电针“足三里”对功能性消化不良大鼠胃排空及自噬信号通路的影响
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摘要
目的:观察电针"足三里"对功能性消化不良(FD)大鼠胃排空、自噬标记物微管相关蛋白1轻链3(LC3)及自噬信号通路分子腺苷酸活化蛋白激酶(AMPK)的影响,探讨电针改善FD胃肠动力障碍的机制。方法:SD大鼠随机分为空白组、模型组、电针组、抑制剂组、电针+抑制剂组,每组8只。采用多因素应激干预法建立FD模型。电针组电针双侧"足三里"穴,每日1次,连续7d;抑制剂组大鼠予以Compound C腹腔注射(20mg/kg),每日1次,连续7d;电针+抑制剂组大鼠予以Compound C腹腔注射和电针干预。检测各组大鼠胃内残留率、小肠推进率;Western blot法检测胃窦组织酪氨酸激酶受体(c-kit)、LC3-Ⅱ/Ⅰ、自噬基因Beclin 1、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、磷酸化自噬相关蛋白1(p-ULK1)的表达。结果:与空白组比较,模型组大鼠胃内残留率升高,小肠推进率降低(P<0.01),胃窦中的c-kit表达明显降低,LC3-Ⅱ/Ⅰ、Beclin 1、p-AMPK、p-ULK1表达明显升高(P<0.01);与模型组比较,电针组、抑制剂组、电针+抑制剂组大鼠胃内残留率降低,小肠推进率升高(P<0.05,P<0.01),胃窦中的c-kit表达明显升高,LC3-Ⅱ/Ⅰ、Beclin 1、p-AMPK、p-ULK1表达明显降低(P<0.01);与抑制剂组比较,电针组、电针+抑制剂组大鼠胃窦中的c-kit表达均明显升高(P<0.05,P<0.01),LC3-Ⅱ/Ⅰ、Beclin 1表达明显降低(P<0.05),p-AMPK、p-ULK1蛋白表达明显降低(P<0.01)。结论:电针"足三里"能够改善FD大鼠胃肠动力障碍,其作用机制可能是通过调控AMPK/ULK1信号通路进而抑制Cajal间质细胞过度自噬水平。
Objective To explore the effect of electroacupuncture(EA)at "Zusanli"(ST36)on gastrointestinal motility and expression of autophagy marker LC3 and autophagy signaling pathway molecule AMP-activated protein kinase(AMPK)in rats with functional dyspepsia(FD),so as to explore its mechanisms underlying improvement of FD.Methods A total of 40 male SD rats were randomly divided into blank control,model,EA,AMPK inhibitor and EA+AMPK inhibitor groups,with 8 rats in each group.The FD model was established by tail-clip(30 min/time,twice daily)+single day feeding,and gavage of normal saline(2 mL/time,twice a day)for 2 successive weeks.For rats of EA and EA+AMPK inhibitor groups,EA(4 Hz,1.0 mA)was applied to bilateral ST36 for 20 min,once daily for 7 successive days.For rats of the AMPK-inhibitor and EA+AMPK inhibitor groups,Compound C(20 mg/kg)solution was administered by intraperitoneal injection before every EA administration.The gastric residual rate and small intestinal transit rate were calculated based on the weight of stomach and length of ink propelling and total small intestine,respectively.The expression levels of c-kit,microtubule-associated protein 1 light chain 3,Beclin 1,phosphorylated(p)-AMPK and p-unc-51 like autophagy activating kinase 1(ULK1)in the gastric antrum tissue were detected by using Western blot.Results Compared with the blank control group,the gastric residual rate and the expression levels of LC3-Ⅱ/LC3Ⅰ,Beclin 1,p-AMPK and p-ULK1 proteins were significantly increased,and the small intestinal transit rate and the expression of c-kit protein obviously decreased in the model group(P<0.01).After EA intervention,modeling-induced increase of gastric residual rate and the expression of LC3-Ⅱ/LC3Ⅰ,Beclin 1,p-AMPK and p-ULK1 proteins,and decrease of small intestinal transit rate and expression of c-kit protein were reversed in the EA,AMPK inhibitor and EA+AMPK inhibitor groups(P<0.05,P<0.01).The therapeutic effect of EA and EA+AMPK was significantly superior to that of AMPK inhibitor in downregulating the expression of LC3Ⅱ/LC3Ⅰ,Beclin 1,p-AMPK and p-ULK1 proteins and in up-regulating the expression of c-kit protein(P<0.05,P<0.01).No significant differences were found among the EA,AMPK inhibitor and EA+AMPK inhibitor groups in lowering gastric residual rate and elevating the small intestinal transit rate(P>0.05).Conclusion EA at ST36 can promote gastrointestinal motility in FD rats,which is possibly mediated by inhibiting excessive autophagy of interstitial cells of Cajal via down-regulating AMPK/ULK1 signaling.
Objective To explore the effect of electroacupuncture(EA)at "Zusanli"(ST36)on gastrointestinal motility and expression of autophagy marker LC3 and autophagy signaling pathway molecule AMP-activated protein kinase(AMPK)in rats with functional dyspepsia(FD),so as to explore its mechanisms underlying improvement of FD.Methods A total of 40 male SD rats were randomly divided into blank control,model,EA,AMPK inhibitor and EA+AMPK inhibitor groups,with 8 rats in each group.The FD model was established by tail-clip(30 min/time,twice daily)+single day feeding,and gavage of normal saline(2 mL/time,twice a day)for 2 successive weeks.For rats of EA and EA+AMPK inhibitor groups,EA(4 Hz,1.0 mA)was applied to bilateral ST36 for 20 min,once daily for 7 successive days.For rats of the AMPK-inhibitor and EA+AMPK inhibitor groups,Compound C(20 mg/kg)solution was administered by intraperitoneal injection before every EA administration.The gastric residual rate and small intestinal transit rate were calculated based on the weight of stomach and length of ink propelling and total small intestine,respectively.The expression levels of c-kit,microtubule-associated protein 1 light chain 3,Beclin 1,phosphorylated(p)-AMPK and p-unc-51 like autophagy activating kinase 1(ULK1)in the gastric antrum tissue were detected by using Western blot.Results Compared with the blank control group,the gastric residual rate and the expression levels of LC3-Ⅱ/LC3Ⅰ,Beclin 1,p-AMPK and p-ULK1 proteins were significantly increased,and the small intestinal transit rate and the expression of c-kit protein obviously decreased in the model group(P<0.01).After EA intervention,modeling-induced increase of gastric residual rate and the expression of LC3-Ⅱ/LC3Ⅰ,Beclin 1,p-AMPK and p-ULK1 proteins,and decrease of small intestinal transit rate and expression of c-kit protein were reversed in the EA,AMPK inhibitor and EA+AMPK inhibitor groups(P<0.05,P<0.01).The therapeutic effect of EA and EA+AMPK was significantly superior to that of AMPK inhibitor in downregulating the expression of LC3Ⅱ/LC3Ⅰ,Beclin 1,p-AMPK and p-ULK1 proteins and in up-regulating the expression of c-kit protein(P<0.05,P<0.01).No significant differences were found among the EA,AMPK inhibitor and EA+AMPK inhibitor groups in lowering gastric residual rate and elevating the small intestinal transit rate(P>0.05).Conclusion EA at ST36 can promote gastrointestinal motility in FD rats,which is possibly mediated by inhibiting excessive autophagy of interstitial cells of Cajal via down-regulating AMPK/ULK1 signaling.
引文
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[11]王煜姣,凌江红,张钰琴,等.复合病因造模法制备功能性消化不良大鼠模型[J].世界华人消化杂志,2014,22(2):210-214.
[12]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:68-70.
[13]肖靓宜,刘未艾,吴清明,等.隔药饼灸对功能性消化不良肝郁脾虚大鼠下丘脑单胺类神经递质表达的影响[J].针刺研究,2016,41(1):60-64.
[14]ALERS S,LOFFLER A S,WESSELBORG S,et al.Role of AMPK-mTOR-Ulkl/2in the regulation of autophagy:cross talk,shortcuts and feedbacks[J].Mol Cell Biol,2012,32(1):2-11.
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[16]唐翠娟,程军平,梅志刚,等.针药结合对糖尿病胃轻瘫小鼠胃窦Cajal间质细胞超微结构的影响[J].中华中医药杂志,2016,31(7):2518-2521.
[17]STREUTKER C J,HUIZINGA J D,CAMPBELL F,et al.Loss of CD117(c-kit)-and CD34-positive ICC and associated CD34-positive fibroblasts defines a subpopulation of chronic intestinal pseudo-obstruction[J].Am J Surg Pathol,2003,27(2):228-235.
[18]戴彦成,张亚利,唐志鹏,等.ICC自噬调控:溃疡性结肠炎肠动力紊乱治疗的新靶点[J].胃肠病学,2015,20(6):377-379.
[19]纪云西,凌江红,庞黎明,等.胃肠道Cajal间质细胞、Ca2+与胃肠动力[J].实用医学杂志,2013,29(11):1872-1873.
[20]WARD S M,SANDERS K M,HIRST G D.Role of interstitial cells of Cajal in neural control of gastrointestinal smooth muscles[J].Neurogastroenterol Motil,2004,16(s1):112-117.
[21]CHIFENTI B,LOCCI M T,LAZZERI G,et al.Autophagyrelated protein LC3and Beclin-1in the first trimester of pregnancy[J].Clin Exp Reprod Med,2013,40(1):33-37.
[22]WIRAWAN E,LIPPENS S,VANDEN BERGHE T,et al.Beclin1:a role in membrane dynamics and beyond[J].Autophagy,2012,8(1):6-17.
[23]EDINGER A L,THOMPSON C B.Defective autophagy leads to cancer[J].Cancer Cell,2003,4(6):422-424.
[24]ZHANG L M,ZENG L J,DENG J,et al.Investigation of autophagy and differentiation of myenteric interstitial cells of cajal in the pathogenesis of gastric motility disorders in rats with functional dyspepsia[J].Biotechnol Appl Biochem,2018,65(4):533-539.
[25]韩永丽,陈松,康朝霞,等.艾灸对功能性消化不良大鼠cajal间质细胞c-kit表达的影响[J].湖北中医药大学学报,2017,19(6):8-11.
[26]肖飞,李伟杰,刘秀兰,等.龙牡壮骨颗粒对功能性消化不良大鼠胃动素水平和cajal间质细胞数量的影响[J].华中科技大学学报(医学版),2014,43(6):684-686.
[27]白杨,郭义.针刺效应/信号调节网络初探[J].针刺研究,2013,38(4):330-333.
[28]李跃兵,张泓,李铁浪,等.电针治疗功能性消化不良大鼠海马体NMDA受体的调节机制[J].医学理论与实践,2015,28(4):421-422.
[2]徐派的,杨云,辛玉,等.电针对功能性消化不良肝郁脾虚型大鼠中枢及外周VIP及其受体VPAC-1的影响[J].中华中医药杂志,2016,31(8):3020-3024.
[3]杨云,徐派的,辛玉,等.神经降压素介导的脑-肠轴在电针治疗功能性消化不良大鼠中的作用[J].针刺研究,2016,41(1):35-39.
[4]房殿春.胃肠起搏治疗胃肠动力紊乱性疾病[J].解放军医学杂志,2004,29(10):850-853.
[5]刘宁,李平,张建英,等.Cajal间质细胞研究进展[J].中国中西医结合消化杂志,2011,19(6):418-419.
[6]潘小丽,康朝霞,毛玮,等.电针对功能性消化不良肝郁脾虚模型大鼠胃电节律及胃窦Cajal间质细胞表达的影响[J].中医杂志,2018,59(17):1503-1506.
[7]丁雪菲,李慧,夏军权.糖尿病胃轻瘫与cajal间质细胞的关系及中医辨证研究[J].时珍国医国药,2015,30(11):2742-2744.
[8]王凯悦,余芝,梁超,等.针刺调节结肠慢传输便秘ICC相关机制的研究进展[J].世界华人消化杂志,2015,23(5):748-753.
[9]DAI Y C,ZHENG L,ZHANG Y L,et al.Jianpi Qingchang decoction regulates intestinal motility of dextran sulfate sodium-induced colitis through reducing autophagy of interstitial cells of Cajal[J].World J Gastroenterol,2017,23(26):4724-4734.
[10]郭海军,林洁,李国成,等.功能性消化不良的动物模型研究[J].中国中西医结合消化杂志,2001,9(3):141-142.
[11]王煜姣,凌江红,张钰琴,等.复合病因造模法制备功能性消化不良大鼠模型[J].世界华人消化杂志,2014,22(2):210-214.
[12]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:68-70.
[13]肖靓宜,刘未艾,吴清明,等.隔药饼灸对功能性消化不良肝郁脾虚大鼠下丘脑单胺类神经递质表达的影响[J].针刺研究,2016,41(1):60-64.
[14]ALERS S,LOFFLER A S,WESSELBORG S,et al.Role of AMPK-mTOR-Ulkl/2in the regulation of autophagy:cross talk,shortcuts and feedbacks[J].Mol Cell Biol,2012,32(1):2-11.
[15]RUSSELL R C,TIAN Y,YUAN H X,et al.ULK1induces autophagy by phosphorylating Beclin-1and activating VPS34lipid kinase[J].Nat Cell Biol,2013,15(7):741-750.
[16]唐翠娟,程军平,梅志刚,等.针药结合对糖尿病胃轻瘫小鼠胃窦Cajal间质细胞超微结构的影响[J].中华中医药杂志,2016,31(7):2518-2521.
[17]STREUTKER C J,HUIZINGA J D,CAMPBELL F,et al.Loss of CD117(c-kit)-and CD34-positive ICC and associated CD34-positive fibroblasts defines a subpopulation of chronic intestinal pseudo-obstruction[J].Am J Surg Pathol,2003,27(2):228-235.
[18]戴彦成,张亚利,唐志鹏,等.ICC自噬调控:溃疡性结肠炎肠动力紊乱治疗的新靶点[J].胃肠病学,2015,20(6):377-379.
[19]纪云西,凌江红,庞黎明,等.胃肠道Cajal间质细胞、Ca2+与胃肠动力[J].实用医学杂志,2013,29(11):1872-1873.
[20]WARD S M,SANDERS K M,HIRST G D.Role of interstitial cells of Cajal in neural control of gastrointestinal smooth muscles[J].Neurogastroenterol Motil,2004,16(s1):112-117.
[21]CHIFENTI B,LOCCI M T,LAZZERI G,et al.Autophagyrelated protein LC3and Beclin-1in the first trimester of pregnancy[J].Clin Exp Reprod Med,2013,40(1):33-37.
[22]WIRAWAN E,LIPPENS S,VANDEN BERGHE T,et al.Beclin1:a role in membrane dynamics and beyond[J].Autophagy,2012,8(1):6-17.
[23]EDINGER A L,THOMPSON C B.Defective autophagy leads to cancer[J].Cancer Cell,2003,4(6):422-424.
[24]ZHANG L M,ZENG L J,DENG J,et al.Investigation of autophagy and differentiation of myenteric interstitial cells of cajal in the pathogenesis of gastric motility disorders in rats with functional dyspepsia[J].Biotechnol Appl Biochem,2018,65(4):533-539.
[25]韩永丽,陈松,康朝霞,等.艾灸对功能性消化不良大鼠cajal间质细胞c-kit表达的影响[J].湖北中医药大学学报,2017,19(6):8-11.
[26]肖飞,李伟杰,刘秀兰,等.龙牡壮骨颗粒对功能性消化不良大鼠胃动素水平和cajal间质细胞数量的影响[J].华中科技大学学报(医学版),2014,43(6):684-686.
[27]白杨,郭义.针刺效应/信号调节网络初探[J].针刺研究,2013,38(4):330-333.
[28]李跃兵,张泓,李铁浪,等.电针治疗功能性消化不良大鼠海马体NMDA受体的调节机制[J].医学理论与实践,2015,28(4):421-422.