推拿改善T2DM大鼠糖脂代谢性炎性损伤的分子机制研究
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摘要
目的:观察推拿对2型糖尿病(Type 2 Diabetes Mellitus,T2DM)大鼠腹直肌组织腺苷酸活化蛋白激酶_(α2)(AMPK_(α2))、核因子-κB(Nuclear Factor-κB,NF-κB)及白细胞介素-6(Interleukin-6,IL-6)基因转录及蛋白表达的影响,探讨其改善T2DM大鼠代谢性炎性损伤的分子机制。方法:选取40只Wistar大鼠,随机取出6只为空白组,其余高脂高糖饮食联合低剂量(35 mg/kg)链脲佐菌素腹腔注射建立T2DM模型,成模大鼠32只随机分入模型组、西药组、推拿组及西药推拿组。空白组及模型组每日灌胃蒸馏水,西药组每天以250 mg/kg标准进行二甲双胍灌胃1次,推拿组每日给予推拿手法治疗1次,西药推拿组在相同给药标准基础上每日加用推拿治疗1次。共干预8周,分别记录第2、4、8周的空腹血糖(Fast Blood Glucose,FBG)值,并运用realtime-PCR测定腹直肌AMPK_(α2)、NF-κB及IL-6基因转录,Western-blot测定腹直肌AMPKα2及NF-κB蛋白含量,ELISA测定IL-6蛋白含量。结果:与模型组比较,西药组及西药推拿组FBG在第2、4、8周时显著下降,差异有统计学意义(P <0. 05,P <0. 01),推拿组FBG在第4、8周时显著下降,差异有统计学意义(P <0. 05);与西药组比较,西药推拿组在第4周降糖效果更为显著,差异有统计学意义(P <0. 05);与推拿组比较,西药推拿组在第4、8周时降糖效果更为显著,差异有统计学意义(P <0. 05);与0周时比较,各观察组FBG在第2、4、8周时,均显著降低,差异有统计学意义(P <0. 05,P <0. 01)。与空白组比较,模型组AMPK_(α2)mRNA转录及蛋白表达均下降,差异有统计学意义(P <0. 05);与模型组比较,西药组、推拿组、西药推拿组AMPK_(α2)mRNA转录及蛋白表达均升高,差异有统计学意义(P <0. 05)。与空白组比较,模型组NF-κBmRNA转录显著升高,差异有统计学意义(P <0. 01),蛋白表达升高,差异有统计学意义(P <0. 05);与模型组比较,西药组NF-κBmRNA转录下降,差异有统计学意义(P <0. 05);与模型组比较,推拿组NF-κBmRNA转录及蛋白表达均下降,差异有统计学意义(P <0. 05);与模型组比较,西药推拿组NF-κBmRNA转录和蛋白表达显著下降,差异有统计学意义(P <0. 01,P <0. 05);与空白组比较,模型组IL-6 mRNA转录升高,差异有统计学意义(P <0. 05),蛋白表达显著升高,差异有统计学意义(P <0. 01);与模型组比较,西药组、推拿组及西药推拿组IL-6基因转录均显著下降,差异有统计学意义(P <0. 05,P <0. 01),蛋白表达均显著下降,差异有统计学意义(P <0. 01)。结论:推拿可改善T2DM大鼠的空腹血糖,并通过上调骨骼肌AMPK_(α2)基因转录与蛋白表达,下调NF-κB、IL-6基因转录与蛋白表达,发挥其改善糖脂代谢性炎性损伤的手法效应。
Objective: To observe the effects of Tuina on the gene transcription and protein expression of 5'AMP-activated protein kinaseα2( AMPKα2),nuclear factor-κB( NF-κB) and interleukin( IL-6) in rectus abdominis muscle of type 2 Diabetes Mellitus( T2DM) rats. To explore the molecular mechanism of Tuina on improving the metabolic inflammatory injury in T2DM. Methods: A total of 6 Wistar rats out of 40 were randomly selected as the blank group( CON). Others were feed with high-fat and high-sugar diets combined with low-dose( 35 mg/kg) streptozotocin to establish a T2DM model. A total of 32 successfully modeled rats were randomly divided into the model group( MOD),western medicine group( MET),and Tuina group( TNA) and western medicine combined Tuina group( MIX). The CON and the MOD were perfused with distilled water daily. The MET was daily given a dose of250 mg/kg for metformin,and the TNA was given daily for manual treatment,and the MIX was given both intervention from MET and TNA. After 8 weeks of intervention,the values of fast blood glucose( FBG) at 2 nd,4 th,and 8 thweek were recorded,and realtime PCR was used to measure the gene transcription of AMPKα2,NF-κB,and IL-6 in the rectus abdominis. Western-blot was used to measure the protein expression of AMPKα2 and NF-κB in the rectus abdominis muscle,and the protein expression of IL-6 was determined by ELISA. Results: Compared with the MOD,the FBG of the MET and the MIX decreased significantly at the 2 nd,4 th,and 8 thweek( P < 0. 05,P < 0. 01),and the FBG of the TNA decreased significantly at the 4 th and 8 th week( P < 0. 05). Compared with the MET,the MIX had a more significant hypoglycemic effect at the 4 th week( P < 0. 05),and compared with the TNA,the MIX had a more significant hypoglycemic effect at the 4 th and 8 th week( P < 0. 05). Compared with 0 weeks,FBG of each treatment group was significantly decreased at the 2 nd,4 th,and 8 thweek( P < 0. 05,P < 0. 01). Compared with the CON,the mRNA and protein expression of AMPKα2 in the MOD was decreased( P < 0. 05). Compared with the MOD,the mRNA and protein expression of AMPKα2 in the MET,the TNA and the MIX were increased( P < 0. 05). Compared with the CON,the transcription of NF-κB mRNA in the MOD was significantly increased( P < 0. 01),and the protein expression was increased( P < 0. 05). Compared with the MOD,the transcription of NF-κB mRNA in the MET was decreased( P < 0. 05). Compared with the MOD,the transcription of NF-κB mRNA in the MET was significantly decreased( P < 0. 01),and the protein expression was decreased( P < 0. 05). Compared with the CON,the IL-6 mRNA expression in the MOD was increased( P < 0. 05),and the protein expression was significantly increased( P < 0. 01). Compared with the MOD,the IL-6 gene transcription in the MET,the TNA and the MIX was all significantly decreased( P < 0. 05,P < 0. 01),and protein expression was also decreased significantly( P < 0. 01). Conclusion: Tuina can improve the FBG of T2DM rats,and up-regulate the gene transcription and protein expression of AMPKα2 in skeletal muscle,downregulate the gene transcription and protein expression of NF-κB and IL-6,in order to improve the metabolic damage caused by glycolipid toxicity.
Objective: To observe the effects of Tuina on the gene transcription and protein expression of 5'AMP-activated protein kinaseα2( AMPKα2),nuclear factor-κB( NF-κB) and interleukin( IL-6) in rectus abdominis muscle of type 2 Diabetes Mellitus( T2DM) rats. To explore the molecular mechanism of Tuina on improving the metabolic inflammatory injury in T2DM. Methods: A total of 6 Wistar rats out of 40 were randomly selected as the blank group( CON). Others were feed with high-fat and high-sugar diets combined with low-dose( 35 mg/kg) streptozotocin to establish a T2DM model. A total of 32 successfully modeled rats were randomly divided into the model group( MOD),western medicine group( MET),and Tuina group( TNA) and western medicine combined Tuina group( MIX). The CON and the MOD were perfused with distilled water daily. The MET was daily given a dose of250 mg/kg for metformin,and the TNA was given daily for manual treatment,and the MIX was given both intervention from MET and TNA. After 8 weeks of intervention,the values of fast blood glucose( FBG) at 2 nd,4 th,and 8 thweek were recorded,and realtime PCR was used to measure the gene transcription of AMPKα2,NF-κB,and IL-6 in the rectus abdominis. Western-blot was used to measure the protein expression of AMPKα2 and NF-κB in the rectus abdominis muscle,and the protein expression of IL-6 was determined by ELISA. Results: Compared with the MOD,the FBG of the MET and the MIX decreased significantly at the 2 nd,4 th,and 8 thweek( P < 0. 05,P < 0. 01),and the FBG of the TNA decreased significantly at the 4 th and 8 th week( P < 0. 05). Compared with the MET,the MIX had a more significant hypoglycemic effect at the 4 th week( P < 0. 05),and compared with the TNA,the MIX had a more significant hypoglycemic effect at the 4 th and 8 th week( P < 0. 05). Compared with 0 weeks,FBG of each treatment group was significantly decreased at the 2 nd,4 th,and 8 thweek( P < 0. 05,P < 0. 01). Compared with the CON,the mRNA and protein expression of AMPKα2 in the MOD was decreased( P < 0. 05). Compared with the MOD,the mRNA and protein expression of AMPKα2 in the MET,the TNA and the MIX were increased( P < 0. 05). Compared with the CON,the transcription of NF-κB mRNA in the MOD was significantly increased( P < 0. 01),and the protein expression was increased( P < 0. 05). Compared with the MOD,the transcription of NF-κB mRNA in the MET was decreased( P < 0. 05). Compared with the MOD,the transcription of NF-κB mRNA in the MET was significantly decreased( P < 0. 01),and the protein expression was decreased( P < 0. 05). Compared with the CON,the IL-6 mRNA expression in the MOD was increased( P < 0. 05),and the protein expression was significantly increased( P < 0. 01). Compared with the MOD,the IL-6 gene transcription in the MET,the TNA and the MIX was all significantly decreased( P < 0. 05,P < 0. 01),and protein expression was also decreased significantly( P < 0. 01). Conclusion: Tuina can improve the FBG of T2DM rats,and up-regulate the gene transcription and protein expression of AMPKα2 in skeletal muscle,downregulate the gene transcription and protein expression of NF-κB and IL-6,in order to improve the metabolic damage caused by glycolipid toxicity.
引文
[1]汪会琴,胡如英,武海滨,等.2型糖尿病报告发病率研究进展[J].浙江预防医学,2016,28(1):37-39,57.
[2]Chatterjee S,Khunti K,Davies MJ.Type 2 diabetes[J].Lancet,2017,389(10085):2239-2251.
[3]Romeo GR,Lee J,Shoelson SE.Metabolic syndrome,insulin resistance,and roles of inflammation--mechanisms and therapeutic targets[J].Arterioscler Thromb Vasc Biol,2012,32(8):1771-1776.
[4]Herzig S,Shaw RJ.AMPK:guardian of metabolism and mitochondrial homeostasis[J].Nat Rev Mol Cell Biol,2018,19(2):121-135.
[5]王康,国生,钮妍,等.振腹推拿对T2DM大鼠骨骼肌AMPK/Glut4信号链的影响[J].环球中医药,2017,10(11):1224-1228.
[6]Kahn SE,Cooper ME,Del PS.Pathophysiology and treatment of type2 diabetes:perspectives on the past,present,and future[J].Lancet,2014,383(9922):1068-1083.
[7]Bensellam M,Laybutt DR,Jonas JC.The molecular mechanisms of pancreaticβ-cell glucotoxicity:recent findings and future research directions[J].Mol Cell Endocrinol,2012,364(1-2):1-27.
[8]唐佳雯,朱涛.核因子κB抑制物在糖尿病周围神经病变中的研究进展[J].医学综述,2017,23(8):1613-1617.
[9]Major B,Rattazzi L,Brod S,Pilipovi'c I,Leposavi'c G,D'Acquisto F.Massage-like stroking boosts the immune system in mice[J].Sci Rep,2015,5:10913.
[10]王朝辉,齐伟,韩东岳,宋柏林,王之虹.经穴推拿对肥胖2型糖尿病患者血清炎症因子的影响[J].南京中医药大学学报,2014,30(2):111-113.
[11]王朝辉,齐伟,韩东岳,等.腹部经穴推拿联合盐酸二甲双胍对肥胖2型糖尿病患者脂联素蛋白表达的影响[J].上海中医药杂志,2014,48(3):45-47.
[12]Lund SS,Tarnow L,Stehouwer CD,et al.Impact of metformin versus repaglinide on non-glycaemic cardiovascular risk markers related to inflammation and endothelial dysfunction in non-obese patients with type 2 diabetes[J].Eur J Endocrinol,2008,158(5):631-641.
[13]Green CJ,Pedersen M,Pedersen BK,et al.Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase[J].Diabetes,2011,60(11):2810-2819.
[14]Garcia D,Shaw RJ.AMPK:Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance[J].Mol Cell,2017,66(6):789-800.
[15]Cokorinos EC,Delmore J,Reyes AR,et al.Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice[J].Cell Metab,2017,25(5):1147-1159.e10.
[16]宋柏林,朴春丽,陈曦,等.推拿配合二甲双胍治疗肥胖2型糖尿病患者80例临床观察[J].世界中西医结合杂志,2011,6(3):206-209.
[17]商德俊,李页,冯科.“减肥八穴”穴位埋线结合健脾祛湿推拿法治疗单纯性肥胖症(脾虚湿盛型)的疗效观察[J].中医药导报,2016,22(1):57-59.
[18]宋柏林,朴春丽,于淼,等.从改善脂肪组织炎症方面探讨推拿手法治疗肥胖2型糖尿病胰岛素抵抗机制[J].中国老年学杂志,2011,31(6):1084-1085.
[19]李欣,刘师伟.肌肉因子Irisin与糖尿病血管并发症的最新研究进展[J].中国药物与临床,2017,17(7):987-989.
[20]张怡,孙易,丁树哲.新的肌肉因子---鸢尾素[J].中国生物化学与分子生物学报,2017,33(5):429-435.
[21]宋威巍,柏友萍.Chemerin、肥胖与运动研究进展[J].安徽师范大学学报:自然科学版,2018,41(1):98-102.
[22]宫计划,徐昭.2型糖尿病患者血清脂肪细胞因子Chemerin表达水平及临床价值[J].中国医学创新,2018,15(27):22-25.
[2]Chatterjee S,Khunti K,Davies MJ.Type 2 diabetes[J].Lancet,2017,389(10085):2239-2251.
[3]Romeo GR,Lee J,Shoelson SE.Metabolic syndrome,insulin resistance,and roles of inflammation--mechanisms and therapeutic targets[J].Arterioscler Thromb Vasc Biol,2012,32(8):1771-1776.
[4]Herzig S,Shaw RJ.AMPK:guardian of metabolism and mitochondrial homeostasis[J].Nat Rev Mol Cell Biol,2018,19(2):121-135.
[5]王康,国生,钮妍,等.振腹推拿对T2DM大鼠骨骼肌AMPK/Glut4信号链的影响[J].环球中医药,2017,10(11):1224-1228.
[6]Kahn SE,Cooper ME,Del PS.Pathophysiology and treatment of type2 diabetes:perspectives on the past,present,and future[J].Lancet,2014,383(9922):1068-1083.
[7]Bensellam M,Laybutt DR,Jonas JC.The molecular mechanisms of pancreaticβ-cell glucotoxicity:recent findings and future research directions[J].Mol Cell Endocrinol,2012,364(1-2):1-27.
[8]唐佳雯,朱涛.核因子κB抑制物在糖尿病周围神经病变中的研究进展[J].医学综述,2017,23(8):1613-1617.
[9]Major B,Rattazzi L,Brod S,Pilipovi'c I,Leposavi'c G,D'Acquisto F.Massage-like stroking boosts the immune system in mice[J].Sci Rep,2015,5:10913.
[10]王朝辉,齐伟,韩东岳,宋柏林,王之虹.经穴推拿对肥胖2型糖尿病患者血清炎症因子的影响[J].南京中医药大学学报,2014,30(2):111-113.
[11]王朝辉,齐伟,韩东岳,等.腹部经穴推拿联合盐酸二甲双胍对肥胖2型糖尿病患者脂联素蛋白表达的影响[J].上海中医药杂志,2014,48(3):45-47.
[12]Lund SS,Tarnow L,Stehouwer CD,et al.Impact of metformin versus repaglinide on non-glycaemic cardiovascular risk markers related to inflammation and endothelial dysfunction in non-obese patients with type 2 diabetes[J].Eur J Endocrinol,2008,158(5):631-641.
[13]Green CJ,Pedersen M,Pedersen BK,et al.Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase[J].Diabetes,2011,60(11):2810-2819.
[14]Garcia D,Shaw RJ.AMPK:Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance[J].Mol Cell,2017,66(6):789-800.
[15]Cokorinos EC,Delmore J,Reyes AR,et al.Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice[J].Cell Metab,2017,25(5):1147-1159.e10.
[16]宋柏林,朴春丽,陈曦,等.推拿配合二甲双胍治疗肥胖2型糖尿病患者80例临床观察[J].世界中西医结合杂志,2011,6(3):206-209.
[17]商德俊,李页,冯科.“减肥八穴”穴位埋线结合健脾祛湿推拿法治疗单纯性肥胖症(脾虚湿盛型)的疗效观察[J].中医药导报,2016,22(1):57-59.
[18]宋柏林,朴春丽,于淼,等.从改善脂肪组织炎症方面探讨推拿手法治疗肥胖2型糖尿病胰岛素抵抗机制[J].中国老年学杂志,2011,31(6):1084-1085.
[19]李欣,刘师伟.肌肉因子Irisin与糖尿病血管并发症的最新研究进展[J].中国药物与临床,2017,17(7):987-989.
[20]张怡,孙易,丁树哲.新的肌肉因子---鸢尾素[J].中国生物化学与分子生物学报,2017,33(5):429-435.
[21]宋威巍,柏友萍.Chemerin、肥胖与运动研究进展[J].安徽师范大学学报:自然科学版,2018,41(1):98-102.
[22]宫计划,徐昭.2型糖尿病患者血清脂肪细胞因子Chemerin表达水平及临床价值[J].中国医学创新,2018,15(27):22-25.