured for assessing the cardiac performance.Histopathological changes of the left ventricular myocardium were observed by HE staining.The expression levels of LC3-Ⅰ,LC3-Ⅱ and p62 proteins of the left ventricle myocardium tissue were detected by Western blot.Results After modeling,the pathological changes of myocardium(as myocardial cell swelling with vacuoles,myocardial fibre breakage,etc.)were obvious,and the HR,LVEDP,LC3-Ⅱand LC3-Ⅱ/Ⅰ protein expression levels were significantly increased in the model group compared with the normal group(P<0.01),while the CO,LVSP,±dp/dtmax,and the expression of p62 protein were significantly down-regulated(P<0.01).Following the interventions,the myocardial injury was reduced,the HR,LVEDP,LC3-Ⅱ and LC3-Ⅱ/Ⅰ levels in both moxibustion and 3-MA groups were significantly decreased(P<0.05,P<0.01),while the CO,LVSP,±dp/dtmax and p62 expression level were significantly increased relevant to the model group(P<0.05,P<0.01).In addition,the ratio of LC3-Ⅱ/Ⅰ was significantly increased,and the expression level of p62 significantly down-regulated in the RAPA group compared with the model group(P<0.01).Conclusion Moxibustion can improve cardiac function in CHF rats,which may be related to its effects in down-regulating the ratio of LC3-Ⅱ/Ⅰ and up-regulating the expression of p62 protein to inhibit cardiomyocyte autophagy.
Objective To observe the effect of moxibustion on cardiac function and the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3(LC3)and selective autophagy receptor signaling adaptor sequestosome 1(SQSTM1/p62)in rats with chronic heart failure(CHF),so as to explore its underlying mechanisms in preventing and treating CHF.Methods Sixty male SD rats were randomly divided into normal,model,moxibustion,autophagy inhibitor 3-methyladenine(3-MA)and autophagy agonist rapamycin(RAPA)groups(n=12 rats/group).The CHF model was established by intraperitoneal injection of adriamycin(ADR,2 mg/kg,once every week for 12 weeks).Mild moxibustion was applied to bilateral"Feishu"(BL13)and"Xinshu"(BL15)for 20 min every time.Rats of the 3-MA group were treated by intraperitoneal injection of 3-MA suspension(15 mg/kg),and those of the RAPA group treated by gavage of RAPA suspension(2 mg/kg).All the treatments were given once a day for 3 weeks.The heart rate(HR),cardiac output(CO),left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure(LVEDP)and maximum rising and lowering rates of left ventricular pressure(±dp/dtmax)were measured for assessing the cardiac performance.Histopathological changes of the left ventricular myocardium were observed by HE staining.The expression levels of LC3-Ⅰ,LC3-Ⅱ and p62 proteins of the left ventricle myocardium tissue were detected by Western blot.Results After modeling,the pathological changes of myocardium(as myocardial cell swelling with vacuoles,myocardial fibre breakage,etc.)were obvious,and the HR,LVEDP,LC3-Ⅱand LC3-Ⅱ/Ⅰ protein expression levels were significantly increased in the model group compared with the normal group(P<0.01),while the CO,LVSP,±dp/dtmax,and the expression of p62 protein were significantly down-regulated(P<0.01).Following the interventions,the myocardial injury was reduced,the HR,LVEDP,LC3-Ⅱ and LC3-Ⅱ/Ⅰ levels in both moxibustion and 3-MA groups were significantly decreased(P<0.05,P<0.01),while the CO,LVSP,±dp/dtmax and p62 expression level were significantly increased relevant to the model group(P<0.05,P<0.01).In addition,the ratio of LC3-Ⅱ/Ⅰ was significantly increased,and the expression level of p62 significantly down-regulated in the RAPA group compared with the model group(P<0.01).Conclusion Moxibustion can improve cardiac function in CHF rats,which may be related to its effects in down-regulating the ratio of LC3-Ⅱ/Ⅰ and up-regulating the expression of p62 protein to inhibit cardiomyocyte autophagy.
引文
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[8]续龙,井蕾,贺琨,等.针刺配合艾灸治疗膝关节骨性关节炎:随机对照研究[J].中国针灸,2013,33(10):871-876.
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[10]王茎,曾永蕾,武凤琴,等.艾灸“肺俞”“心俞”对慢性心力衰竭大鼠心肌组织髓样分化因子、半胱氨酸天冬氨基酸特异性蛋白酶-3表达水平的影响[J].针刺研究,2016,41(5):429-434.
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[14]徐攀,徐海顺,陈京,等.慢性心力衰竭心阳虚证大鼠模型的建立与评价研究[J].中华中医药学刊,2016,34(8):1957-1960.
[15]赵美,臧伟进.巨自噬在心脏疾病调节中的双向作用[J].生理科学进展,2011,42(2):81-85.
[16]MENGHINI R,CASAGRANDE V,MARINO A,et al.MiR-216a:a link between endothelial dysfunction and autophagy[J].Cell Death Dis,2014,5(1):1029-1038.
[17]吕丹丹.细胞自噬与肿瘤的关系[J].世界临床医学,2015,9(2):66-68.
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[19]朱少飞,唐群新.自噬在雷帕霉素逆转脊髓吗啡耐受形成机制研究[J].实用药物与临床,2015,7(18):772-776.
[20]WAN G,XIE W,LIU Z,et al.Hypoxia induced MiR-155is apotent autophagy inducer by targeting multiple players in the MTOR pathway[J].Autophagy,2014,10(1):70-79.
[21]QI L,ZHANG Y.The micro RNA 132regulates fluid shear stress induced differentiation in periodontal ligament cells through m TOR signaling pathway[J].Cell Physiol Biochem,2014,33(2):433-445.
[22]刘晓,秦海红,徐金华.自噬与系统性红斑狼疮发病机制的研究进展[J].国际皮肤性病学杂志,2017,43(3):146-149.
[23]武婧,仇锦波.细胞自噬及其与肿瘤关系的研究[J].世界最新医学信息文摘,2016,18(16):74-75.
[24]郝刚,孙天胜,李绍光.哺乳动物自噬检测技术的研究进展[J].中华临床医师杂志,2012,6(6):1531-1533.
[25]袁召,姚淮芳,张叶祥.中医药治疗慢性心力衰竭研究进展[J].中西医结合心脑血管病杂志,2016,10(14):1110-1112.
[26]王茎,曾永蕾,江莉,等.艾灸对慢性心衰模型大鼠心功能及ET的影响[J].中医药临床杂志,2012,24(4):326-328.
[27]贾学昭,李丹,王茎,等.艾灸对慢性心力衰竭大鼠心功能及心肌组织TNF-α、NF-κB表达水平的影响[J].中国中西医结合杂志,2017,37(10):1220-1225.
[2]林超,刘兆国,钱星,等.自噬在心血管疾病中的作用研究进展[J].中国药理学通报,2014,30(10):1347-1349.
[3]杨方,何曦,姚雪,等.自噬在卵巢癌发生发展及化疗耐药中的作用[J].中国现代医学杂志,2014,24(34):45-48.
[4]陈向华,何海明.艾灸“肺俞”“肾俞”对哮喘大鼠外周血T细胞和血清白介素的影响[J].针刺研究,2017,42(2):159-162.
[5]LIANG Y L,XU X K,ZHOU X H,et al.Study on the effect of moxibustion at“Shénquè”on the immune system of rats taking long-term exhaustive exercise[J].World J AcuMoxi,2017,27(1):43-47.
[6]冯菊凤,张伶俐,张倪雁.硫酸镁湿敷联合艾灸对心血管疾病合并急性痛风治疗效果的影响[J].护理实践与研究,2017,14(2):54-56.
[7]杨锦湄.艾灸治疗老年慢性心力衰竭的护理体会[J].实用中西医结合临床,2014,14(7):80-81.
[8]续龙,井蕾,贺琨,等.针刺配合艾灸治疗膝关节骨性关节炎:随机对照研究[J].中国针灸,2013,33(10):871-876.
[9]王茎,曾永蕾,武凤琴,等.艾灸肺俞和心俞对慢性心力衰竭大鼠心室质量指数及心肌组织TNF-α、IL-6mRNA表达水平的影响[J].安徽中医药大学学报,2014,33(4):48-50.
[10]王茎,曾永蕾,武凤琴,等.艾灸“肺俞”“心俞”对慢性心力衰竭大鼠心肌组织髓样分化因子、半胱氨酸天冬氨基酸特异性蛋白酶-3表达水平的影响[J].针刺研究,2016,41(5):429-434.
[11]KARL-HEINZ D,ROBIN H,MORTON D,et al.A good practice guide to the administration of substances and removal of blood,including routes and volumes[J].J Appl Toxicol,2001,21:15-23.
[12]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:327-329.
[13]刘海涛,张立德,刘玉丽.大鼠“肺俞”“心俞”穴定位研究[J].山西中医,2016,32(2):38-40.
[14]徐攀,徐海顺,陈京,等.慢性心力衰竭心阳虚证大鼠模型的建立与评价研究[J].中华中医药学刊,2016,34(8):1957-1960.
[15]赵美,臧伟进.巨自噬在心脏疾病调节中的双向作用[J].生理科学进展,2011,42(2):81-85.
[16]MENGHINI R,CASAGRANDE V,MARINO A,et al.MiR-216a:a link between endothelial dysfunction and autophagy[J].Cell Death Dis,2014,5(1):1029-1038.
[17]吕丹丹.细胞自噬与肿瘤的关系[J].世界临床医学,2015,9(2):66-68.
[18]李皓.异丙酚后处理对抗心肌缺血的促生存效应机制研究[D].上海:第二军医大学,2011.
[19]朱少飞,唐群新.自噬在雷帕霉素逆转脊髓吗啡耐受形成机制研究[J].实用药物与临床,2015,7(18):772-776.
[20]WAN G,XIE W,LIU Z,et al.Hypoxia induced MiR-155is apotent autophagy inducer by targeting multiple players in the MTOR pathway[J].Autophagy,2014,10(1):70-79.
[21]QI L,ZHANG Y.The micro RNA 132regulates fluid shear stress induced differentiation in periodontal ligament cells through m TOR signaling pathway[J].Cell Physiol Biochem,2014,33(2):433-445.
[22]刘晓,秦海红,徐金华.自噬与系统性红斑狼疮发病机制的研究进展[J].国际皮肤性病学杂志,2017,43(3):146-149.
[23]武婧,仇锦波.细胞自噬及其与肿瘤关系的研究[J].世界最新医学信息文摘,2016,18(16):74-75.
[24]郝刚,孙天胜,李绍光.哺乳动物自噬检测技术的研究进展[J].中华临床医师杂志,2012,6(6):1531-1533.
[25]袁召,姚淮芳,张叶祥.中医药治疗慢性心力衰竭研究进展[J].中西医结合心脑血管病杂志,2016,10(14):1110-1112.
[26]王茎,曾永蕾,江莉,等.艾灸对慢性心衰模型大鼠心功能及ET的影响[J].中医药临床杂志,2012,24(4):326-328.
[27]贾学昭,李丹,王茎,等.艾灸对慢性心力衰竭大鼠心功能及心肌组织TNF-α、NF-κB表达水平的影响[J].中国中西医结合杂志,2017,37(10):1220-1225.