电针对高脂诱导胰岛素抵抗大鼠肝脏腺苷酸活化蛋白激酶信号转导通路相关蛋白表达的影响
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摘要
目的:观察电针对胰岛素抵抗(IR)大鼠肝组织腺苷酸活化蛋白激酶(AMPK)、p38丝裂原活化蛋白激酶(p38MAPK)、过氧化物酶体增殖物活化受体-γ(PPARγ)蛋白的影响,探讨电针治疗IR的作用机制。方法:从40只雄性SD大鼠中随机选取8只做为空白组,其余通过高脂饮食诱导IR模型,选取24只造模成功的大鼠按随机区组原则分为模型组(8只)、西药组(8只)和电针组(8只)。西药组按大鼠体质量以吡格列酮10mg·kg-1·d-1灌胃,电针组取双侧"丰隆""三阴交"穴电针治疗,1次/d,均连续治疗2周。透射电镜观察大鼠肝组织线粒体结构,ELISA法检测大鼠血清C肽(C-P)、脂联素(ADP)、瘦素(LEP)、抵抗素(RES)含量,免疫蛋白印迹法检测肝组织AMPK、p38MAPK、PPARγ的蛋白表达。结果:电镜观察显示模型组肝细胞线粒体形态、结构受损;而西药、电针组线粒体结构得到恢复融合。与空白组比较,模型组大鼠血清C-P、LEP、RES含量显著升高(P<0.01),ADP含量显著降低(P<0.01),肝组织AMPK、PPARγ蛋白表达水平显著降低(P<0.01),p38MAPK蛋白表达水平显著升高(P<0.05)。与模型组比较,电针组、西药组大鼠血清C-P、LEP、RES含量显著降低(P<0.05,P<0.01),ADP含量显著升高(P<0.01,P<0.05),肝组织AMPK、PPARγ蛋白表达水平显著升高(P<0.01),p38MAPK蛋白表达水平显著降低(P<0.01)。电针组与西药组各指标比较差异无统计学意义(P>0.05)。结论:电针可明显改善IR大鼠的脂质代谢紊乱,其作用机制可能与电针调节肝组织AMPK/p38MAPK/PPARγ通路相关,由此下调脂肪酸合成相关酶的活性,使肝组织内合成甘油三酯、胆固醇减少,改善肝组织IR。
Objective To observe the effect of eletroacupuncture(EA)intervention on lipid metabolism and expression of AMP-activated kinase(AMPK),p38 mitogen-activated protein kinase(p38 MAPK)and peroxisome proliferator-activated receptorγ(PPARγ)protein in the liver in rats with insulin resistance(IR),so as to reveal its mechanisms underlying improvement of IR.Methods Forty male SD rats were randomly divided into blank control,model,medication,and EA groups(n=8 in each).The IR model was established by feeding the rats with high-fat diet for 12 weeks.After successful establishment of model,the rats in the blank control group and model group were fixed in the self-made rat bag without receiving any treatment.The rats in the medication group were treated by gavage of pioglitazone(10 mL/kg).EA(2 Hz/100 Hz,1 mA)was applied to bilateral"Fenglong"(ST40)and"Sanyinjiao"(SP6)for 20 min,once a day,for continuous 14 days for rats in the EA group.The ultrastructure of the liver tissue was observed by transmission electron microscope(TEM).Blood samples were taken from the abdominal aorta for detecting serum C-peptide(C-P),adiponectin(ADP),leptin(LEP)and resistin(RES)contents using enzyme linked immunosorbent assay(ELISA).The expression levels of AMPK,p38 MAPK and PPARγproteins in the liver tissue were detected by Western blot.Results After modeling,the contents of serum C-P,LEP and RES,and the expression of liver p38 MAPK protein were significantly up-regulated(P<0.01,P<0.05),and the content of ADP and expression of AMPK and PPARγsignificantly down-regulated in the model group compared with the blank control group(P<0.01).The increased contents of C-P,LEP and RES,and p38 MAPK protein expression and the decreased serum ADP and hepatic AMPK and PPARγexpression levels were completely reversed in both the EA and medication groups relevant to the model group(P<0.01,P<0.05).No significant differences were found between the EA and medication groups in up-regulating the levels of ADP,AMPK and PPARγand in downregulating the levels of C-P,LEP,RES and p38 MAPK(P>0.05).Outcomes of TEM showed that morphological structure of liver mitochondria was damaged,including a large number of lipid droplets,being blur in appearance,rupture of partial membrane,dissapearance of partial mitochondrial crests with vacuolus-like appearance and decrease of rough endoplasmic reticulum in the model group,which was relatively milder in both EA and medication groups.Conclusion EA intervention is able to improve the disorder of lipid metabolism of IR rats,which may be associated with its effects in lowering the activity of fatty acid synthesis-related enzymes and regulating AMPK/p38 MAPK/PPARγsignaling to improve IR in the liver tissue.
Objective To observe the effect of eletroacupuncture(EA)intervention on lipid metabolism and expression of AMP-activated kinase(AMPK),p38 mitogen-activated protein kinase(p38 MAPK)and peroxisome proliferator-activated receptorγ(PPARγ)protein in the liver in rats with insulin resistance(IR),so as to reveal its mechanisms underlying improvement of IR.Methods Forty male SD rats were randomly divided into blank control,model,medication,and EA groups(n=8 in each).The IR model was established by feeding the rats with high-fat diet for 12 weeks.After successful establishment of model,the rats in the blank control group and model group were fixed in the self-made rat bag without receiving any treatment.The rats in the medication group were treated by gavage of pioglitazone(10 mL/kg).EA(2 Hz/100 Hz,1 mA)was applied to bilateral"Fenglong"(ST40)and"Sanyinjiao"(SP6)for 20 min,once a day,for continuous 14 days for rats in the EA group.The ultrastructure of the liver tissue was observed by transmission electron microscope(TEM).Blood samples were taken from the abdominal aorta for detecting serum C-peptide(C-P),adiponectin(ADP),leptin(LEP)and resistin(RES)contents using enzyme linked immunosorbent assay(ELISA).The expression levels of AMPK,p38 MAPK and PPARγproteins in the liver tissue were detected by Western blot.Results After modeling,the contents of serum C-P,LEP and RES,and the expression of liver p38 MAPK protein were significantly up-regulated(P<0.01,P<0.05),and the content of ADP and expression of AMPK and PPARγsignificantly down-regulated in the model group compared with the blank control group(P<0.01).The increased contents of C-P,LEP and RES,and p38 MAPK protein expression and the decreased serum ADP and hepatic AMPK and PPARγexpression levels were completely reversed in both the EA and medication groups relevant to the model group(P<0.01,P<0.05).No significant differences were found between the EA and medication groups in up-regulating the levels of ADP,AMPK and PPARγand in downregulating the levels of C-P,LEP,RES and p38 MAPK(P>0.05).Outcomes of TEM showed that morphological structure of liver mitochondria was damaged,including a large number of lipid droplets,being blur in appearance,rupture of partial membrane,dissapearance of partial mitochondrial crests with vacuolus-like appearance and decrease of rough endoplasmic reticulum in the model group,which was relatively milder in both EA and medication groups.Conclusion EA intervention is able to improve the disorder of lipid metabolism of IR rats,which may be associated with its effects in lowering the activity of fatty acid synthesis-related enzymes and regulating AMPK/p38 MAPK/PPARγsignaling to improve IR in the liver tissue.
引文
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[14]MATSUNAMI T,SATO Y,ARIGA S,et al.Regulation of synthesis and oxidation of fatty acids by adiponectin receptors(AdipoR1/R2)and insulin receptor substrate isoforms(IRS-1/-2)of the liver in a nonalcoholic steatohepatitis animal model[J].Metabolism,2011,60(6):805-814.
[15]HOTTA K,FUNAHASHI T,ARITA Y,et al.Plasma concentrations of a novel,adipose-specific protein,adiponectin,in type 2diabetic patients[J].Arterioscler Thromb Vasc Biol,2000,20(6):1595-1599.
[16]MOONISHAA T M,NANDA S K,SHAMRAJ M,et al.Evaluation of leptin as a marker of insulin resistance in type 2diabetes mellitus[J].Int J Appl Basic Med Res,2017,7(3):176.
[17]STEPPAN C M,LAZAR M A.Resistin and obesity-associated insulin resistance[J].Trends Endocrinol Metab,2002,13(1):18-23.
[18]宋青,苟文丽.吡格列酮对胰岛素抵抗型多囊卵巢综合征患者脂肪细胞因子的影响[J].西安交通大学学报(医学版),2010,31(2):227-230.
[19]SAHA A K,AVILUCEA P R,YE J M,et al.Pioglitazone treatment activates AMP-activated protein kinase in rat liver and adipose tissue in vivo[J].Biochem Biophys Res Commun,2004,314(2):580-585.
[20]HRESKO R C,HEIMBERG H,CHI M M,et al.Glucosamine-induced insulin resistance in 3T3-L1adipocytes is caused by depletion of intracellular ATP[J].J Biol Chem,1998,273(32):20658-20668.
[21]LOWELL B B,SHULMAN G I.Mitochondrial dysfunction and type 2diabetes[J].Science,2005,307(5708):384-387.
[22]HUYPENS P,MOENS K,HEIMBERG H,et al.Adiponectin-mediated stimulation of AMP-activated protein kinase(AMPK)in pancreatic beta cells[J].Life Sci,2005,77(11):1273-1282.
[23]HOMMES D W,PEPPELENBOSCH M P,van DEVENTER SJ.Mitogen activated protein(MAP)kinase signal transduction pathways and novel anti-inflammatory targets[J].Gut,2003,52(1):144-151.
[24]LI T,WANG W,ZHAO J H,et al.Pseudolaric acid B inhibits T-cell mediated immune response in vivo via p38MAPKsignal cascades and PPARγactivation[J].Life Sci,2015,121:88-96.
[25]PANG X,LIU J,LI Y,et al.Emodin inhibits homocysteineinduced C-reactive protein generation in vascular smooth muscle cells by regulating PPARγexpression and ROS-ERK1/2/p38signal pathway[J].PLoS One,2014,10(7):e0131295.
[26]DAHABREH D F,MEDH J D.Activation of peroxisome proliferator activated receptor-gamma results in an atheroprotective apolipoprotein profile in HepG2cells[J].Adv Biol Chem,2012,2(3):218-225.
[27]GUERRE-MILLO M.Adiponectin:an update[J].Diabetes Metab,2008,34(1):12-18.
[2]KUBOTA N,TOBE K,TERAUCHI Y,et al.Disruption of insulin receptor substrate 2causes type 2diabetes because of liver insulin resistance and lack of compensatory beta-cell hyperplasia[J].Diabetes,2000,49(11):1880-1889.
[3]WATT M J,DZAMKO N,THOMAS W G,et al.CNTFreverses obesity-induced insulin resistance by activating skeletal muscle AMPK[J].Nat Med,2006,12(5):541-548.
[4]RUDERMAN N B,CARLING D,PRENTKI M,et al.AMPK,insulin resistance,and the metabolic syndrome[J].JClin Invest,2013,123(7):2764-2772.
[5]VIOLETT B,ANDREELLI F,JORGENSEN S B,et al.The AMP-activated protein kinaseα2catalytic subunit controls whole-body insulin sensitivity[J].J Clin Invest,2003,111(1):91-98.
[6]李知行,蓝丹纯,张弘弢,等.针刺对高脂饲养胰岛素抵抗大鼠葡萄糖输注率的影响[J].中国老年学杂志,2016,36(19):4676-4678.
[7]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:334-336.
[8]李知行,蓝丹纯,张海华,等.基于数据挖掘的针灸治疗胰岛素抵抗用穴规律研究[J].针刺研究,2016,41(6):545-549.
[9]李知行,张海华,蓝丹纯,等.电针对高脂诱导胰岛素抵抗大鼠肝脏固醇调节元件结合蛋白-1c、脂肪酸合成酶的影响[J].针刺研究,2018,43(1):8-13.
[10]SHOELSON S E,HERRERO L,NAAZ A.Obesity,inflammation,and insulin resistance[J].Gastroenterology,2007,132(6):2169-2180.
[11]NAVAB M,GHARAVI N,WATSON A D.Inflammation and metabolic disorders[J].Curr Opin Clin Nutr Metab Care,2008,11(4):459-464.
[12]王国洪,许瑞吉,张中书,等.血清C肽浓度测定的临床意义探讨[J].放射免疫学杂志,2006,19(1):15-17.
[13]XU W,YU L,ZHOU W,et al.Resistin increases lipid accumulation and CD36expression in human macrophages[J].Biochem Biophys Res Commun,2006,351(2):376-382.
[14]MATSUNAMI T,SATO Y,ARIGA S,et al.Regulation of synthesis and oxidation of fatty acids by adiponectin receptors(AdipoR1/R2)and insulin receptor substrate isoforms(IRS-1/-2)of the liver in a nonalcoholic steatohepatitis animal model[J].Metabolism,2011,60(6):805-814.
[15]HOTTA K,FUNAHASHI T,ARITA Y,et al.Plasma concentrations of a novel,adipose-specific protein,adiponectin,in type 2diabetic patients[J].Arterioscler Thromb Vasc Biol,2000,20(6):1595-1599.
[16]MOONISHAA T M,NANDA S K,SHAMRAJ M,et al.Evaluation of leptin as a marker of insulin resistance in type 2diabetes mellitus[J].Int J Appl Basic Med Res,2017,7(3):176.
[17]STEPPAN C M,LAZAR M A.Resistin and obesity-associated insulin resistance[J].Trends Endocrinol Metab,2002,13(1):18-23.
[18]宋青,苟文丽.吡格列酮对胰岛素抵抗型多囊卵巢综合征患者脂肪细胞因子的影响[J].西安交通大学学报(医学版),2010,31(2):227-230.
[19]SAHA A K,AVILUCEA P R,YE J M,et al.Pioglitazone treatment activates AMP-activated protein kinase in rat liver and adipose tissue in vivo[J].Biochem Biophys Res Commun,2004,314(2):580-585.
[20]HRESKO R C,HEIMBERG H,CHI M M,et al.Glucosamine-induced insulin resistance in 3T3-L1adipocytes is caused by depletion of intracellular ATP[J].J Biol Chem,1998,273(32):20658-20668.
[21]LOWELL B B,SHULMAN G I.Mitochondrial dysfunction and type 2diabetes[J].Science,2005,307(5708):384-387.
[22]HUYPENS P,MOENS K,HEIMBERG H,et al.Adiponectin-mediated stimulation of AMP-activated protein kinase(AMPK)in pancreatic beta cells[J].Life Sci,2005,77(11):1273-1282.
[23]HOMMES D W,PEPPELENBOSCH M P,van DEVENTER SJ.Mitogen activated protein(MAP)kinase signal transduction pathways and novel anti-inflammatory targets[J].Gut,2003,52(1):144-151.
[24]LI T,WANG W,ZHAO J H,et al.Pseudolaric acid B inhibits T-cell mediated immune response in vivo via p38MAPKsignal cascades and PPARγactivation[J].Life Sci,2015,121:88-96.
[25]PANG X,LIU J,LI Y,et al.Emodin inhibits homocysteineinduced C-reactive protein generation in vascular smooth muscle cells by regulating PPARγexpression and ROS-ERK1/2/p38signal pathway[J].PLoS One,2014,10(7):e0131295.
[26]DAHABREH D F,MEDH J D.Activation of peroxisome proliferator activated receptor-gamma results in an atheroprotective apolipoprotein profile in HepG2cells[J].Adv Biol Chem,2012,2(3):218-225.
[27]GUERRE-MILLO M.Adiponectin:an update[J].Diabetes Metab,2008,34(1):12-18.