木香烃内酯抑制乙醇诱导的肝细胞损伤与脂肪变性
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摘要
探究木香烃内酯体外对乙醇诱导肝细胞损伤及脂肪变性的影响。建立乙醇导致人LO2肝细胞损伤模型,检测木香烃内酯对细胞活力、ALT和AST释放、脂质生成、脂质调控因子表达及AMPK活性的影响。发现乙醇在高于100 mM浓度时显著抑制肝细胞活力,据此将100 mM浓度的乙醇作为体外刺激肝细胞的实验浓度。木香烃内酯能够逆转乙醇对肝细胞活力的抑制作用,并降低乙醇导致的肝细胞ALT、AST的释放。木香烃内酯能够降低乙醇诱导的肝细胞脂质成分集聚,降低细胞内TG、TC水平。此外,乙醇导致肝细胞中重要的脂质调控转录因子SREBP-1c的表达显著上调,使PPARα的表达显著下调;而木香烃内酯能够减少SREBP-1c的表达并增加PPARα的表达。进一步发现,木香烃内酯显著促进肝细胞中AMPK的磷酸化,且AMPK抑制剂BML-275能够显著削弱木香烃内脂对SREBP-1c和PPARα的调控作用。综上,木香烃内酯体外显著改善乙醇诱导的肝细胞损伤与脂肪变性,该作用与激活AMPK进而调控SREBP-1c与PPARα的表达有关。本研究为将木香烃内酯作为抗酒精性脂肪肝候选药物研究提供实验依据。
To investigate the effects of costunolide on ethanol-induced hepatocyte injury and steatosis in vitro and the underlying mechanism.Ethanol-induced LO2 hepatocyte cell injury model was established in vitro.We examined the effects of costunolide on the release of alanine aminotransferase(ALT) and aspartate aminotransferase(AST),the intracellular contents of total cholesterol(TG) and triglyceride(TC),the mRNA and protein expression of lipogenesis-related transcription factors and the activity of AMP-activated protein kinase(AMPK) in ethanol-stimulated LO2 cells.The results showed that ethanol at concentrations higher than 100 mM significantly suppressed the viability of LO2 cells,and ethanol at 100 mM was selected for establishing hepatocyte injury model in vitro.Costunolide restored the ethanol-induced inhibition of LO2 cell viability,and costunolide at 20 μM exerted significant effects.Costunolide also reduced the supernatant levels of ALT and AST in ethanol-stimulated LO2 cells.Furthermore,costunolide concentration-dependently decreased the accumulation of lipid components in ethanol-stimulated LO2 cells,and reduced the intracellular levels of TG and TC.Molecular examinations showed that ethanol significantly upregulated the mRNA and protein expression of SREBP-1 c and downregulated the mRNA and protein expression of PPARα in LO2 cells.However,costunolide concentration-dependently reduced SREBP-1 c expression and restored PPARα expression at both mRNA and protein levels in ethanol-stimulated LO2 cells.Furthermore,costunolide significantly promoted AMPK phosphorylation,and AMPK specific inhibitor BML-275 remarkably abrogated the regulatory effects of costunolide on SREBP-1 c and PPARα.In conclusion,costunolide could significantly attenuate ethanol-induced hepatocyte injury and steatosis in vitro.These effects were associated with activation of AMPK and modulation of lipid metabolism-related transcription factors.These data provided experimental evidence for developing costunolide as a therapeutic option for alcohol fatty liver disease.
To investigate the effects of costunolide on ethanol-induced hepatocyte injury and steatosis in vitro and the underlying mechanism.Ethanol-induced LO2 hepatocyte cell injury model was established in vitro.We examined the effects of costunolide on the release of alanine aminotransferase(ALT) and aspartate aminotransferase(AST),the intracellular contents of total cholesterol(TG) and triglyceride(TC),the mRNA and protein expression of lipogenesis-related transcription factors and the activity of AMP-activated protein kinase(AMPK) in ethanol-stimulated LO2 cells.The results showed that ethanol at concentrations higher than 100 mM significantly suppressed the viability of LO2 cells,and ethanol at 100 mM was selected for establishing hepatocyte injury model in vitro.Costunolide restored the ethanol-induced inhibition of LO2 cell viability,and costunolide at 20 μM exerted significant effects.Costunolide also reduced the supernatant levels of ALT and AST in ethanol-stimulated LO2 cells.Furthermore,costunolide concentration-dependently decreased the accumulation of lipid components in ethanol-stimulated LO2 cells,and reduced the intracellular levels of TG and TC.Molecular examinations showed that ethanol significantly upregulated the mRNA and protein expression of SREBP-1 c and downregulated the mRNA and protein expression of PPARα in LO2 cells.However,costunolide concentration-dependently reduced SREBP-1 c expression and restored PPARα expression at both mRNA and protein levels in ethanol-stimulated LO2 cells.Furthermore,costunolide significantly promoted AMPK phosphorylation,and AMPK specific inhibitor BML-275 remarkably abrogated the regulatory effects of costunolide on SREBP-1 c and PPARα.In conclusion,costunolide could significantly attenuate ethanol-induced hepatocyte injury and steatosis in vitro.These effects were associated with activation of AMPK and modulation of lipid metabolism-related transcription factors.These data provided experimental evidence for developing costunolide as a therapeutic option for alcohol fatty liver disease.
引文
1 Lívero FA,Acco A.Molecular basis of alcoholic fatty liver disease:From incidence to treatment[J].Hepatol Res,2016,46:111-123.
2 Ju C,Mandrekar P.Macrophages and alcohol-related liver inflammation[J].Alcohol Res,2015,37:251-262.
3 Lin X,Peng Z,Su C.Potential anti-cancer activities and mechanisms of costunolide and dehydrocostuslactone[J].Int J Mol Sci,2015,16:10888-10906.
4 Wang Y,Zhang X,Zhao L,et al.Costunolide protects lipopolysaccharide/d-galactosamine-induced acute liver injury in mice by inhibiting NF-κB signaling pathway[J].J Surg Res,2017,220:40-45.
5 Ferré P,Foufelle F.Hepatic steatosis:a role for de novo lipogenesis and the transcription factor SREBP-1c[J].Diabetes Obes Metab,2010,12(S2):83-92.
6 Kersten S.Integrated physiology and systems biology of PPARα[J].Mol Metab,2014,3:354-371.
7 Sid B,Verrax J,Calderon PB.Role of AMPK activation in oxidative cell damage:Implications for alcohol-induced liver disease[J].Biochem Pharmacol,2013,86:200-209.
8 Wang S,Pacher P,De Lisle RC,et al.A mechanistic review of cell death in alcohol-induced liver injury[J].Alcohol Clin Exp Res,2016,40:1215-1223.
9 Matsuda H,Shimoda H,Ninomiya K,et al.Inhibitory mechanism of costunolide,a sesquiterpene lactone isolated from Laurus nobilis,on blood-ethanol elevation in rats:Involvement of inhibition of gastric emptying and increase in gastric juice secretion[J].Alcohol Alcoholism,2002,37:121-127.
10 Chen HC,Chou CK,Lee SD,et al.Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells[J].Antiviral Res,1995,27:99-109.
11 Eliza J,Daisy P,Ignacimuthu S.Antioxidant activity of costunolide and eremanthin isolated from Costus speciosus(Koen ex.Retz) Sm[J].Chem Biol Interact,2010,188:467-472.
12 Liu CY,Chang HS,Chen IS,et al.Costunolide causes mitotic arrest and enhances radiosensitivity in human hepatocellular carcinoma cells[J].Radiat Oncol,2011,6:56.
13 Park HJ,Lee SJ,Song Y,et al.Schisandra chinensis prevents alcohol-induced fatty liver disease in rats[J].J Med Food,2014,17:103-110.
14 Ferré P,Foufelle F.SREBP-1c transcription factor and lipid homeostasis:Clinical perspective[J].Horm Res,2007,68(2):72-82.
15 Lin Y,Xia Z,Yang F,et al.Research progress in the natural agonists for PPA receptors[J].Nat Prod Res Dev(天然产物研究与开发),2012,24:998-1005.
16 Kong L,Ren W,Li W,et al.Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol induced steatohepatitis in mice[J].Lipids Health Dis,2011,10:246.
17 Gadji M,Crous AM,Fortin D,et al.EGF receptor inhibitors in the treatment of glioblastoma multiform:Old clinical allies and newly emerging therapeutic concepts[J].Eur J Pharmacol,2009,625(1-3):23-30.
18 Cantó C,Auwerx J.AMP-activated protein kinase and its downstream transcriptional pathways[J].Cell Mol Life Sci,2010,67:3407-3423.
2 Ju C,Mandrekar P.Macrophages and alcohol-related liver inflammation[J].Alcohol Res,2015,37:251-262.
3 Lin X,Peng Z,Su C.Potential anti-cancer activities and mechanisms of costunolide and dehydrocostuslactone[J].Int J Mol Sci,2015,16:10888-10906.
4 Wang Y,Zhang X,Zhao L,et al.Costunolide protects lipopolysaccharide/d-galactosamine-induced acute liver injury in mice by inhibiting NF-κB signaling pathway[J].J Surg Res,2017,220:40-45.
5 Ferré P,Foufelle F.Hepatic steatosis:a role for de novo lipogenesis and the transcription factor SREBP-1c[J].Diabetes Obes Metab,2010,12(S2):83-92.
6 Kersten S.Integrated physiology and systems biology of PPARα[J].Mol Metab,2014,3:354-371.
7 Sid B,Verrax J,Calderon PB.Role of AMPK activation in oxidative cell damage:Implications for alcohol-induced liver disease[J].Biochem Pharmacol,2013,86:200-209.
8 Wang S,Pacher P,De Lisle RC,et al.A mechanistic review of cell death in alcohol-induced liver injury[J].Alcohol Clin Exp Res,2016,40:1215-1223.
9 Matsuda H,Shimoda H,Ninomiya K,et al.Inhibitory mechanism of costunolide,a sesquiterpene lactone isolated from Laurus nobilis,on blood-ethanol elevation in rats:Involvement of inhibition of gastric emptying and increase in gastric juice secretion[J].Alcohol Alcoholism,2002,37:121-127.
10 Chen HC,Chou CK,Lee SD,et al.Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells[J].Antiviral Res,1995,27:99-109.
11 Eliza J,Daisy P,Ignacimuthu S.Antioxidant activity of costunolide and eremanthin isolated from Costus speciosus(Koen ex.Retz) Sm[J].Chem Biol Interact,2010,188:467-472.
12 Liu CY,Chang HS,Chen IS,et al.Costunolide causes mitotic arrest and enhances radiosensitivity in human hepatocellular carcinoma cells[J].Radiat Oncol,2011,6:56.
13 Park HJ,Lee SJ,Song Y,et al.Schisandra chinensis prevents alcohol-induced fatty liver disease in rats[J].J Med Food,2014,17:103-110.
14 Ferré P,Foufelle F.SREBP-1c transcription factor and lipid homeostasis:Clinical perspective[J].Horm Res,2007,68(2):72-82.
15 Lin Y,Xia Z,Yang F,et al.Research progress in the natural agonists for PPA receptors[J].Nat Prod Res Dev(天然产物研究与开发),2012,24:998-1005.
16 Kong L,Ren W,Li W,et al.Activation of peroxisome proliferator activated receptor alpha ameliorates ethanol induced steatohepatitis in mice[J].Lipids Health Dis,2011,10:246.
17 Gadji M,Crous AM,Fortin D,et al.EGF receptor inhibitors in the treatment of glioblastoma multiform:Old clinical allies and newly emerging therapeutic concepts[J].Eur J Pharmacol,2009,625(1-3):23-30.
18 Cantó C,Auwerx J.AMP-activated protein kinase and its downstream transcriptional pathways[J].Cell Mol Life Sci,2010,67:3407-3423.