人参皂苷Compound K通过AMPK及SREBP1途径改善非酒精性脂肪肝的机制研究
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摘要
目的:研究人参皂苷Compound K(CK)对非酒精性脂肪肝的改善作用及其与腺苷酸激活蛋白激酶(AMPK)及固醇调节元件结合蛋白(SREBP1)信号转导通路的关系。方法:采用高脂饲料喂养C57BL/6J雄性小鼠9周,建立非酒精性脂肪肝动物模型;9周后随机分为5组,正常饲料组(RD),高脂饲料组(HFD),给药组(HFD+CK 3,9,27 mg·kg~(-1)),每天灌胃给药1次,每周测2次体质量,连续11周。实验结束后,分别称量小鼠体质量和肝质量;检测三酰甘油(TG)、胆固醇(cholesterol)、非游离脂肪酸(NEFA)、谷草转氨酶(AST)和谷丙转氨酶(ALT)水平。苏木素-伊红(HE)染色观察肝脏病理变化,Western blot法检测AMPK和乙酰辅酶A羧化酶(ACC)的磷酸化表达,PCR法检测脂肪合成转录因子SREBP1及其靶基因(FAS、SCD1)的基因表达。结果:治疗11周后,与RD组比较, HFD组小鼠肝质量及肝质量与体质量的比值显著增加(P<0.001,P<0.05),HE染色显示HFD组较RD组肝细胞明显增大且伴有大泡性脂肪变,说明高脂饲料诱导非酒精性脂肪肝模型的建立。与HFD组比较,给药组小鼠体质量、肝质量显著降低(P<0.05),血脂(TG、CHO、NEFA)和肝功(sAST和sALT)指标也显著降低(P<0.05,P<0.01,P<0.001);HE染色也说明给药组能够明显改善肝脏病理状态,从而改善肝细胞脂肪变性;Western blot结果显示HFD组较RD组,AMPK和ACC磷酸化均被抑制,给药组较HFD组,AMPK和ACC均被磷酸化,且随药物浓度的增加,磷酸化越明显;PCR法结果显示HFD较RD组,脂肪合成转录因子SREBP1及其靶基因表达显著增强,而在给药组上述基因表达均显著被抑制。结论:CK对非酒精性脂肪肝具有改善作用,其机制之一可能是通过激活AMPK和ACC的磷酸化、抑制脂肪合成转录因子SREBP1及其靶基因的表达来实现的。
OBJECTIVE To investigate effect of CK on nonalcoholic fatty liver and its relationship with adenylate activated protein kinase(AMPK) and sterol regulatory element binding proteins 1(SREBP1) signal transduction pathway. METHODS C57BL/6J male mice were fed with high fat diet for 9 weeks to establish non-alcoholic fatty liver animal model. After 9 weeks, they were randomly divided into 5 groups, regular diet(RD) group, high fat diet(HFD) group, HFD+CK(3, 9, 27 mg·kg~(-1)) group. Administration of gastric infusion was executed once daily, and their weight were measured twice a week for 11 weeks. After the end of the experiment, the weight of liver tissue and body were measured, and the index of triglyceride(TG), cholesterol(Cholesterol), free fatty acid(NEFA) and aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were detected. Hematoxylin eosin(HE) staining was used to observe the pathological changes of liver. The phosphorylation of AMPK and acetyl coenzyme A carboxylase(ACC) were detected by western blot and the gene expression of the transcription factor SREBP1 and its target genes(FAS, SCD1, GPAT) were detected by RT-PCR. RESULTS After 11 weeks of treatment, compared with group RD, the HFD mice liver weight and the ratio of liver weight and body weight increased significantly(P<0.001, P<0.05). HE staining showed that the hepatocytes in the HFD group were significantly larger than those in the RD group with macrovesicular fatty change, indicating the establishment of the non-alcoholic fatty liver model induced by high-fat diet. Compared with HFD group, the body weight and liver weight of mice in treatment group were significantly decreased(P<0.05), and the indexes of blood lipid(TG, CHO, NEFA) and liver function(sAST and sALT) were also significantly decreased(P<0.05, P<0.01, P<0.001); HE staining also indicated that the treatment group could significantly improve the pathological state of liver and thus improve hepatocyte steatosis; Western blot results showed that AMPK and ACC phosphorylation were inhibited in HFD group compared with RD group, AMPK and ACC phosphorylation were both inhibited in HFD group compared with HFD group, and the phosphorylation was more significant with the increase of drug concentration; PCR results showed that the expression of lipogenesis transcription factor SREBP1 and its target gene was significantly increased in HFD group compared with RD group, but significantly inhibited in treatment group. CONCLUSION CK could ameliorate the nonalcoholic fatty liver disease, which may be achieved by activating phosphorylation of AMPK and ACC and inhibiting the expression of transcription factor SREBP1 and its target genes in adipose tissue.
OBJECTIVE To investigate effect of CK on nonalcoholic fatty liver and its relationship with adenylate activated protein kinase(AMPK) and sterol regulatory element binding proteins 1(SREBP1) signal transduction pathway. METHODS C57BL/6J male mice were fed with high fat diet for 9 weeks to establish non-alcoholic fatty liver animal model. After 9 weeks, they were randomly divided into 5 groups, regular diet(RD) group, high fat diet(HFD) group, HFD+CK(3, 9, 27 mg·kg~(-1)) group. Administration of gastric infusion was executed once daily, and their weight were measured twice a week for 11 weeks. After the end of the experiment, the weight of liver tissue and body were measured, and the index of triglyceride(TG), cholesterol(Cholesterol), free fatty acid(NEFA) and aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were detected. Hematoxylin eosin(HE) staining was used to observe the pathological changes of liver. The phosphorylation of AMPK and acetyl coenzyme A carboxylase(ACC) were detected by western blot and the gene expression of the transcription factor SREBP1 and its target genes(FAS, SCD1, GPAT) were detected by RT-PCR. RESULTS After 11 weeks of treatment, compared with group RD, the HFD mice liver weight and the ratio of liver weight and body weight increased significantly(P<0.001, P<0.05). HE staining showed that the hepatocytes in the HFD group were significantly larger than those in the RD group with macrovesicular fatty change, indicating the establishment of the non-alcoholic fatty liver model induced by high-fat diet. Compared with HFD group, the body weight and liver weight of mice in treatment group were significantly decreased(P<0.05), and the indexes of blood lipid(TG, CHO, NEFA) and liver function(sAST and sALT) were also significantly decreased(P<0.05, P<0.01, P<0.001); HE staining also indicated that the treatment group could significantly improve the pathological state of liver and thus improve hepatocyte steatosis; Western blot results showed that AMPK and ACC phosphorylation were inhibited in HFD group compared with RD group, AMPK and ACC phosphorylation were both inhibited in HFD group compared with HFD group, and the phosphorylation was more significant with the increase of drug concentration; PCR results showed that the expression of lipogenesis transcription factor SREBP1 and its target gene was significantly increased in HFD group compared with RD group, but significantly inhibited in treatment group. CONCLUSION CK could ameliorate the nonalcoholic fatty liver disease, which may be achieved by activating phosphorylation of AMPK and ACC and inhibiting the expression of transcription factor SREBP1 and its target genes in adipose tissue.
引文
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[1] Povero D,Feldstein AE.Novel Molecular Mechanisms in the Development of Non-Alcoholic Steatohepatitis [J].Diabetes Metab J,2016,40:1-11.
[2] Guzman G,Brunt EM,Petrovic LM,et al.Does nonalcoholic fatty liver disease predispose patients to hepatocellular carcinoma in the absence of cirrhosis?[J].Arch PatholLab Med,2008,132(11):1761-1766.
[3] Loomba R,Sanyal,AJ.The global NAFLD epidemic [J].Nat Rev Gastroenterol Hepatol,2013,10:686-690.
[4] Masarone M,Federico A,Abenavoli L,et al.Non alcoholic fatty liver:epidemiology and natural history [J].Rev Recent Clin Trials,2014,9:126-133.
[5] Li Z,Xue J,Chen P,et al.Prevalence of nonalcoholic fatty liver disease in mainland of China:a meta-analysis of published studies [J].J Gastroenterol Hepatol,2014,29:42-51.
[6] Rinella ME.Nonalcoholic fatty liver disease:a systematic review [J].JAMA,2015,313(22):2263-2273.
[7] Yang XD,Yang YY,Ouyang DS,et al.A review of biotransformation and pharmacology of ginsenoside compound K [J].Fitoterapia,2015,100:208-220.
[8] Wei S,Li Wl,Yu Y,et al.Ginsenoside Compound K suppresses the hepatic gluconeogenesis via activating adenosine-5′ monophosphate kinase:A study in vitro and in vivo [J].Life Sci,2015,139:8-15.
[9] Zhang GM,Zhu T,Chen GH,et al.The effect of ginsenoside Compound K on the formation of atherosclerosis [J].J Cadiovascular Pulmonary Dis (心肺血管病杂志),2017,5(36):408-413.
[10] Li XC,Zhang XY,Xu S,et al.Establishment of C57BL/6J mouse nonalcoholic fatty liver model [J].Chin J Veter Med (中国兽医杂志),2013,1(49):6-8.
[11] Day CP.Non-alcoholic fatty liver disease:a massive problem [J].Clin Med(Lond),2011,11(2):176-178.
[12] Guan QW,Jiang HL,Pan QW.Advances in the molecular mechanism and treatment of nonalcoholic fatty liver disease [J].J Mil Surg Southwest Chin(西南军医),2016,15(5):467-470.
[13] Mu Q,Fang X,Li X,et al.Ginsenoside Rb1 promotes browning through regulation of PPARγ in 3T3-L1 adipocytes [J].Biochem Biophys Res Commun,2015,466(3):530.
[14] Yu X,Ye L,Zhang H,et al.Ginsenoside Rb1 ameliorates liver fat accumulation by upregulating perilipin expression in adipose tissue of db/db obese mice [J].J Ginseng Res,2015,39 (3):199-205.
[15] Lin ZJ,Zhang B,Liu XQ.Research progress of AMPK-ACC signaling pathway and related metaolic diseases [J].Chin J Diabetes(中国糖尿病杂志),2013,21(5):474-477.
[16] Viollet B,Mounier R,Leclerc J,et al.Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders [J].Diabetes Metab,2007,33(6):395-402.
[17] Zhao WX,Yan L,Shao MY.Effect of huatan clearing damp huoxue recipe on ADPN/AMPK/ACC pathway in rats with nonalcoholic steatohepatitis [J].J Clin Hepatol (临床肝胆病杂志),2017,33(5):932-936.
[18] Brownsey RW,Boone AN,Elliott JE,et al.Regulation of acetyl-CoA carboxylase [J].Biochem Soc Trans,2006,34(Pt2):223-227.
[19] Shimano H.SREBPs:physiology and pathophysiology of the SREBP family [J].FEBS J,2009,276(3):616-621.
[1] Povero D,Feldstein AE.Novel Molecular Mechanisms in the Development of Non-Alcoholic Steatohepatitis [J].Diabetes Metab J,2016,40:1-11.
[2] Guzman G,Brunt EM,Petrovic LM,et al.Does nonalcoholic fatty liver disease predispose patients to hepatocellular carcinoma in the absence of cirrhosis?[J].Arch PatholLab Med,2008,132(11):1761-1766.
[3] Loomba R,Sanyal,AJ.The global NAFLD epidemic [J].Nat Rev Gastroenterol Hepatol,2013,10:686-690.
[4] Masarone M,Federico A,Abenavoli L,et al.Non alcoholic fatty liver:epidemiology and natural history [J].Rev Recent Clin Trials,2014,9:126-133.
[5] Li Z,Xue J,Chen P,et al.Prevalence of nonalcoholic fatty liver disease in mainland of China:a meta-analysis of published studies [J].J Gastroenterol Hepatol,2014,29:42-51.
[6] Rinella ME.Nonalcoholic fatty liver disease:a systematic review [J].JAMA,2015,313(22):2263-2273.
[7] Yang XD,Yang YY,Ouyang DS,et al.A review of biotransformation and pharmacology of ginsenoside compound K [J].Fitoterapia,2015,100:208-220.
[8] Wei S,Li Wl,Yu Y,et al.Ginsenoside Compound K suppresses the hepatic gluconeogenesis via activating adenosine-5′ monophosphate kinase:A study in vitro and in vivo [J].Life Sci,2015,139:8-15.
[9] Zhang GM,Zhu T,Chen GH,et al.The effect of ginsenoside Compound K on the formation of atherosclerosis [J].J Cadiovascular Pulmonary Dis (心肺血管病杂志),2017,5(36):408-413.
[10] Li XC,Zhang XY,Xu S,et al.Establishment of C57BL/6J mouse nonalcoholic fatty liver model [J].Chin J Veter Med (中国兽医杂志),2013,1(49):6-8.
[11] Day CP.Non-alcoholic fatty liver disease:a massive problem [J].Clin Med(Lond),2011,11(2):176-178.
[12] Guan QW,Jiang HL,Pan QW.Advances in the molecular mechanism and treatment of nonalcoholic fatty liver disease [J].J Mil Surg Southwest Chin(西南军医),2016,15(5):467-470.
[13] Mu Q,Fang X,Li X,et al.Ginsenoside Rb1 promotes browning through regulation of PPARγ in 3T3-L1 adipocytes [J].Biochem Biophys Res Commun,2015,466(3):530.
[14] Yu X,Ye L,Zhang H,et al.Ginsenoside Rb1 ameliorates liver fat accumulation by upregulating perilipin expression in adipose tissue of db/db obese mice [J].J Ginseng Res,2015,39 (3):199-205.
[15] Lin ZJ,Zhang B,Liu XQ.Research progress of AMPK-ACC signaling pathway and related metaolic diseases [J].Chin J Diabetes(中国糖尿病杂志),2013,21(5):474-477.
[16] Viollet B,Mounier R,Leclerc J,et al.Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders [J].Diabetes Metab,2007,33(6):395-402.
[17] Zhao WX,Yan L,Shao MY.Effect of huatan clearing damp huoxue recipe on ADPN/AMPK/ACC pathway in rats with nonalcoholic steatohepatitis [J].J Clin Hepatol (临床肝胆病杂志),2017,33(5):932-936.
[18] Brownsey RW,Boone AN,Elliott JE,et al.Regulation of acetyl-CoA carboxylase [J].Biochem Soc Trans,2006,34(Pt2):223-227.
[19] Shimano H.SREBPs:physiology and pathophysiology of the SREBP family [J].FEBS J,2009,276(3):616-621.