电针“足三里”穴对脾气虚大鼠心肌PGC-1α/NRFs/TFAM信号通路表达的影响
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摘要
目的:观察电针"足三里"穴对脾气虚模型大鼠心肌PGC-1α/NRFs/TFAM信号通路相关分子表达的影响。方法:将32只SD雄性大鼠随机分为正常组、模型组、针刺组、非经非穴组。除正常组外,其余3组均采用劳倦过度和不规则饮食复合法建立脾气虚证大鼠模型。造模成功后,针刺组给予电针双侧"足三里"穴治疗,非经非穴组予以电针双侧非经非穴点治疗,每日1次,每次20 min,共治疗7 d。采用荧光定量PCR法检测大鼠心肌组织PGC-1α、NRF1、NRF2、TFAM的mRNA表达水平。采用Western Blot检测大鼠心肌组织PGC-1α及NRF1的蛋白表达水平。结果:与正常组相比,模型组和非经非穴组大鼠心肌组织PGC-1α、NRF1、NRF2、TFAM的mRNA表达以及PGC-1α和NRF1的蛋白表达水平均明显降低(P<0.05);与模型组相比,针刺组大鼠心肌组织PGC-1α、NRF1、NRF2、TFAM的mRNA表达以及PGC-1α和NRF1的蛋白表达水平有所升高(P<0.05),非经非穴组未见显著性差异(P>0.5)。结论:电针"足三里"穴可以上调脾气虚大鼠心肌组织PGC-1α、NRF1、NRF2、TFAM基因以及PGC-1α和NRF1蛋白的表达水平,可以通过促进线粒体生物合成,提高线粒体呼吸功能,进而调控能量代谢而发挥健脾益气作用。
Objecte: To observe the effect of electroacupuncture oat Zusanli(ST36) on expression of PGC-1 alpha/NRFs/TFAM signaling pathway in myocardium of rats with spleen Qi deficiency. Methods: Thirty-two male SD rats were randomly devided into 4 groups:normal group, model of spleen Qi deficiencygroup, acupuncture group and sham acupuncture group(n=8ineachgroup). Except the normal group, the other 3 groups were established the model of spleen deficiency rats by the complex factors of improper diet and overstrain.Electroacupuncture(2Hz/15 Hz, 0.5mA) was applied to bilateral Zusanli(ST36) in acupuncture group and sham acupoints in sham acupuncture group for 20 min, once a day for 7 days. The level of mRNA expression of PGC-1 alpha, NRF1, NRF2 and TFAM in rat myocardium was detected by fluorescence quantitative PCR.Western blot was used to detect the protein expression levels of PGC-1 alpha and NRF1 in rat myocardium. Results: Compared with the control group, PGC-1 alpha, NRF1, NRF2 and TFAM mRNA, PGC-1 alpha and NRF1 protein expressiondecreased obviously in rats myocardial tissue compared with those of model group and sham acupuncture group(P<0.05). Compared with the model group, acupuncture group's PGC-1 alpha, NRF1, NRF2 and TFAM mRNA, PGC-1 alpha and NRF1 protein expression increased significantly in rat myocardial tissue(P<0.05) and there was no obviously changed increase in gene and protein of rats in the sham acupuncture group(P>0.05).Conclusion:Electroacupuncture at Zusanli(ST36) may increase PGC-1 alpha, NRF1, NRF2 and TFAM mRNA, PGC-1 alpha and NRF1 protein expression in the myocardium of rats with spleen Qi deficiency and promote the mitochondrial biosynthesis, improve the respiratory function of mitochondria, and then regulate the energy metabolism, so as to play the role of invigorating spleen and replenishing Qi.
Objecte: To observe the effect of electroacupuncture oat Zusanli(ST36) on expression of PGC-1 alpha/NRFs/TFAM signaling pathway in myocardium of rats with spleen Qi deficiency. Methods: Thirty-two male SD rats were randomly devided into 4 groups:normal group, model of spleen Qi deficiencygroup, acupuncture group and sham acupuncture group(n=8ineachgroup). Except the normal group, the other 3 groups were established the model of spleen deficiency rats by the complex factors of improper diet and overstrain.Electroacupuncture(2Hz/15 Hz, 0.5mA) was applied to bilateral Zusanli(ST36) in acupuncture group and sham acupoints in sham acupuncture group for 20 min, once a day for 7 days. The level of mRNA expression of PGC-1 alpha, NRF1, NRF2 and TFAM in rat myocardium was detected by fluorescence quantitative PCR.Western blot was used to detect the protein expression levels of PGC-1 alpha and NRF1 in rat myocardium. Results: Compared with the control group, PGC-1 alpha, NRF1, NRF2 and TFAM mRNA, PGC-1 alpha and NRF1 protein expressiondecreased obviously in rats myocardial tissue compared with those of model group and sham acupuncture group(P<0.05). Compared with the model group, acupuncture group's PGC-1 alpha, NRF1, NRF2 and TFAM mRNA, PGC-1 alpha and NRF1 protein expression increased significantly in rat myocardial tissue(P<0.05) and there was no obviously changed increase in gene and protein of rats in the sham acupuncture group(P>0.05).Conclusion:Electroacupuncture at Zusanli(ST36) may increase PGC-1 alpha, NRF1, NRF2 and TFAM mRNA, PGC-1 alpha and NRF1 protein expression in the myocardium of rats with spleen Qi deficiency and promote the mitochondrial biosynthesis, improve the respiratory function of mitochondria, and then regulate the energy metabolism, so as to play the role of invigorating spleen and replenishing Qi.
引文
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[5] 郭胜男,丛培玮,王艳杰,等.脾气虚模型大鼠心脏组织葡萄糖转运蛋白-1和水通道蛋白-1表达水平[J].中国老年学杂志,2018,38(7):1686-1688.
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[8] 谢俊,陈泽斌,吴松,等.标本配穴法电针对慢性心肌缺血大鼠心肌线粒体结构及线粒体DNA相关调控因子表达的影响[J].中华中医药杂志,2017,32(3):1251-1255.
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[14] 张月,张国山,刘雨儿,等.电针足三里对功能性消化不良大鼠胃电节律的影响[J].世界华人消化杂志,2014,22(26):3953-3957.
[15] 上官豪,柳维林,陈文列,等.电针曲池、足三里穴对缺血再灌注损伤大鼠线粒体Caspase-3途径诱导细胞凋亡的影响[J].中国康复理论与实践,2015,21(8):900-904.
[16] 高巍,黄裕新,陈洪,等.电针“足三里”对大鼠脑肠肽含量的影响及其对免疫系统的调控作用[J].针刺研究,2002,27(1):50-55.
[17] 勇入琳,曲怡,李欣欣,等.电针“足三里”对脾气虚大鼠空肠组织胃生长激素释放激素/环磷酸腺苷/蛋白激酶A表达的影响[J].针刺研究,2016,41(6):497-501.
[18] 薛亚楠,曲怡,王建波,等.电针双侧“足三里”穴对脾气虚模型大鼠小肠及下丘脑组织胃促生长素和血管活性肠肽表达影响的研究[J].中华中医药学刊,2017,35(8):2081-2084.
[19] 冯晓帆,孙延娜,张立德,等.补脾方药对脾虚大鼠心肌细胞ATP 酶活性和Ca2 +浓度的影响[J].辽宁中医杂志,2011,38(6):1223-1224.
[20] 李思琦,张哲,杨关林,等.益气健脾方对脾气虚证模型大鼠心肌细胞线粒体呼吸链酶复合物活性的影响[J].中国医科大学学报,2017,46(6):515-518.
[21] 程岩岩,贾连群,宋囡,等.基于PCRarray技术探讨脾虚痰浊AS对心肌线粒体能量代谢通路相关基因mRNA表达的影响[J].辽宁中医杂志,2016,43(5):8,1069-1071.
[22] 刘友章,王昌俊,周俊亮,等.长期脾虚模型大鼠细胞线粒体的研究[J].中医药学刊,2006,24(3):391-394.
[23] 程岩岩. 健脾祛痰化瘀法对脾虚痰浊AS巴马小型猪心肌线粒体作用及机制研究[D].沈阳:辽宁中医药大学,2016.
[24] Rowe GC,Jiang A,Arany Z.PGC-1 coactivators in cardiac delelopmentand disease[J]. Circ Res,2010,107(7): 825-838.
[25] 曹琼丹,杨育红,于胜男,等.黄芪甲苷对Ⅰ型糖尿病大鼠心肌细胞PGC-1α和NRF-1表达的影响[J].中国药理学通报,2015,31(8):1096-1101.
[26] 陈锐,刘友生.核呼吸因子研究进展[J].国外医学(生理、病理科学与临床分册),2005(1):59-61.
[27] Kelly DP, Scarpulla RC. Transcriptional regulatory circuits controllingmitochondrial biogenesis and function [J]. Genes Dev, 2004, 18(4):357-368.
[28] Brennan K, Smith Kazutaka, Mukai James S, et al. AMP-activated protein kinase isrequired for exercise-induced peroxisome proliferator-activated receptor co-activator 1 translocation to subsarcolemmal mitochondria in skeletal muscle[J]. The Journal of physiology, 2013,591(Pt6): 1551-1561.
[2] 汪永锋,杨丽霞,康开彪,等.基于现代医学理论剖析中医脾的解剖位置及脏腑功能[J].中医研究,2016,29(2):1-3.
[3] 姜晓琳.脾气虚证模型大鼠心功能变化及其机制的研究[D].沈阳:辽宁中医药大学,2016.
[4] 姜晓琳,张立德.脾气虚证模型大鼠中BNP介导的cAMP-PKA信号通路对bFGF表达的影响[J].中国病理生理杂志,2016,32(2):251-255.
[5] 郭胜男,丛培玮,王艳杰,等.脾气虚模型大鼠心脏组织葡萄糖转运蛋白-1和水通道蛋白-1表达水平[J].中国老年学杂志,2018,38(7):1686-1688.
[6] Hood DA,Irrcher I,Ljubicic V,et al.Coordination ofmetabolic plasticity in skeletalmuscle[J].JExp Biol,2006,209:2265-2275.
[7] 赵沐霖. 运动预适应对力竭大鼠心肌线粒体生物发生及呼吸功能的影响[D].石家庄:河北大学,2017.
[8] 谢俊,陈泽斌,吴松,等.标本配穴法电针对慢性心肌缺血大鼠心肌线粒体结构及线粒体DNA相关调控因子表达的影响[J].中华中医药杂志,2017,32(3):1251-1255.
[9] 郑小伟.中医实验动物模型方法学[M]. 上海:上海中医药大学出版社,1999:63-111.
[10] 李忠仁.实验针灸学[M]. 北京:中国中医药出版社,2003:327.
[11] 林莺,纪峰,黄桂榕,等.针刺实验研究中大鼠非经非穴选取方法及思考[J].针刺研究,2013(4): 334-338.
[12] 邢俊标.足三里穴研究进展[J].现代中西医结合杂志,2007(34):5226-5228.
[13] 公维军,张通,崔利华,等.电针足三里改善脑卒中偏瘫痉挛期患者下肢运动功能的临床研究[J].中国康复理论与实践,2008,14(11):1057-1058.
[14] 张月,张国山,刘雨儿,等.电针足三里对功能性消化不良大鼠胃电节律的影响[J].世界华人消化杂志,2014,22(26):3953-3957.
[15] 上官豪,柳维林,陈文列,等.电针曲池、足三里穴对缺血再灌注损伤大鼠线粒体Caspase-3途径诱导细胞凋亡的影响[J].中国康复理论与实践,2015,21(8):900-904.
[16] 高巍,黄裕新,陈洪,等.电针“足三里”对大鼠脑肠肽含量的影响及其对免疫系统的调控作用[J].针刺研究,2002,27(1):50-55.
[17] 勇入琳,曲怡,李欣欣,等.电针“足三里”对脾气虚大鼠空肠组织胃生长激素释放激素/环磷酸腺苷/蛋白激酶A表达的影响[J].针刺研究,2016,41(6):497-501.
[18] 薛亚楠,曲怡,王建波,等.电针双侧“足三里”穴对脾气虚模型大鼠小肠及下丘脑组织胃促生长素和血管活性肠肽表达影响的研究[J].中华中医药学刊,2017,35(8):2081-2084.
[19] 冯晓帆,孙延娜,张立德,等.补脾方药对脾虚大鼠心肌细胞ATP 酶活性和Ca2 +浓度的影响[J].辽宁中医杂志,2011,38(6):1223-1224.
[20] 李思琦,张哲,杨关林,等.益气健脾方对脾气虚证模型大鼠心肌细胞线粒体呼吸链酶复合物活性的影响[J].中国医科大学学报,2017,46(6):515-518.
[21] 程岩岩,贾连群,宋囡,等.基于PCRarray技术探讨脾虚痰浊AS对心肌线粒体能量代谢通路相关基因mRNA表达的影响[J].辽宁中医杂志,2016,43(5):8,1069-1071.
[22] 刘友章,王昌俊,周俊亮,等.长期脾虚模型大鼠细胞线粒体的研究[J].中医药学刊,2006,24(3):391-394.
[23] 程岩岩. 健脾祛痰化瘀法对脾虚痰浊AS巴马小型猪心肌线粒体作用及机制研究[D].沈阳:辽宁中医药大学,2016.
[24] Rowe GC,Jiang A,Arany Z.PGC-1 coactivators in cardiac delelopmentand disease[J]. Circ Res,2010,107(7): 825-838.
[25] 曹琼丹,杨育红,于胜男,等.黄芪甲苷对Ⅰ型糖尿病大鼠心肌细胞PGC-1α和NRF-1表达的影响[J].中国药理学通报,2015,31(8):1096-1101.
[26] 陈锐,刘友生.核呼吸因子研究进展[J].国外医学(生理、病理科学与临床分册),2005(1):59-61.
[27] Kelly DP, Scarpulla RC. Transcriptional regulatory circuits controllingmitochondrial biogenesis and function [J]. Genes Dev, 2004, 18(4):357-368.
[28] Brennan K, Smith Kazutaka, Mukai James S, et al. AMP-activated protein kinase isrequired for exercise-induced peroxisome proliferator-activated receptor co-activator 1 translocation to subsarcolemmal mitochondria in skeletal muscle[J]. The Journal of physiology, 2013,591(Pt6): 1551-1561.