电针“足三里”穴对脾气虚大鼠肌肉组织内NRF1及NRF2表达的影响
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摘要
目的:观察电针双侧"足三里"穴对脾气虚模型大鼠肌肉组织内核呼吸因子1(NRF1)和2(NRF2)基因及蛋白表达的影响。方法:将32只SD雄性大鼠随机分为正常组、脾气虚组、足三里组、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,对足三里组、非经非穴组大鼠分别电针"足三里"穴、非经非穴点干预处理7 d。采用蛋白质免疫印迹法(WB)检测大鼠胃及骨骼肌组织内NRF1及NRF2蛋白含量变化,荧光定量PCR法检测大鼠胃及骨骼肌组织NRF1及NRF2 mRNA表达水平。结果:脾气虚组和非经非穴组大鼠胃及骨骼肌组织内NRF1及NRF2蛋白和基因表达水平均明显低于正常组(P<0.01);足三里组大鼠胃及骨骼肌组织内NRF1及NRF2蛋白和基因表达水平相比于脾气虚组有所升高(P<0.01),非经非穴组未见显著性差异(P<0.01)。结论:电针"足三里"穴可以调节脾气虚大鼠肌肉组织内NRF1及NRF2基因及蛋白的异常表达,参与线粒体能量代谢的调控作用进而改善脾气虚证。
Objective: To observe the effect of electroacupuncture at bilateral "Zusanli(ST36)" on the expressions of nuclear respiratory factor 1(NRF1) and 2(NRF2) gene and protein in muscle tissue of rats with spleen Qi deficiency model. Methods: Thirty-two male SD rats were randomly divided into normal group, spleen Qi deficiency group, Zusanli(ST36) group and non-acupoint group, and the model of spleen Qi deficiency syndrome was established by the composite methods of improper diet and overstrain except the normal group. The rats were treated with electroacupuncture at the bilateral "Zusanli(ST36)" and non-acupoint intervention seven days after the model was successfully established. The changes of NRF1 and NRF2 mRNA and protein expression in skeletal muscle and gastric smooth muscle were detected by fluorescence quantitative PCR method and Western blot(WB). Results: The expressions of NRF1 and NRF2 protein and mRNA in skeletal muscle and gastric smooth muscle of spleen Qi deficiency group were significantly lower than those of normal group, and the level of its' expression had increased in Zusanli(ST36) group, there was no significant difference between spleen Qi deficiency group and non-acupoint group(P<0.01). Conclusion: Electroacupuncture at "Zusanli(ST36)" can improve spleen Qi deficiency by participating in the regulation of mitochondrial energy metabolism through regulating the abnormal expressions of NRF1 and NRF2 genes and proteins in muscle tissue of rats with spleen Qi deficiency.
Objective: To observe the effect of electroacupuncture at bilateral "Zusanli(ST36)" on the expressions of nuclear respiratory factor 1(NRF1) and 2(NRF2) gene and protein in muscle tissue of rats with spleen Qi deficiency model. Methods: Thirty-two male SD rats were randomly divided into normal group, spleen Qi deficiency group, Zusanli(ST36) group and non-acupoint group, and the model of spleen Qi deficiency syndrome was established by the composite methods of improper diet and overstrain except the normal group. The rats were treated with electroacupuncture at the bilateral "Zusanli(ST36)" and non-acupoint intervention seven days after the model was successfully established. The changes of NRF1 and NRF2 mRNA and protein expression in skeletal muscle and gastric smooth muscle were detected by fluorescence quantitative PCR method and Western blot(WB). Results: The expressions of NRF1 and NRF2 protein and mRNA in skeletal muscle and gastric smooth muscle of spleen Qi deficiency group were significantly lower than those of normal group, and the level of its' expression had increased in Zusanli(ST36) group, there was no significant difference between spleen Qi deficiency group and non-acupoint group(P<0.01). Conclusion: Electroacupuncture at "Zusanli(ST36)" can improve spleen Qi deficiency by participating in the regulation of mitochondrial energy metabolism through regulating the abnormal expressions of NRF1 and NRF2 genes and proteins in muscle tissue of rats with spleen Qi deficiency.
引文
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[19] 刘景玉,刘治中,马俊楠,等.针刺大椎、足三里穴对大鼠运动性疲劳影响的实验研究[J].中国中医基础医学杂志,2015,21(11):1435-1437.
[20] 李全,张源,梁跃伟,等.Nrf2抗氧化应激保护内皮细胞损伤的研究进展[J].昆明理工大学学报(自然科学版),2017,42(2):77-81.
[21] Biswas M, Chan JY. Role of Nrf1 in antioxidant response element -mediated gene expression and beyond[J]. Toxicol Appl Pharmacol ,2010,244:16-20.
[22] 张书韬,任鹏,官大威.Nrf2在大鼠骨骼肌挫伤愈合中表达及分布的研究[J].哈尔滨医科大学学报,2015,49(5):379-384.
[23] 秦国华,王瑞霞,徐志芳,等.核呼吸因子1在心肌细胞损伤中的保护作用[J].中国环境科学,2017,37(4):1556-1561.
[24] 邵明璐,李洋,崔华峰,等.针刺预处理对大鼠心肌缺血再灌注氧化应激损伤的保护作用[J].中国针灸,2017,37(3):285-290.
[25] 崔俣,马海英,孔力.Nrf2/ARE通路与机体抗氧化机制的研究进展[J].吉林大学学报(医学版),2011,37(1):187-190.
[26] 于漫,王彩霞,刘俊兰,等.脾气虚证模型大鼠能量代谢异常及脂质过氧化损伤机理的实验研究[J].辽宁中医药大学学报,2017,19(7):28-31.
[27] 成映霞,段云燕,段永强,等.脾气虚大鼠骨骼肌组织钙调蛋白信号通路中CaM及CaMKⅡ基因表达变化[J].中国老年学杂志,2015,35(13):3489-3492.
[2] 徐建兴,吕斌. 呼吸链作为“生命引擎”的工作原理和中医阴阳五行理论的阴阳元素[J]. 科学中国人,2011(19):76.
[3] 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.
[4] 李忠仁.实验针灸学[M]. 北京:中国中医药出版社,2003:327.
[5] 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.
[6] 刘小云,陈松林,张为西. 健脾益肾法联合西药治疗耐受糖皮质激素重症肌无力临床研究[J]. 中国中西医结合杂志,2010(3):271.
[7] 何利雷,陈逊文,朱永展.健脾益气法对严重创伤软组织损伤创面组织中碱性成纤维细胞生长因子和表皮生长因子的影响[J]. 中国骨伤,2010(7):530.
[8] 吕国雄,徐秀梅. 健脾益气法治疗眼肌型重症肌无力26例[J]. 江西中医药,2010(6):58.
[9] Wang Z, Yi T, Long M, et al. Electro-Acupuncture at Zusanli Acupoint (ST36) Suppresses Inflammation in Allergic Contact Dermatitis Via Triggering Local IL-10 Production and Inhibiting p38 MAPK Activation[J]. Inflammation,2017,40(4):1351.
[10] Wang H, Wang CY, Zhang JS, et al. Acupuncture therapy for experimental stomach ulcer and c-Fos expression in rats[J]. World J Gastroenterol,2005,11(35):5517.
[11] Song Q, Hu S, Wang H, et al. Electroacupuncturing at Zusanli point (ST36) attenuates pro-inflammatory cytokine release and organ dysfunction by activating cholinergic anti-inflammatory pathway in rat with endotoxin challenge[J]. Afr J Tradit Complement Altern Med,2014,11(2):469.
[12] Scarpulla RC. Transcriptional paradigms in mammalian mitochondrial biogenesis and function[J]. Physiol Rev,2008,88(2):611.
[13] 曹思思,颜苗,张毕奎,等. 以Nrf2为靶点防治肾脏疾病的研究进展[J]. 中国药理学通报,2017(3):301.
[14] 余璐.AMPK介导大鼠视皮质神经元兴奋依赖性PGC-1α和NRF-1表达及线粒体能量代谢调节[D].西安:第四军医大学,2010.
[15] 胡齐,孙莹,宋雅芳,等.四君子汤对脾虚大鼠线粒体氧化损伤及能量代谢的影响[J].中华中医药学刊,2017,35(8):1972-1976.
[16] 琚星萌,宋雅芳,雷孝文,等.四君子汤对脾虚大鼠骨骼肌SDH活性及PGC-1α基因和蛋白表达的影响[J].时珍国医国药,2017,28(4):802-804.
[17] 战丽彬.脾虚与物质能量代谢[J].大连医科大学学报,2014,36(1):1-6.
[18] 刘继东,苗嘉芮,李宁,等.四君子汤调节脾气虚大鼠能量代谢相关基因的研究[J].中华中医药杂志,2016,31(3):985-987.
[19] 刘景玉,刘治中,马俊楠,等.针刺大椎、足三里穴对大鼠运动性疲劳影响的实验研究[J].中国中医基础医学杂志,2015,21(11):1435-1437.
[20] 李全,张源,梁跃伟,等.Nrf2抗氧化应激保护内皮细胞损伤的研究进展[J].昆明理工大学学报(自然科学版),2017,42(2):77-81.
[21] Biswas M, Chan JY. Role of Nrf1 in antioxidant response element -mediated gene expression and beyond[J]. Toxicol Appl Pharmacol ,2010,244:16-20.
[22] 张书韬,任鹏,官大威.Nrf2在大鼠骨骼肌挫伤愈合中表达及分布的研究[J].哈尔滨医科大学学报,2015,49(5):379-384.
[23] 秦国华,王瑞霞,徐志芳,等.核呼吸因子1在心肌细胞损伤中的保护作用[J].中国环境科学,2017,37(4):1556-1561.
[24] 邵明璐,李洋,崔华峰,等.针刺预处理对大鼠心肌缺血再灌注氧化应激损伤的保护作用[J].中国针灸,2017,37(3):285-290.
[25] 崔俣,马海英,孔力.Nrf2/ARE通路与机体抗氧化机制的研究进展[J].吉林大学学报(医学版),2011,37(1):187-190.
[26] 于漫,王彩霞,刘俊兰,等.脾气虚证模型大鼠能量代谢异常及脂质过氧化损伤机理的实验研究[J].辽宁中医药大学学报,2017,19(7):28-31.
[27] 成映霞,段云燕,段永强,等.脾气虚大鼠骨骼肌组织钙调蛋白信号通路中CaM及CaMKⅡ基因表达变化[J].中国老年学杂志,2015,35(13):3489-3492.