黄连碱改善脂肪肝SD大鼠模型的效应及机制研究
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摘要
目的:观察黄连碱对脂肪肝SD大鼠模型脂代谢指标的影响,探讨作用机制。方法:采用高脂饲养构建SD大鼠脂肪肝模型,随机分组为正常对照组、高脂模型组、黄连碱低、高剂量组(10 mg/kg、50 mg/kg),给药30 d,分析大鼠体重、肝指数、三酰甘油(TG)、肝功能(ALT,AST)及肝脏病理等指标;采用人肝癌HepG2细胞系,蛋白免疫印迹法(WB)观察黄连碱对AMP-活化蛋白激酶(AMPK)蛋白的表达,反转录荧光定量PCR(RT-PCR)方法分析肉碱脂酰转移酶1(CPT-1) mRNA、羟甲基戊二酰辅酶A(HMG-Coa) mRNA的表达,高效液相色谱(HPLC)测定细胞内AMP的含量。结果:与高脂饮食组比较,黄连碱低、高剂量组均降低了TG水平、改善AST以及肝脏组织学形态,且黄连碱高剂量组效果优于低剂量组。在HepG2细胞中,黄连碱显著提高AMP的浓度,激活AMPK蛋白磷酸化表达,上调CPT-1 mRNA的表达,抑制HMG-Coa mRNA的表达。结论:黄连碱具有显著改善大鼠脂肪肝的效应,作用机制可能与提高AMP的浓度,激活AMPK信号通路有关。
Objective: To observe the effect of coptisine on the fatty liver disea-se of SD (Sprague Dawley) rats and investigate the mechanism. Methods: The SD fatty liver rats were induced by high fat diet. Rats were randomly divided into normal control group,high fat model group,coptisine low and high dose groups (10 mg/kg or 50 mg/kg). After 30 days of administration,the body weight,liver index,triglyceride (TG),liver function (alanine transaminase (ALT),aspartate aminotransferase (AST) and liver pathology of each group were analyzed. The expression of coptisine to adenosine monophosphate activated protein kinase (AMPK)protein in human Hep G 2 cell line was estimated by Western Blot (WB),the expression of carnitine palmitoyltransferase-1 messenger Ribose Nucleic Acid (CPT-1 mRNA) and hydroxy-methylglutaryl coenzyme A messenger Ribose Nucleic Acid (HMG-CoA mRNA) was analyzed by Real Time-Poly Polymerase Chain Reaction (RT-PCR),and the content of adenosine monophosphate (AMP) in the cell was determined by high performance liquid chromatography (HPLC). Results: Compared with the high-fat diet group,the two groups of coptisine intervention reduced the TG level of blood lipid,AST,and triglyceride content in liver,and significantly improved the histological morphology of liver in high-fat feeding. The high-dose group was better than the low-dose group.The Hep G2 cells were treated with coptisine,AMP concentration was significantly increased,the activation of AMPK protein phosphorylation was increased,the expression of CPT-1 mRNA expression was also increased,and the expression of HMG-CoA mRNA was inhibited (P < 0. 05). Conclusion: The coptisine can remarkably improve fatty liver in rats,and its mechanism may be related to the increasing of AMP concentration and the activation of AMPK signaling pathway.
Objective: To observe the effect of coptisine on the fatty liver disea-se of SD (Sprague Dawley) rats and investigate the mechanism. Methods: The SD fatty liver rats were induced by high fat diet. Rats were randomly divided into normal control group,high fat model group,coptisine low and high dose groups (10 mg/kg or 50 mg/kg). After 30 days of administration,the body weight,liver index,triglyceride (TG),liver function (alanine transaminase (ALT),aspartate aminotransferase (AST) and liver pathology of each group were analyzed. The expression of coptisine to adenosine monophosphate activated protein kinase (AMPK)protein in human Hep G 2 cell line was estimated by Western Blot (WB),the expression of carnitine palmitoyltransferase-1 messenger Ribose Nucleic Acid (CPT-1 mRNA) and hydroxy-methylglutaryl coenzyme A messenger Ribose Nucleic Acid (HMG-CoA mRNA) was analyzed by Real Time-Poly Polymerase Chain Reaction (RT-PCR),and the content of adenosine monophosphate (AMP) in the cell was determined by high performance liquid chromatography (HPLC). Results: Compared with the high-fat diet group,the two groups of coptisine intervention reduced the TG level of blood lipid,AST,and triglyceride content in liver,and significantly improved the histological morphology of liver in high-fat feeding. The high-dose group was better than the low-dose group.The Hep G2 cells were treated with coptisine,AMP concentration was significantly increased,the activation of AMPK protein phosphorylation was increased,the expression of CPT-1 mRNA expression was also increased,and the expression of HMG-CoA mRNA was inhibited (P < 0. 05). Conclusion: The coptisine can remarkably improve fatty liver in rats,and its mechanism may be related to the increasing of AMP concentration and the activation of AMPK signaling pathway.
引文
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[20]王修婧.不同比率DHA/EPA干预高脂小鼠脂质代谢作用及机制[D].武汉:武汉轻工大学,2016.
[21]黄德强,罗凌玉,王丽丽,等.AMPK在胰岛素信号转导通路中的作用[J].中国细胞生物学学报,2011,33(11):1220-1229.
[22]Alam M A,Subhan N,Rahman M M,et al.Effect of Citrus Flavonoids,Naringin and Naringenin,on Metabolic Syndrome and Their Mechanisms of A-ction[J].Advances in Nutrition,2014,5(4):404-417.
[2]潘晓丽,向晖,谢运飞,等.黄连碱基于NF-κB信号通道的体外抗炎实验研究[J].中国抗生素杂志,2013,38(12):951-954.
[3]Jing G,Shou-Bao W,Tian-Yi Y,et al.Coptisine protects rat heart against myocardial ischemia/reperfusion injury by suppressing myocardial apoptosis and inflammation[J].Atherosclerosis,2013,231(2):384-391.
[4]Zhang Z H,Zhang H J,Deng A J,et al.Synthesis and Structure-Activity Relationships of Quaternary Coptisine Derivatives as Potential Anti-ulcerative Colitis Agents[J].Journal of Medicinal Chemistry,2015,58(18):7557-71.
[5]Jin Y,Liu S,Ma Q,et al.Berberine enhances the AMPK activation and autophagy and mitigates high glucose-induced apoptosis of mouse podocytes[J].European Journal of Pharmacology,2016,794:106-114.
[6]Chernyak B V,Antonenko Y N,Galimov E R,et al.Novel mitochondria-targeted compounds composed of natural constituents:conjugates of plant alkalo-ids berberine and palmatine with plastoquinone[J].Biochemistry Biokhimii⌒a,2012,77(9):983.
[7]杨红燕,秦兴华,杨兴斌,等.小檗碱通过减少O-GlcNAc修饰降低高糖诱导的HUVECs氧化应激并调节线粒体功能[J].中国病理生理杂志,2015,31(10):1822.
[8]Fan H,Chen Y Y,Bei W J,et al.In Vitro Screening for Antihepatic Ste-atosis Active Components within Coptidis Rhizoma Alkaloids Extract Using Li-ver Cell Extraction with HPLC Analysis and a Free Fatty Acid-Induced Hepatic Steatosis HepG2 Cell Assay[J].EvidenceBased Complementray and Alternat-ive Medicine,2013,2013(6):459390.
[9]Sotoacosta R,Bautistacarbajal P,Cervantessalazar M,et al.DENV upregu-lates the HMG-CoA reductase activity through the impairment of AMPK phos-phorylation:A potential antiviral target[J].Plos Pathogens,2017,13(4):e1006257.
[10]Li D,Zheng J,Hu Y.Amelioration of Intestinal Barrier Dysfunction by Berberine in the Treatment of Nonalcoholic Fatty Liver Disease in Rats[J].Ph-armacognosy Magazine,2017,13(52):677-682.
[11]Chen Z,Li C,Yang C,et al.Lipid Regulation Effects of Raw and Proc-essed Notoginseng Radix Et Rhizome on Steatotic Hepatocyte L02 Cell:[J].Bi-omed Res Int,2016,2016(11):2919034.
[12]刘玲,黄紫乐.齐墩果酸经线粒体损伤诱导SMMC-7721细胞凋亡的作用[J].中国临床药理学杂志,2017,33(2):144-148.
[13]Gong LL,Fang LH,Wang SB,et al.Coptisine exert cardioprotective eff-ect through anti-oxidative and inhibition of RhoA/Rho kinase pathway on isopr-oterenol-induced myocardial infarction in rats[J].Atherosclerosis,2012,222(1):50-58.
[14]Wang Z,Wang J,Chan P.Treating Type 2 Diabetes Mellitus with Tradit-ional Chinese and Indian Medicinal Herbs[J].EvidenceBased Complementray and Alternative Medicine,2013,2013(6):343594.
[15]Cheng P,Wang B,Liu X,et al.Facile synthesis of tetrahydroprotoberber-ine and protoberberine alkaloids from protopines and study on their antibacteri-al activities[J].Natural Product Research,2014,28(7):413-419.
[16]Tang F,Mei W,Tian D,et al.An Evidence-based Perspective of Coptis-Chinensis,(Chinese Goldthread)for Cancer Patients[M].Evidence-based Antic-ancer Materia Medica.Springer Netherlands,2011:111-130.
[17]Zhou L,Yang F,Li G,et al.Coptisine Induces Apoptosis in Human Hepatoma Cells Through Activating 67-kDa Laminin Receptor/c GMP Signaling[J].Frontiers in Pharmacology,2018,9:00517.
[18]杨凡,李欣,章婷婷,等.黄连碱经活性氧中介物-线粒体途径诱导肺癌NCI-H1650细胞凋亡[J].实用医学杂志,2017,33(24):4033-4037.
[19]孙常磊.PH区带逆流色谱分离纯化石蒜、黄连、豆豉姜和三颗针中的生物碱类化合物[D].济南:山东中医药大学,2016.
[20]王修婧.不同比率DHA/EPA干预高脂小鼠脂质代谢作用及机制[D].武汉:武汉轻工大学,2016.
[21]黄德强,罗凌玉,王丽丽,等.AMPK在胰岛素信号转导通路中的作用[J].中国细胞生物学学报,2011,33(11):1220-1229.
[22]Alam M A,Subhan N,Rahman M M,et al.Effect of Citrus Flavonoids,Naringin and Naringenin,on Metabolic Syndrome and Their Mechanisms of A-ction[J].Advances in Nutrition,2014,5(4):404-417.