基于AMPK/ACC信号通路探讨夏枯草提取物调节ZDF大鼠脂代谢的机制
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摘要
目的:观察夏枯草提取物(Prunellae Spica extracts,PS)对Zucker糖尿病肥胖(ZDF)模型大鼠肝腺苷酸活化蛋白激酶(AMPK)/乙酰辅酶A羧化酶(ACC)信号通路的影响以探讨其改善大鼠脂代谢的机制。方法:32只雄性ZDF(fa/fa)2型糖尿病模型大鼠随机分为模型组、二甲双胍组(180 mg·kg~(-1)·d~(-1))和PS低、高剂量组(12.25,24.5 mg·kg~(-1)·d~(-1)),每组8只。8只Zuker Lean(ZL)大鼠设为正常组。于给药0,4,8周测体质量及空腹血糖。给药8周后,腹主动脉取血,离心抽取血清-20℃冻存,取肝组织-80℃冻存,及4%多聚甲醛固定,石蜡包埋。放射免疫法测血清甘油三酯(TG),胆固醇(CHO),低密度脂蛋白胆固醇(LDL-C)及游离脂肪酸(FFA)。油红O染色观察肝细胞脂滴含量。实时荧光定量聚合酶链式反应(Real-time PCR)检测肝脏AMPKα_2及ACC mRNA表达。免疫组化法测肝细胞磷酸化(p)-AMPKα蛋白表达。结果:与正常组比较,模型组大鼠血清TG,CHO,LDL-C及FFA升高,肝细胞脂滴增加,肝AMPKα_2 mRNA表达减少而ACC1和ACC2 mRNA表达增加,p-AMPKα蛋白表达减少(P<0.05,P<0.01)。与模型组比较,PS低、高剂量组可明显降低ZDF大鼠血清TG,CHO,LDL-C及FFA,减少肝细胞脂滴,上调肝AMPKα_2 mRNA且下调ACC1和ACC2 mRNA表达,上调p-AMPKα蛋白表达(P<0.05,P<0.01)。结论:PS能有效改善2型糖尿病ZDF大鼠肝脏脂代谢紊乱,其机制可能与调节肝AMPK/ACC信号通路有关。
Objective: To observe the effect of Prunellae Spica extracts( PS) on the lipid metabolism in Zuker Diabetes Fatty( ZDF) rats based on AMP-activated protein kinase/acetyl Co A carboxylase( AMPK/ACC)signaling pathway. Method: The 32 male ZDF( fa/fa) type 2 diabetic rats were randomly divided into model group,metformin group( 180 mg·kg~(-1)·d~(-1)),and low and high-dose PS groups( 12. 25,24. 5 mg·kg~(-1)·d~(-1)),with 8 in each group. 8 male Zuker Lean( ZL) rats were selected as normal group. Body weight and fasting blood glucose were monitored at the 0~(th),4~(th) and 8~(th) weeks after administration. After 8 weeks,abdominal aorta blood was collected,serum was frozen at -20 ℃ by centrifugation,liver tissue was frozen at -80 ℃,fixed with 4%paraformaldehyde and embedded in paraffin. Serum triglyceride( TG),cholesterol( CHO),low density lipoprotein cholesterol( LDL-C) and free fatty acid( FFA) levels were measured by radioimmunoassay. Fat droplets in hepatocytes were measured by oil red O staining. Gene expressions of AMP-activated protein kinase-alpha 2( AMPKα_2),Acetyl Co A carboxylase( ACC) in liver were detected by real-time polymerase chain reaction( Realtime PCR). Protein expressions of p-AMPKα were observed by immuno-histochemical( IHC) method. Result:Compared with the normal group,the T2DM model group showed significant increases in serum levels of TG,CHO,LDL-C,FFA and lipid droplets in hepatocytes. AMPKα_2 mRNA expression was decreased,while ACC1 and ACC2 mRNA expressions were increased significantly. p-AMPKα protein expression in liver was decreased significantly( P < 0. 05,P < 0. 01). Compared with the model group,low and high-dose Ps groups showed significant decreases in serum levels of TG,CHO,LDL-C,FFA and lipid droplets in hepatocytes in ZDF rats,up-regulation in mRNA expression of AMPKα_2,down-regulation in mRNA expressions of ACC1 and ACC2,and up-regulation in protein expression of p-AMPKα( P < 0. 05,P < 0. 01). Conclusion: PS can effectively improve liver lipid metabolism in ZDF rats. Its mechanism may be related to the regulation of AMPK/ACC signaling pathway in liver.
Objective: To observe the effect of Prunellae Spica extracts( PS) on the lipid metabolism in Zuker Diabetes Fatty( ZDF) rats based on AMP-activated protein kinase/acetyl Co A carboxylase( AMPK/ACC)signaling pathway. Method: The 32 male ZDF( fa/fa) type 2 diabetic rats were randomly divided into model group,metformin group( 180 mg·kg~(-1)·d~(-1)),and low and high-dose PS groups( 12. 25,24. 5 mg·kg~(-1)·d~(-1)),with 8 in each group. 8 male Zuker Lean( ZL) rats were selected as normal group. Body weight and fasting blood glucose were monitored at the 0~(th),4~(th) and 8~(th) weeks after administration. After 8 weeks,abdominal aorta blood was collected,serum was frozen at -20 ℃ by centrifugation,liver tissue was frozen at -80 ℃,fixed with 4%paraformaldehyde and embedded in paraffin. Serum triglyceride( TG),cholesterol( CHO),low density lipoprotein cholesterol( LDL-C) and free fatty acid( FFA) levels were measured by radioimmunoassay. Fat droplets in hepatocytes were measured by oil red O staining. Gene expressions of AMP-activated protein kinase-alpha 2( AMPKα_2),Acetyl Co A carboxylase( ACC) in liver were detected by real-time polymerase chain reaction( Realtime PCR). Protein expressions of p-AMPKα were observed by immuno-histochemical( IHC) method. Result:Compared with the normal group,the T2DM model group showed significant increases in serum levels of TG,CHO,LDL-C,FFA and lipid droplets in hepatocytes. AMPKα_2 mRNA expression was decreased,while ACC1 and ACC2 mRNA expressions were increased significantly. p-AMPKα protein expression in liver was decreased significantly( P < 0. 05,P < 0. 01). Compared with the model group,low and high-dose Ps groups showed significant decreases in serum levels of TG,CHO,LDL-C,FFA and lipid droplets in hepatocytes in ZDF rats,up-regulation in mRNA expression of AMPKα_2,down-regulation in mRNA expressions of ACC1 and ACC2,and up-regulation in protein expression of p-AMPKα( P < 0. 05,P < 0. 01). Conclusion: PS can effectively improve liver lipid metabolism in ZDF rats. Its mechanism may be related to the regulation of AMPK/ACC signaling pathway in liver.
引文
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[2]佚名.中国2型糖尿病防治指南(2017年版)[J].中国实用内科杂志,2018,38(4):292-344.
[3]李咏梅,肖冰梅.夏枯草的药用研究概述[J].中国医药指南,2013,11(19):479-480.
[4]ZHENG J,HE J,JI B,et al.Antihyperglycemic activity of Prunella vulgaris L.in streptozotocin-induced diabetic mice[J].Asia Pac J Clin Nutr,2007,16(S1):427-431.
[5]吴慧平,陈美娟,郜明,等.夏枯草水提物延缓正常ICR小鼠单糖吸收作用研究[J].中药材,2010,33(5):782-785.
[6]谭剑斌,赵敏,杨杏芬,等.夏枯草对氧化应激损伤的保护作用研究[J].中国实验方剂学杂志,2016,22(4):89-94.
[7]张明发,沈雅琴.齐墩果酸和熊果酸调血脂、抗肥胖药理作用研究进展[J].药物评价研究,2015,38(1):90-97.
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[9]陆付耳.中医治疗糖尿病从强调“益气养阴”到兼顾“解毒扶阳”[J].中国中西医结合杂志,2009,29(4):293-295.
[10]庞博,赵进喜,王颖辉,等.糖尿病清热解毒治法探讨[J].中华中医药杂志,2011,26(7):1471-1474.
[11]于淼,朴春丽,南征.2型糖尿病胰岛素抵抗从毒损肝络论治的理论初探[J].上海中医药杂志,2007,41(3):18-20.
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[13]王海焱,姜月滢,宋亚南,等.糖耐康颗粒干预糖尿病患者前期糖耐量异常临床观察[J].世界科学技术—中医药现代,2016,18(11):1839-1844.
[14]李林忆,吴欣莉,秦灵灵,等.基于能量限制效应考察中药复方糖耐康干预db/db小鼠胰岛素抵抗作用机制[J].中国科学:生命科学,2016,46(8):949-958.
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[16]Yokoi N,Hoshino M,Hidaks S,et al.A novel rat model of type 2 diabetes:the zucker fatty diabetes mellitus zfdm rat[J].J Diabetes Res,2013,doi:10.1155/2013/103731.
[17]Hardie D G.AMP-activated protein kinase:maintaining energy homeostasis at the cellular and whole-body levels[J].Annu Rev Nutr,2014,34:31-55.
[18]QIU S L.AMP-activated protein kinase alpha 2 protects against liver injury from metastasized tumors via reduced glucose deprivation-induced oxidative stress[J].J Biol Chem,2014,289(13):9449-9459.
[19]Grahame H D.AMP-activated protein kinase:a key regulator of energy balance with many roles in human disease[J].Intern Med,2014,276(6):543-559.
[20]Wakil S J,Abu-Elheiga L A.Fatty acid metabolism:target for metabolic syndrome[J].J Lipid Res,2009,50:S138-S143.
[21]Kim K H.Regulation of mammalian acetyl-coenzyme A carboxylase[J].Annu Rev Nutr,1997,17:77-99.
[22]Bianchi A.Identification of an isozymic form of acetyl Co A carboxylase[J].J Biol Chem,1990,265(3):1502-1509.
[23]Harwood H J.Jr Treating the metabolic syndrome:Acetyl-Co A carboxylase inhibition[J].Expert Opin Ther Targets,2005,9(2):267-281.
[24]Mc Garry J D,Brown N F.The mitochondrial carnitine palmitoyltransferase system.From concept to molecular analysis[J].Eur J Biochem,1997,244(1):1-14.
[25]Harriman G,Greenwood J,Bhat S,et al.Acetyl-Co A carboxylase inhibition by ND-630 reduces hepatic steatosis,improves insulin sensitivity,and modulates dyslipidemia in rats[J].Proc Natl Acad Sci USA,2016,113(13):E1796-E1805.
[26]Hawley S A,Fullerton M D,Ross F A,et al.The ancient drug salicylate directly activates AMP-activated protein kinase[J].Science,2012,336(6083):918-922.
[27]朱伟芬.宫内营养不良及早期GH干预对大鼠AMPK-α1/SREBP-1c/ACC-1通路的影响及机制[D].杭州:浙江大学,2015.
[28]Cho K,Kim S J,Park S H,et al.Protective effect of codonopsis lanceolata root extract against alcoholic fatty liver in the rat[J].J Med Food,2009,12(6):1293-1301.
[29]潘洪彬,赵素梅,黄英,等.饲粮能量水平对乌金猪脂肪组织脂类合成代谢相关基因表达的影响[J].动物营养学报,2011,23(5):781-788.
[30]LI J,Grigoryev D N,Ye S Q,et al.Chronic intermittent hypoxia upregulates genes of lipid biosynthesis in obese mice[J].J Appl Physiol,2005,99(5):1643-1648.
[31]许光远,孙文,郭璇,等.青钱柳总皂苷对游离脂肪酸诱导的H4-ⅡE细胞脂肪代谢的影响及作用机制[J].中国实验方剂学杂志,2017,23(15):124-129.
[2]佚名.中国2型糖尿病防治指南(2017年版)[J].中国实用内科杂志,2018,38(4):292-344.
[3]李咏梅,肖冰梅.夏枯草的药用研究概述[J].中国医药指南,2013,11(19):479-480.
[4]ZHENG J,HE J,JI B,et al.Antihyperglycemic activity of Prunella vulgaris L.in streptozotocin-induced diabetic mice[J].Asia Pac J Clin Nutr,2007,16(S1):427-431.
[5]吴慧平,陈美娟,郜明,等.夏枯草水提物延缓正常ICR小鼠单糖吸收作用研究[J].中药材,2010,33(5):782-785.
[6]谭剑斌,赵敏,杨杏芬,等.夏枯草对氧化应激损伤的保护作用研究[J].中国实验方剂学杂志,2016,22(4):89-94.
[7]张明发,沈雅琴.齐墩果酸和熊果酸调血脂、抗肥胖药理作用研究进展[J].药物评价研究,2015,38(1):90-97.
[8]黎梅桂,魏刚,黄敏怡.夏枯草对肥胖小鼠糖脂代谢的影响[J].北方药学,2016,13(3):118-120.
[9]陆付耳.中医治疗糖尿病从强调“益气养阴”到兼顾“解毒扶阳”[J].中国中西医结合杂志,2009,29(4):293-295.
[10]庞博,赵进喜,王颖辉,等.糖尿病清热解毒治法探讨[J].中华中医药杂志,2011,26(7):1471-1474.
[11]于淼,朴春丽,南征.2型糖尿病胰岛素抵抗从毒损肝络论治的理论初探[J].上海中医药杂志,2007,41(3):18-20.
[12]田硕,刘铜华,孙文,等.夏枯草提取物对2型糖尿病ZDF大鼠肝糖原代谢的影响[J].中国实验方剂学杂志,2018,24(10):101-106.
[13]王海焱,姜月滢,宋亚南,等.糖耐康颗粒干预糖尿病患者前期糖耐量异常临床观察[J].世界科学技术—中医药现代,2016,18(11):1839-1844.
[14]李林忆,吴欣莉,秦灵灵,等.基于能量限制效应考察中药复方糖耐康干预db/db小鼠胰岛素抵抗作用机制[J].中国科学:生命科学,2016,46(8):949-958.
[15]刘敬顺等.复方夏枯草降糖胶囊治疗2型糖尿病阴虚热盛证的临床研究[D].南京:南京中医药大学,2002.
[16]Yokoi N,Hoshino M,Hidaks S,et al.A novel rat model of type 2 diabetes:the zucker fatty diabetes mellitus zfdm rat[J].J Diabetes Res,2013,doi:10.1155/2013/103731.
[17]Hardie D G.AMP-activated protein kinase:maintaining energy homeostasis at the cellular and whole-body levels[J].Annu Rev Nutr,2014,34:31-55.
[18]QIU S L.AMP-activated protein kinase alpha 2 protects against liver injury from metastasized tumors via reduced glucose deprivation-induced oxidative stress[J].J Biol Chem,2014,289(13):9449-9459.
[19]Grahame H D.AMP-activated protein kinase:a key regulator of energy balance with many roles in human disease[J].Intern Med,2014,276(6):543-559.
[20]Wakil S J,Abu-Elheiga L A.Fatty acid metabolism:target for metabolic syndrome[J].J Lipid Res,2009,50:S138-S143.
[21]Kim K H.Regulation of mammalian acetyl-coenzyme A carboxylase[J].Annu Rev Nutr,1997,17:77-99.
[22]Bianchi A.Identification of an isozymic form of acetyl Co A carboxylase[J].J Biol Chem,1990,265(3):1502-1509.
[23]Harwood H J.Jr Treating the metabolic syndrome:Acetyl-Co A carboxylase inhibition[J].Expert Opin Ther Targets,2005,9(2):267-281.
[24]Mc Garry J D,Brown N F.The mitochondrial carnitine palmitoyltransferase system.From concept to molecular analysis[J].Eur J Biochem,1997,244(1):1-14.
[25]Harriman G,Greenwood J,Bhat S,et al.Acetyl-Co A carboxylase inhibition by ND-630 reduces hepatic steatosis,improves insulin sensitivity,and modulates dyslipidemia in rats[J].Proc Natl Acad Sci USA,2016,113(13):E1796-E1805.
[26]Hawley S A,Fullerton M D,Ross F A,et al.The ancient drug salicylate directly activates AMP-activated protein kinase[J].Science,2012,336(6083):918-922.
[27]朱伟芬.宫内营养不良及早期GH干预对大鼠AMPK-α1/SREBP-1c/ACC-1通路的影响及机制[D].杭州:浙江大学,2015.
[28]Cho K,Kim S J,Park S H,et al.Protective effect of codonopsis lanceolata root extract against alcoholic fatty liver in the rat[J].J Med Food,2009,12(6):1293-1301.
[29]潘洪彬,赵素梅,黄英,等.饲粮能量水平对乌金猪脂肪组织脂类合成代谢相关基因表达的影响[J].动物营养学报,2011,23(5):781-788.
[30]LI J,Grigoryev D N,Ye S Q,et al.Chronic intermittent hypoxia upregulates genes of lipid biosynthesis in obese mice[J].J Appl Physiol,2005,99(5):1643-1648.
[31]许光远,孙文,郭璇,等.青钱柳总皂苷对游离脂肪酸诱导的H4-ⅡE细胞脂肪代谢的影响及作用机制[J].中国实验方剂学杂志,2017,23(15):124-129.