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
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摘要
目的:观察电针"足三里"穴对脾气虚大鼠空肠组织胃生长激素释放激素(Ghrelin)/环磷酸腺苷(cAMP)/蛋白激酶A(PKA)信号通路的影响,探讨电针"足三里"穴改善脾气虚大鼠能量代谢的作用机制。方法:雄性SD大鼠随机分为空白组、脾气虚组、电针组和非经非穴组,每组10只。采用劳倦过度及饮食不节的复合因素复制大鼠脾气虚证候模型。电针组电针双侧"足三里"穴,非经非穴组电针双侧非经非穴点(髂嵴连线上方15mm,后正中线左右旁开20mm处),每日1次,每次20min,共治疗6d。HE染色法观察各组大鼠空肠组织病理形态学改变;ELISA法测定各组大鼠空肠组织中Ghrelin、三磷酸腺苷(ATP)、cAMP含量;Western blot法检测空肠组织PKA蛋白表达。结果:脾气虚组大鼠肠绒毛肿胀、缩短、增粗,顶端部分破损或断裂,经电针穴位治疗后其结构基本恢复正常。脾气虚组大鼠空肠组织中的Ghrelin、ATP、cAMP含量及PKA蛋白表达较空白组显著降低(P<0.05),而电针组显著高于脾气虚组(P<0.05),其中cAMP含量及PKA蛋白表达电针组显著高于非经非穴组(P<0.05)。结论:电针"足三里"穴可通过改善脾气虚大鼠空肠组织病理形态学变化,增加脾气虚大鼠空肠组织中Ghrelin、ATP、cAMP含量,上调PKA表达,从而增强大鼠能量代谢,发挥其补益脾气的作用。
Objective To observe the effect of electroacupuncture(EA)at"Zusanli"(ST 36)on Ghrelin/cAMP/PKA expression in the jejunum in rats with spleen qi deficiency syndrome,so as to reveal its underlying mechanism in improving energy metabolism.Methods Forty male SD rats were randomly divided into 4groups:normal group,spleen qi deficiency syndrome(model)group,EA group and non-acupoint group(n=10in each group).The model of spleen qi deficiency syndrome was established by improper diet and overstrain.EA(2Hz/15 Hz,0.5mA)was applied to bilateral"Zusanli"(ST 36)in the EA group and non-acupoint in non-acupoint group for 20 min,once a day for 6days.The pathologic changes of the jejunum tissue were detected by H&E staining.Ghrelin,ATP and cAMP levels in jejunum tissue were determined by ELISA.The expression levels of PKA protein in jejunum tissue were determined by Western blot.Results H&E staining showed that the intestinal villi of the model group were swelling,shortening and thickening,with a damaged or broken top-part in the model group,and basically restored to normal after EA treatment.ELISA results showed that the contents of Ghrelin,ATP and cAMP in the jejunum tissue were significantly lower in the model group than in the normal group(P<0.05),while significantly higher in the EA group than in the model group(P<0.05).Western blot results showed that the expression of PKA protein in the jejunum tissue was significantly lower in the model group than in the normal group(P<0.05),and significantly higher in the EA group than in the model group and non-acupoint group(P<0.05).Conclusion EA at ST 36 can improve the morphological changes in the jejunum of spleen qi deficiency rats,which may be associated with its effects in increasing Ghrelin,ATP and cAMP contents,and up-regulating PKA expression,leading to an increase of energy metabolism and spleen qi at last.
Objective To observe the effect of electroacupuncture(EA)at"Zusanli"(ST 36)on Ghrelin/cAMP/PKA expression in the jejunum in rats with spleen qi deficiency syndrome,so as to reveal its underlying mechanism in improving energy metabolism.Methods Forty male SD rats were randomly divided into 4groups:normal group,spleen qi deficiency syndrome(model)group,EA group and non-acupoint group(n=10in each group).The model of spleen qi deficiency syndrome was established by improper diet and overstrain.EA(2Hz/15 Hz,0.5mA)was applied to bilateral"Zusanli"(ST 36)in the EA group and non-acupoint in non-acupoint group for 20 min,once a day for 6days.The pathologic changes of the jejunum tissue were detected by H&E staining.Ghrelin,ATP and cAMP levels in jejunum tissue were determined by ELISA.The expression levels of PKA protein in jejunum tissue were determined by Western blot.Results H&E staining showed that the intestinal villi of the model group were swelling,shortening and thickening,with a damaged or broken top-part in the model group,and basically restored to normal after EA treatment.ELISA results showed that the contents of Ghrelin,ATP and cAMP in the jejunum tissue were significantly lower in the model group than in the normal group(P<0.05),while significantly higher in the EA group than in the model group(P<0.05).Western blot results showed that the expression of PKA protein in the jejunum tissue was significantly lower in the model group than in the normal group(P<0.05),and significantly higher in the EA group than in the model group and non-acupoint group(P<0.05).Conclusion EA at ST 36 can improve the morphological changes in the jejunum of spleen qi deficiency rats,which may be associated with its effects in increasing Ghrelin,ATP and cAMP contents,and up-regulating PKA expression,leading to an increase of energy metabolism and spleen qi at last.
引文
[1]陈文,顾红卫,马维平,等.针刺足三里、悬钟对缺血性中风患者脑血管功能的影响:多中心随机对照研究[J].中国针灸,2006,26(12):851-853.
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[3]孙华,赵慧,张捷,等.针刺百会、足三里对抑郁症患者血清炎性细胞因子水平的影响[J].中国针灸,2010,30(3):195-199.
[4]李聪,谢鸣,赵荣华.柴疏四君汤对肝郁-脾虚-肝郁脾虚不同证候模型大鼠消化系统功能的影响[J].中华中医药杂志,2014,29(1):72-75.
[5]赵荣华,谢鸣,李聪,等.肝郁、脾虚和肝郁脾虚证模型大鼠的免疫功能变化[J].北京中医药大学学报,2013,36(12):821-824.
[6]彭艳,彭芬,易受乡,等.艾灸对脾虚大鼠小肠运动吸收功能及ATP含量的影响[J].中国针灸,2012,32(3):246-250.
[7]王昕,赵明亮,吉长福,等.针刺“足三里”穴对脾虚证大鼠血清中睾酮和雌二醇水平的影响[J].针刺研究,2011,36(4):268-271.
[8]刘继东,苗嘉芮,李宁,等.脾虚模型大鼠能量代谢相关性基因研究[J].中华中医药杂志,2015,30(9):3304-3306.
[9]Dickson L M,Gandhi S,Layden B T,et al.Protein kinase A induces UCP 1expression in specific adipose depots to increase energy expenditure and improve metabolic health[J].Am J Physiol Regul Integr Comp Physiol,2016,311(7):79-88.
[10]王晶,许智博,田慧,等.PKA信号通路参与调控肝细胞cAMP反应元件结合蛋白与PGC-1α的表达[J].生物医学工程与临床,2011,15(3):277-279.
[11]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.
[12]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:327.
[13]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,2013,38(4):334-338.
[14]陈玉龙,张海艇,李茹柳,等.脾虚大鼠唾液淀粉酶分泌障碍与cAMP-PKA信号通路关系的研究[J].中华中医药杂志,2011,26(8):836-839.
[15]宋囡,贾连群,王俊岩,等.脾虚证大鼠模型胃组织cAMPPKA信号传导通路变化的研究[J].中国中医基础医学杂志,2015,21(6):653-655.
[16]Kojima M,Hosoda H,Date Y,et al.Ghrelin is a growthhormone-releasing acylated peptide from stomach[J].Nature,1999,402(6762):656-660.
[17]Asakawa A,Inui A,Kaga T,et al.Antagonism of ghrelin receptor reduces food intake and body weight gain in mice[J].Gut,2003,52(7):947-952.
[18]Cheung C K,Wu J C.Role of ghrelin in the pathophysiology of gastrointestinal disease[J].Gut Liver,2013,7(5):505-512.
[19]Bacha F,Arslanian S A.Ghrelin suppression in overweight children:a manifestation of insulin resistance[J].J Clin Endocrinol Metab,2005,90(5):2725-2730.
[20]Langenberg C,Bergstrom J,Laughlin G A,et al.Ghrelin and the metabolic syndrome in older adults[J].J Clin Endocrinol Metab,2005,90(12):6448-6453.
[21]Beavo J A,Brunton L L.Cyclic nucleotide research——still expanding after half a century[J].Nat Rev Mol Cell Biol,2002,3(9):710-718.
[22]Bossis I,Stratakis C A.Minireview:PRKAR 1A:normal and abnormal functions[J].Endocrinology,2004,145(12):5452-5458.
[23]何丹娟,黄光英,张明敏.针刺对胚胎着床障碍大鼠黄体功能的影响[J].中国针灸,2009,29(11):910-913.
[24]吴巧凤,杨阳,赵纪岚,等.基于-1HNMR代谢组学技术研究针刺经穴与非经穴治疗溃疡性结肠炎的脑代谢物质基础[J].北京中医药大学学报,2014,37(8):572-576.
[25]白增华,吴兆利,苏妆,等.电针“内关”穴对心肌缺血大鼠心肌氯离子通道相关基因表达的影响[J].针刺研究,2015,40(6):439-443.
[26]邓雪,任路,李静,等.电针“肝俞”穴对抑郁型胃溃疡大鼠胃窦黏膜、下丘脑组织P物质和海马5-羟色胺的影响[J].针刺研究,2014,39(2):124-129.
[27]王颖,李迪,戴俭宇,等.电针内关穴及列缺穴对心肌缺血大鼠心肌细胞蛋白激酶表达的影响[J].中国中西医结合杂志,2015,35(3):338-342.
[28]黄桂榕,李沛,林莺,等.电针“命门”穴对绝经后骨质疏松症大鼠骨形成蛋白-2的影响[J].针刺研究,2014,39(2):130-135.
[2]陈志娟,曲怡,刘宇,等.电针刺激曲池、足三里穴对自发性高血压大鼠血压和肾功能的影响[J].中国中医急症,2012,21(9):1423-1424.
[3]孙华,赵慧,张捷,等.针刺百会、足三里对抑郁症患者血清炎性细胞因子水平的影响[J].中国针灸,2010,30(3):195-199.
[4]李聪,谢鸣,赵荣华.柴疏四君汤对肝郁-脾虚-肝郁脾虚不同证候模型大鼠消化系统功能的影响[J].中华中医药杂志,2014,29(1):72-75.
[5]赵荣华,谢鸣,李聪,等.肝郁、脾虚和肝郁脾虚证模型大鼠的免疫功能变化[J].北京中医药大学学报,2013,36(12):821-824.
[6]彭艳,彭芬,易受乡,等.艾灸对脾虚大鼠小肠运动吸收功能及ATP含量的影响[J].中国针灸,2012,32(3):246-250.
[7]王昕,赵明亮,吉长福,等.针刺“足三里”穴对脾虚证大鼠血清中睾酮和雌二醇水平的影响[J].针刺研究,2011,36(4):268-271.
[8]刘继东,苗嘉芮,李宁,等.脾虚模型大鼠能量代谢相关性基因研究[J].中华中医药杂志,2015,30(9):3304-3306.
[9]Dickson L M,Gandhi S,Layden B T,et al.Protein kinase A induces UCP 1expression in specific adipose depots to increase energy expenditure and improve metabolic health[J].Am J Physiol Regul Integr Comp Physiol,2016,311(7):79-88.
[10]王晶,许智博,田慧,等.PKA信号通路参与调控肝细胞cAMP反应元件结合蛋白与PGC-1α的表达[J].生物医学工程与临床,2011,15(3):277-279.
[11]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.
[12]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:327.
[13]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,2013,38(4):334-338.
[14]陈玉龙,张海艇,李茹柳,等.脾虚大鼠唾液淀粉酶分泌障碍与cAMP-PKA信号通路关系的研究[J].中华中医药杂志,2011,26(8):836-839.
[15]宋囡,贾连群,王俊岩,等.脾虚证大鼠模型胃组织cAMPPKA信号传导通路变化的研究[J].中国中医基础医学杂志,2015,21(6):653-655.
[16]Kojima M,Hosoda H,Date Y,et al.Ghrelin is a growthhormone-releasing acylated peptide from stomach[J].Nature,1999,402(6762):656-660.
[17]Asakawa A,Inui A,Kaga T,et al.Antagonism of ghrelin receptor reduces food intake and body weight gain in mice[J].Gut,2003,52(7):947-952.
[18]Cheung C K,Wu J C.Role of ghrelin in the pathophysiology of gastrointestinal disease[J].Gut Liver,2013,7(5):505-512.
[19]Bacha F,Arslanian S A.Ghrelin suppression in overweight children:a manifestation of insulin resistance[J].J Clin Endocrinol Metab,2005,90(5):2725-2730.
[20]Langenberg C,Bergstrom J,Laughlin G A,et al.Ghrelin and the metabolic syndrome in older adults[J].J Clin Endocrinol Metab,2005,90(12):6448-6453.
[21]Beavo J A,Brunton L L.Cyclic nucleotide research——still expanding after half a century[J].Nat Rev Mol Cell Biol,2002,3(9):710-718.
[22]Bossis I,Stratakis C A.Minireview:PRKAR 1A:normal and abnormal functions[J].Endocrinology,2004,145(12):5452-5458.
[23]何丹娟,黄光英,张明敏.针刺对胚胎着床障碍大鼠黄体功能的影响[J].中国针灸,2009,29(11):910-913.
[24]吴巧凤,杨阳,赵纪岚,等.基于-1HNMR代谢组学技术研究针刺经穴与非经穴治疗溃疡性结肠炎的脑代谢物质基础[J].北京中医药大学学报,2014,37(8):572-576.
[25]白增华,吴兆利,苏妆,等.电针“内关”穴对心肌缺血大鼠心肌氯离子通道相关基因表达的影响[J].针刺研究,2015,40(6):439-443.
[26]邓雪,任路,李静,等.电针“肝俞”穴对抑郁型胃溃疡大鼠胃窦黏膜、下丘脑组织P物质和海马5-羟色胺的影响[J].针刺研究,2014,39(2):124-129.
[27]王颖,李迪,戴俭宇,等.电针内关穴及列缺穴对心肌缺血大鼠心肌细胞蛋白激酶表达的影响[J].中国中西医结合杂志,2015,35(3):338-342.
[28]黄桂榕,李沛,林莺,等.电针“命门”穴对绝经后骨质疏松症大鼠骨形成蛋白-2的影响[J].针刺研究,2014,39(2):130-135.