siRNA抑制饥饿素-O-乙酰基转移酶对肝细胞脂肪变性的影响及作用机制
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摘要
目的探讨siRNA抑制饥饿素-O-乙酰基转移酶(GOAT)对肝脂肪变性影响及作用机制。方法游离脂肪酸(FFA)作用于人肝细胞系LO2细胞以诱导肝细胞脂肪变性。FFA与siRNA-GOAT单独或联合处理LO2细胞,分为4组,即空白对照(NC)组:仅用PBS处理;siRNA-GOAT组:仅以终浓度为10 nmol/L的siRNA-GOAT处理;FFA组:仅以终浓度为1 mmol/L的FFA处理;FFA+siRNA-GOAT组:采用上述浓度FFA和siRNA-GOAT混合处理。肝细胞油红O染色检测脂肪滴形成情况;TG检测试剂盒检测LO2细胞脂质水平;免疫印迹法(Western Blot)、实时定量逆转录聚合酶链反应(RT-PCR)、免疫荧光染色、电子显微镜检测自噬活性;酶联免疫吸附测定法、RT-PCR检测TNFα、IL-6水平。Western Blot检测雷帕霉素(m TOR)、磷酸化m TOR(pm TOR)、AMP-活化蛋白激酶(AMPK)以及磷酸化AMPK(p-AMPK)的蛋白水平变化。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果 FFA+siRNA-GOAT组脂肪滴形成较FFA组明显减轻,TG水平明显低于FFA组,差异有统计学意义(P<0.001)。FFA+siRNA-GOAT组TNFα和IL-6蛋白、mRNA表达水平较FFA组均明显下降,差异均有统计学意义(P值均<0.005)。siRNA-GOTA组LC3-Ⅱ、Beclin-1 mRNA表达水平明显高于NC组,差异均有统计学意义(P值均<0.001)。FFA+siRNA-GOAT组LC3-Ⅱ、Beclin-1 mRNA表达水平明显高于FFA组,差异均有统计学意义(P值均<0.001)。siRNA-GOAT组LC3-Ⅱ、Beclin-1蛋白水平明显高于NC组,差异均有统计学意义(P值均<0.05)。FFA+siRNA-GOAT组LC3-Ⅱ、Beclin-1蛋白水平明显高于FFA组,差异均有统计学意义(P值均<0.05)。免疫荧光染色示FFA+siRNA-GOAT组LO2细胞中内源性LC3-Ⅱ表达水平较FFA组、siRNA-GOAT组升高。电子显微镜观察结果示FFA+siRNA-GOAT组自噬体表达较FFA组增加。自噬抑制剂siRNA-ATG5、3-MA或刺激剂雷帕霉素处理LO2细胞后,FFA+siRNA-ATG5组、FFA+3-MA组TG水平分别与FFA+siRNA-GOAT组、FFA+雷帕霉素组比较,差异均有统计学意义(P值均<0.001)。FFA+siRNA-GOAT组、FFA+雷帕霉素组LC3-Ⅰ/Ⅱ水平明显高于FFA+siRNA-ATG5组、FFA+3-MA组,差异均有统计学意义(P值均<0.05)。FFA+siRNA-GOAT组p-AMPK蛋白表达水平明显高于于FFA组、p-m TOR蛋白表达水平明显低于FFA组,差异均有统计学意义(P值均<0.05)。结论 siRNA抑制GOAT可通过调节AMPK/m TOR通路,上调自噬活性,进而减轻肝细胞内脂肪变性。
Objective To investigate the effect of inhibition of ghrelin O-acyltransferase( GOAT) by small interfering RNA( siRNA) on hepatocyte fatty degeneration and related mechanism of action. Methods Human LO2 hepatocytes were treated with free fatty acid( FFA)to induce hepatocyte fatty degeneration. LO2 hepatocytes were treated with FFA and siRNA-GOAT alone or in combination and then divided into normal control( NC) group( treated with phosphate buffered saline alone),siRNA-GOAT group( treated with siRNA-GOAT at a final concentration of 10 nm),FFA group( treated with FFA at a final concentration of 1 mm),and FFA + siRNA-GOAT group( treated with FFA at a final concentration of 1 mm and siRNA-GOAT at a final concentration of 10 nm). Oil red O staining was performed for hepatocytes to identify lipid droplets; the triglyceride( TG) test kit was used to measure the lipid level in LO2 hepatocytes; Western blot,qRT-PCR,immunofluorescent staining,and electron microscopy were used to measure autophagy; ELISA and RT-PCR were used to measure the levels of tumor necrosis factor-α( TNFα) and interleukin-6( IL-6); ELISA was used to measure the changes in the levels of mammalian target of rapamycin( m TOR),phosphorylated m TOR( p-m TOR),AMP-activated protein kinase( AMPK),and phosphorylated AMPK. A one-way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between any two groups. Results Compared with the FFA group,the FFA + siRNA-GOAT group had a significant reduction in the formation of lipid droplets and a significantly lower TG level( P < 0. 001). Compared with the FFA group,the FFA +siRNA-GOAT group had significant reductions in the protein and mRNA expression of TNFα and IL-6( all P < 0. 005). The siRNA +GOAT group had significantly higher mRNA expression of LC3-II and Beclin-1 than the NC group( all P < 0. 001). The FFA + siRNA-GOAT group had significantly higher mRNA expression of LC3-II and Beclin-1 than the FFA group( all P < 0. 001). The siRNA + GOAT group had significantly higher protein expression of LC3-II and Beclin-1 than the NC group( all P < 0. 05). The FFA + siRNA-GOAT group had significantly higher protein expression of LC3-II and Beclin-1 than the FFA group( all P < 0. 05). Immunofluorescent staining showed that compared with the FFA group and the siRNA-GOAT group,the FFA + siRNA-GOAT group had a significant increase in the expression of endogenous LC3-II in LO2 hepatocytes. Electron microscopy showed that compared with the FFA group,the FFA + siRNA-GOAT group had a significant increase in the expression of autophagosome. After the LO2 hepatocytes were treated by autophagy inhibitors siRNA-ATG5 and 3-MA or an autophagy stimulant,rapamycin,there was a significant difference in TG level between the FFA + siRNA-ATG5 group and the FFA + siRNA-GOAT group( P < 0. 001),as well as between the FFA + 3-MA group and the FFA + rapamycin group( P < 0. 001). The FFA + siRNA-GOAT group had a significantly higher level of LC3-I/II than the FFA + siRNA-ATG5 group( P <0. 05),and the FFA + rapamycin group had a significantly higher level of LC3-I/II than the FFA + 3-MA group( P < 0. 05). Compared with the FFA group,the FFA + siRNA-GOAT group had significantly higher protein expression of p-AMPK( P < 0. 05) and significantly lower protein expression of p-m TOR( P < 0. 05). Conclusion GOAT inhibition by siRNA can upregulate autophagy and alleviate hepatocyte fatty degeneration,possibly by regulating the AMPK/m TOR pathway.
Objective To investigate the effect of inhibition of ghrelin O-acyltransferase( GOAT) by small interfering RNA( siRNA) on hepatocyte fatty degeneration and related mechanism of action. Methods Human LO2 hepatocytes were treated with free fatty acid( FFA)to induce hepatocyte fatty degeneration. LO2 hepatocytes were treated with FFA and siRNA-GOAT alone or in combination and then divided into normal control( NC) group( treated with phosphate buffered saline alone),siRNA-GOAT group( treated with siRNA-GOAT at a final concentration of 10 nm),FFA group( treated with FFA at a final concentration of 1 mm),and FFA + siRNA-GOAT group( treated with FFA at a final concentration of 1 mm and siRNA-GOAT at a final concentration of 10 nm). Oil red O staining was performed for hepatocytes to identify lipid droplets; the triglyceride( TG) test kit was used to measure the lipid level in LO2 hepatocytes; Western blot,qRT-PCR,immunofluorescent staining,and electron microscopy were used to measure autophagy; ELISA and RT-PCR were used to measure the levels of tumor necrosis factor-α( TNFα) and interleukin-6( IL-6); ELISA was used to measure the changes in the levels of mammalian target of rapamycin( m TOR),phosphorylated m TOR( p-m TOR),AMP-activated protein kinase( AMPK),and phosphorylated AMPK. A one-way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between any two groups. Results Compared with the FFA group,the FFA + siRNA-GOAT group had a significant reduction in the formation of lipid droplets and a significantly lower TG level( P < 0. 001). Compared with the FFA group,the FFA +siRNA-GOAT group had significant reductions in the protein and mRNA expression of TNFα and IL-6( all P < 0. 005). The siRNA +GOAT group had significantly higher mRNA expression of LC3-II and Beclin-1 than the NC group( all P < 0. 001). The FFA + siRNA-GOAT group had significantly higher mRNA expression of LC3-II and Beclin-1 than the FFA group( all P < 0. 001). The siRNA + GOAT group had significantly higher protein expression of LC3-II and Beclin-1 than the NC group( all P < 0. 05). The FFA + siRNA-GOAT group had significantly higher protein expression of LC3-II and Beclin-1 than the FFA group( all P < 0. 05). Immunofluorescent staining showed that compared with the FFA group and the siRNA-GOAT group,the FFA + siRNA-GOAT group had a significant increase in the expression of endogenous LC3-II in LO2 hepatocytes. Electron microscopy showed that compared with the FFA group,the FFA + siRNA-GOAT group had a significant increase in the expression of autophagosome. After the LO2 hepatocytes were treated by autophagy inhibitors siRNA-ATG5 and 3-MA or an autophagy stimulant,rapamycin,there was a significant difference in TG level between the FFA + siRNA-ATG5 group and the FFA + siRNA-GOAT group( P < 0. 001),as well as between the FFA + 3-MA group and the FFA + rapamycin group( P < 0. 001). The FFA + siRNA-GOAT group had a significantly higher level of LC3-I/II than the FFA + siRNA-ATG5 group( P <0. 05),and the FFA + rapamycin group had a significantly higher level of LC3-I/II than the FFA + 3-MA group( P < 0. 05). Compared with the FFA group,the FFA + siRNA-GOAT group had significantly higher protein expression of p-AMPK( P < 0. 05) and significantly lower protein expression of p-m TOR( P < 0. 05). Conclusion GOAT inhibition by siRNA can upregulate autophagy and alleviate hepatocyte fatty degeneration,possibly by regulating the AMPK/m TOR pathway.
引文
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[6]TEUFFEL P,WANG L,PRINZ P,et al.Treatment with the ghrelin-O-acyltransferase(GOAT)inhibitor GO-Co A-Tat reduces food intake by reducing meal frequency in rats[J].J Physiol Pharmacol,2015,66(4):493-503.
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[9]AMIR M,CZAJA MJ.Autophagy in nonalcoholic steatohepatitis[J].Expert Rev Gastroenterol Hepatol,2011,5(2):159-166.
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[12]DING ZY,PU L,LU HY,et al.Correlation of liver fat content with serum vitamin A level and insulin resistace in patients with nonalcoholic fatty liver disease[J].J Clin Hepatol,2017,33(12):2361-2365.(in Chinese)丁智勇,卜乐,鲁鸿燕,等.非酒精性脂肪性肝病患者血清维生素A水平、肝脏脂肪含量与胰岛素抵抗的相关性分析[J].临床肝胆病杂志,2017,33(12):2361-2365.
[13]NI MM,ZHANG YQ,CHEN YX,et al.Prevalence of nonalcoholic fatty liver disease and its prognostic factors[J].J Clin Hepatol,2016,32(3):453-458.(in Chinese)倪曼曼,张颖秋,陈岳祥,等.非酒精性脂肪性肝病患者发病和预后的影响因素分析[J].临床肝胆病杂志,2016,32(3):453-458.
[14]ALKHOURI N,CARTER-KENT C,FELDSTEIN AE.Apoptosis in nonalcoholic fatty liver disease:diagnostic and therapeutic implications[J].Expert Rev Gastroenterol Hepatol,2011,5(2):201-212.
[15]KOEK GH,LIEDORP PR,BAST A.The role of oxidative stress in non-alcoholic steatohepatitis[J].Clin Chim Acta,2011,412(15-16):1297-1305.
[16]YUAN RS,LI H,SUN JH,et al.Lipid-lowering effect and antioxidant activity of polysaccharide from Schisandra Chinensis in rats with non-alcoholic fatty liver disease induced by high-fat diet[J].J Jilin Univ:Med Edit,2017,43(6):1103-1108.(in Chinese)苑荣爽,李贺,孙靖辉,等.北五味子多糖对高脂诱导非酒精性脂肪性肝病大鼠的降血脂作用及其抗氧化活性[J].吉林大学学报:医学版,2017,43(6):1103-1108.
[17]JORNAYVAZ FR,SHULMAN GI.Diacylglycerol activation of protein kinase C and hepatic insulin resistance[J].Cell Metabolism,2012,15(5):574-584.
[18]OZCELIK F,YUKSEL C,ARSLAN E,et al.Relationship between visceral adipose tissue and adiponectin,inflammatory markers and thyroid hormones in obese males with hepatosteatosis and insulin resistance[J].Arch Med Res,2013,44(4):273-280.
[19]MUSSO G,GAMBINO R,BO S,et al.Should non-alcoholic fatty liver disease be included in the definition of metabolic syndrome?A cross-sectional comparison with adult treatment Panel III criteria in nonobese nondiabetic subjects[J].Diabetes Care,2008,31(3):562-568.
[20]RAVIKUMAR B,BERGER Z,RUBINSZTEIN DC,et al.Rapamycin pretreatment protects against apoptosis[J].Hum Mol Genet,2006,15(7):1209-1216.
[21]YU L,Mc PHEE CK,ZHENG L,et al.Termination of autophagy and reformation of lysosomes regulated by Mtor[J].Nature,2010,465(7300):942-946.
[22]LIU HY,HAN J,CAO SY,et al.Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia:inhibition of Fox O1-dependent expression of key autophagy genes by insulin[J].J Biol Chem,2009,284(45):31484-31492.
[23]SINHA RA,FARAH BL,SINGH BK,et al.Caffeine stimulates hepatic lipid metabolism by the autophagy-lysosomal pathway in mice[J].Hepatology,2014,59(4):1366-1380.
[24]SINGH R,KAUSHIK S,WANG Y,et al.Autophagy regulates lipid metabolism[J].Nature,2009,458(7242):1131-1135.
[25]DING WX,LI M,CHEN X,et al.Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice[J].Gastroenterology,2010,139(5):1740-1752.
[26]LIU K,CZAJA MJ.Regulation of lipid stores and metabolism by lipophagy[J].Cell Death Differ,2013,20(1):3-11.
[27]LI Y,HAI J,LI L,et al.Administration of ghrelin improves inflammation,oxidative stress,and apoptosis during and after nonalcoholic fatty liver disease development[J].Endocrine,2013,43(2):376-386.
[28]WINDER WW,HARDIE DG.AMP-activated protein kinase,a metabolic master switch:possible roles in type 2 diabetes[J].Am J Physiol,1999,277(1 Pt 1):e1-e10.
[29]YIN XM,DING WX,GAO W.Autophagy in the liver[J].Hepatology,2008,47(5):1773-1785.
[30]BARAZZONI R,SEMOLIC A,CATTIN MR,et al.Acylated ghrelin limits fat accumulation and improves redox state and inflammation markers in the liver of high-fat-fed rats[J].Obesity(Silver Spring),2014,22(1):170-177.
[31]SHOJI-KAWATA S,SUMPTER R,LEVENO M,et al.Identification of a candidate therapeutic autophagy-inducing peptide[J].Nature,2013,494(7436):201-206.
[32]LIN CW,ZHANG H,LI M,et al.Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in Mice[J].J Hepatol,2013,58(5):993-999.
[33]SINHA RA,YOU SH,ZHOU J,et al.Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy[J].J Clin Invest,2012,122(7):2428-2438.
[34]NI HM,WILLIAMS JA,YANG H,et al.Targeting autophagy for the treatment of liver diseases[J].Pharmacol Res,2012,66(6):463-474.
[35]CODOGNO P,MEIJER AJ.Autophagy in the liver[J].J Hepatol,2013,59(2):389-391.
[36]DING WX.Induction of autophagy,a promising approach for treating liver injury[J].Hepatology,2014,59(1):340-343.
[37]LOMONACO R,SUNNY NE,BRIL F,et al.Nonalcoholic fatty liver disease:current issues and novel treatment approaches[J].Drugs,2013,73(1):1-14.
[2]BERLANGA A,GUIU-JURADO E,PORRAS JA,et al.Molecular pathways in non-alcoholic fatty liver disease[J].Clin Exp Gastroenterol,2014,7:221-239.
[3]FERRE P,FOUFELLE F.Hepatic steatosis:a role for de novo lipogenesis and the transcription factor SREBP-1c[J].Diabetes Obes Metab,2010,12(Suppl 2):83-92.
[4]INUI A,ASAKAWA A,BOWERS CY,et al.Ghrelin,appetite,and gastric motility:the emerging role of the stomach as an endocrine organ[J].FASEB J,2004,18(3):439-456.
[5]BARNETT BP,HWANG Y,TAYLOR MS,et al.Glucose and weight control in mice with a designed ghrelin O-acyltransferase inhibitor[J].Science,2010,330(6011):1689-1692.
[6]TEUFFEL P,WANG L,PRINZ P,et al.Treatment with the ghrelin-O-acyltransferase(GOAT)inhibitor GO-Co A-Tat reduces food intake by reducing meal frequency in rats[J].J Physiol Pharmacol,2015,66(4):493-503.
[7]YANG J,BROWN MS,LIANG G,et al.Identification of the acyltransferase that octanoylates ghrelin,an appetite-stimulating peptide hormone[J].Cell,2008,132(3):387-396.
[8]GUALILLO O,LAGO F,DIEGUEZ C.Introducing GOAT:a target for obesity and anti-diabetic drugs?[J].Trends Pharmacol Sci,2008,29(8):398-401.
[9]AMIR M,CZAJA MJ.Autophagy in nonalcoholic steatohepatitis[J].Expert Rev Gastroenterol Hepatol,2011,5(2):159-166.
[10]NOUREDDIN M,YATES KP,VAUGHN IA,et al.Clinical and histological determinants of nonalcoholic steatohepatitis and advanced fibrosis in elderly patients[J].Hepatology,2013,58(5):1644-1654.
[11]KIM D,CHOI SY,PARK EH,et al.Nonalcoholic fatty liver disease is associated with coronary artery calcification[J].Hepatology,2012,56(2):605-613.
[12]DING ZY,PU L,LU HY,et al.Correlation of liver fat content with serum vitamin A level and insulin resistace in patients with nonalcoholic fatty liver disease[J].J Clin Hepatol,2017,33(12):2361-2365.(in Chinese)丁智勇,卜乐,鲁鸿燕,等.非酒精性脂肪性肝病患者血清维生素A水平、肝脏脂肪含量与胰岛素抵抗的相关性分析[J].临床肝胆病杂志,2017,33(12):2361-2365.
[13]NI MM,ZHANG YQ,CHEN YX,et al.Prevalence of nonalcoholic fatty liver disease and its prognostic factors[J].J Clin Hepatol,2016,32(3):453-458.(in Chinese)倪曼曼,张颖秋,陈岳祥,等.非酒精性脂肪性肝病患者发病和预后的影响因素分析[J].临床肝胆病杂志,2016,32(3):453-458.
[14]ALKHOURI N,CARTER-KENT C,FELDSTEIN AE.Apoptosis in nonalcoholic fatty liver disease:diagnostic and therapeutic implications[J].Expert Rev Gastroenterol Hepatol,2011,5(2):201-212.
[15]KOEK GH,LIEDORP PR,BAST A.The role of oxidative stress in non-alcoholic steatohepatitis[J].Clin Chim Acta,2011,412(15-16):1297-1305.
[16]YUAN RS,LI H,SUN JH,et al.Lipid-lowering effect and antioxidant activity of polysaccharide from Schisandra Chinensis in rats with non-alcoholic fatty liver disease induced by high-fat diet[J].J Jilin Univ:Med Edit,2017,43(6):1103-1108.(in Chinese)苑荣爽,李贺,孙靖辉,等.北五味子多糖对高脂诱导非酒精性脂肪性肝病大鼠的降血脂作用及其抗氧化活性[J].吉林大学学报:医学版,2017,43(6):1103-1108.
[17]JORNAYVAZ FR,SHULMAN GI.Diacylglycerol activation of protein kinase C and hepatic insulin resistance[J].Cell Metabolism,2012,15(5):574-584.
[18]OZCELIK F,YUKSEL C,ARSLAN E,et al.Relationship between visceral adipose tissue and adiponectin,inflammatory markers and thyroid hormones in obese males with hepatosteatosis and insulin resistance[J].Arch Med Res,2013,44(4):273-280.
[19]MUSSO G,GAMBINO R,BO S,et al.Should non-alcoholic fatty liver disease be included in the definition of metabolic syndrome?A cross-sectional comparison with adult treatment Panel III criteria in nonobese nondiabetic subjects[J].Diabetes Care,2008,31(3):562-568.
[20]RAVIKUMAR B,BERGER Z,RUBINSZTEIN DC,et al.Rapamycin pretreatment protects against apoptosis[J].Hum Mol Genet,2006,15(7):1209-1216.
[21]YU L,Mc PHEE CK,ZHENG L,et al.Termination of autophagy and reformation of lysosomes regulated by Mtor[J].Nature,2010,465(7300):942-946.
[22]LIU HY,HAN J,CAO SY,et al.Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia:inhibition of Fox O1-dependent expression of key autophagy genes by insulin[J].J Biol Chem,2009,284(45):31484-31492.
[23]SINHA RA,FARAH BL,SINGH BK,et al.Caffeine stimulates hepatic lipid metabolism by the autophagy-lysosomal pathway in mice[J].Hepatology,2014,59(4):1366-1380.
[24]SINGH R,KAUSHIK S,WANG Y,et al.Autophagy regulates lipid metabolism[J].Nature,2009,458(7242):1131-1135.
[25]DING WX,LI M,CHEN X,et al.Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice[J].Gastroenterology,2010,139(5):1740-1752.
[26]LIU K,CZAJA MJ.Regulation of lipid stores and metabolism by lipophagy[J].Cell Death Differ,2013,20(1):3-11.
[27]LI Y,HAI J,LI L,et al.Administration of ghrelin improves inflammation,oxidative stress,and apoptosis during and after nonalcoholic fatty liver disease development[J].Endocrine,2013,43(2):376-386.
[28]WINDER WW,HARDIE DG.AMP-activated protein kinase,a metabolic master switch:possible roles in type 2 diabetes[J].Am J Physiol,1999,277(1 Pt 1):e1-e10.
[29]YIN XM,DING WX,GAO W.Autophagy in the liver[J].Hepatology,2008,47(5):1773-1785.
[30]BARAZZONI R,SEMOLIC A,CATTIN MR,et al.Acylated ghrelin limits fat accumulation and improves redox state and inflammation markers in the liver of high-fat-fed rats[J].Obesity(Silver Spring),2014,22(1):170-177.
[31]SHOJI-KAWATA S,SUMPTER R,LEVENO M,et al.Identification of a candidate therapeutic autophagy-inducing peptide[J].Nature,2013,494(7436):201-206.
[32]LIN CW,ZHANG H,LI M,et al.Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in Mice[J].J Hepatol,2013,58(5):993-999.
[33]SINHA RA,YOU SH,ZHOU J,et al.Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy[J].J Clin Invest,2012,122(7):2428-2438.
[34]NI HM,WILLIAMS JA,YANG H,et al.Targeting autophagy for the treatment of liver diseases[J].Pharmacol Res,2012,66(6):463-474.
[35]CODOGNO P,MEIJER AJ.Autophagy in the liver[J].J Hepatol,2013,59(2):389-391.
[36]DING WX.Induction of autophagy,a promising approach for treating liver injury[J].Hepatology,2014,59(1):340-343.
[37]LOMONACO R,SUNNY NE,BRIL F,et al.Nonalcoholic fatty liver disease:current issues and novel treatment approaches[J].Drugs,2013,73(1):1-14.