| BackgroundExcessive intake of alcohol has caused healthy and social problems in the worldwide,such as mental disorders,pancreatic injury,myocardial damage and so on.Alcohol is mainly metabolized in liver,which can cause liver damage and develop into alcoholic liver disease(ALD).The pathogenesis procession of ALD includes alcoholic fatty liver,alcoholic hepatitis,liver fibrosis,cirrhosis and even hepatica carcinoma.Up to now,the pathogenesis of ALD is still not very clear and the treatment of ALD is limited.Ethanol-induced fatty liver,the early stage of ALD,is marked by triglyceride(TG)accumulation in liver cells,the process of which is reversible.It has been reported that abnormal expression of AMP-activated protein kinases(AMPK),sterol regulatory element-binding protein-1 c(SERBR-1c)and acetyl-CoA carboxylase(ACC)may play an important role in the development of ethanol-caused fatty liver disease.Alcoholic fatty liver can develop into alcoholic hepatitis and abnormal hepatocyte apoptosis is important in the process.Recently it has been revealed that mitogen-activated protein kinase(MAPK)signaling pathway and autophagy take part in ethanol-induced hepatocyte apoptosis.The MAPK family,including p38 and JNK,is vital in signal transduction in cells,which can deliver extracellular signals into the cell by altering phosphorylation of p38 and/or JNK.Series of studies have found that normal level of autophagy in hepatocytes is very important in maintaining homeostasis.Autophagy participates in numerous activities in hepatocytes such as destruction of intracellular pathogens,degradation of inefficient organelles and abnormal protein and helps in the defense against stressful conditions.Corosolic acid,the extracts of leaf of the banana tree,belongs to triterpenoids and has shown the potential in modulation of oxidative stress,lipid metabolism and cell apoptosis.Corosolic acid has shown promise in the treatment of diabetes mellitus,hyperlipidemia and atherosclerosis without adverse effects in various studies.While the influence of corosolic acid on ethanol-induced liver damage is still not clear and the related mechanism is underlying.And In this study,we try to investigate the effect of corosolic acid on ethanol-induced abnormal lipid metabolism and hepatocyte apoptosis and the related intrinsic mechanism.Part ?:The effect of corosolic acid on ethanol-induced abnormal lipid metabolism and the mechanism in BRL-3A and HepG2 cellsObjectiveTo explore the value of corosolic acid in alcohol-impaired lipid homeostasis and the underlying mechanism in BRL-3 A and HepG2 cells.Methods1.Cell culture and treatmentsAccording to the requirement of experiments,BRL-3A and HepG2 cells were treated with medium and incubated with different concentration of ethanol alone plus or not corosolic acid or compound C for indicated time.2.Triglyceride measurementCollected the cells of control group,ethanol group,ethanol+corosolic acid group and ethanol+corosolic acid +compound C group.And then we measured the expression of triglyceride(TG)in these cells by using triglyceride assay kit.3.Real-Time PCRRNA was isolated from control and treated BRL-3A and HepG2 cells.The mRNA expression of ?-actin and SREBP-lc were measured by Real-Time PCR.4.Western-blotProtein examples were isolated from cells of different treated groups.Then the protein expression of phosphorylated-AMPK(p-AMPK),AMPK,phosphorylated-ACC(p-ACC)and ACC was measured by western-blot and analyzed by Image J software.Results1.Effects of ethanol incubation on lipid synthesis in BRL-3A and HepG2 cellsCompared with the control group,ethanol increased the content of triglyceride(TG)dose-dependently in the BRL-3A and HepG2 cells.2.Effects of corosolic acid on lipid synthesis in ethanol-incubated BRL-3A and HepG2 cellsCompared with ethanol-treated group,corosolic acid decreased the TG content in dose dependent manners in BRL-3 A and HepG2 cells.3.Effects of corosolic acid on AMPK/ACC pathway in ethanol-incubated BRL-3A and HepG2 cellsCompared with the control group,ethanol increased p-AMPK/AMPK and p-ACC/ACC ratio in BRL-3A and HepG2 cells.Compared with ethanol-treated group,corosolic acid decreased p-AMPK/AMPK and p-ACC/ACC ratio in the cells.It indicated that corosolic acid up-regulated ethanol-suppressed AMPK pathway and inhibited ethanol-activated ACC pathway.All the above effects of corosolic acid could be abolished by compound C,a inhibitor of AMPK pathway.4.Effects of corosolic acid on mRNA expression of SREBP-lc in ethanol-incubated BRL-3 A and HepG2 cellsCompared with the control,ethanol increased the expression of SREBP-1c mRNA significantly in BRL-3A and HepG2 cells.Compared with ethanol-treated group,corosolic acid decreased expression of SREBP-1c mRNA dose-dependently in the cells.5.Compound C affected the influence of corosolic acid on lipid metabolism in ethanol-incubated BRL-3 A and HepG2 cellsIn the same condition of ethanol-stimulated,compared with corosolic acid-treatment group,compound C increased the content of triglycerides and enhanced the mRNA expression of SREBP-1c in BRL-3 A and HepG2 cells.Conclusion1.In BRL-3 A and HepG2 cells,ethanol could induce triglyceride accumulation.2.In BRL-3A and HepG2 cells,corosolic acid could alleviate ethanol-caused triglyceride accumulation through activating AMPK pathway,inhibiting ACC signaling and suppressing SREBP-lc expression.Part ?:The influence of corosolic acid on ethanol-induced apoptosis and the mechanism in BRL-3A and HepG2 cellsObjectiveTo explore the effect of corosolic acid on ethanol-induced apoptosis and the underlying mechanism in BRL-3 A and HepG2 cell.Methods1.Cell treatmentBRL-3 A and HepG2 cells were treated for indicated time and divided as follows:control group,ethanol group,ethanol+corosolic acid group,ethanol+corosolic acid+bafilomycins A1 group,ethanol+corosolic acid +3-MA group,ethanol+corosolic acid+compound C group,ethanol+corosolic acid+BAPTA-AM group,ethanol+corosolic acid +thapsigargin group.2.MTT assayThe cells were planted into 96 well plates and incubated with ethanol or corosolic acid and so on,according to the experimental requirements.After incubation for 24 hour,MTT was used to detect the survival of cells in each group.3.Western-blot assayProtein examples were extracted from treated BRL-3A and HepG2 cells.Then the protein expression of ?-actin,bax,bcl-2,p38,phosphorylated-p38(p-p38),JNK,phosphorylated-JNK(p-JNK),beclin-1 and LC3?/? was detected by western-blot.Image J was used to analyze the results.4.ELISA assayCollect the cell culture supernatant.And detect the concentration of TNF-a according to the kit instructions.5.Detection of intracellular reactive oxygen species(ROS)and calciumThe cells were collected from each group.The concentration of intracellular ROS and calcium was detected by ROS fluorescent probe,calcium fluorescent probe and flow cytometry.The data were analyzed Flow Jo software.Results1.Effect of ethanol incubation on viability of BRL-3A and HepG2 cellsCompared with the control group,ethanol suppressed the survival of BRL-3A and HepG2 cells dose-dependently.2.Effect of corosolic acid on viability of ethanol-incubated BRL-3A and HepG2 cellsCompared with ethanol-treated group,corosolic acid increased cell viability of BRL-3 A and HepG2 in a dose-dependent manner.3.Effect of corosolic acid on apoptosis of ethanol-incubated BRL-3A and HepG2 cellsCompared with the control,ethanol increased bax/bcl-2 ratio in BRL-3A and HepG2 cells.Compared with ethanol-treated group,corosolic acid significantly decreased bax/bcl-2 ratio dose-dependently in the cells.It indicated that corosolic acid suppressed ethanol-induced hepatocyte apoptosis.4.Effect of corosolic acid on inflammation of ethanol-incubated BRL-3 A cellsCompared with the control,ethanol increased the content of TNF-a in BRL-3A cells.Compared with ethanol-treated group,corosolic acid significantly decreased TNF-a content dose-dependently in the cells.It indicated that corosolic acid suppressed ethanol-induced hepatocyte inflammation.5.Effect of corosolic acid on ROS expression of ethanol-incubated BRL-3A and HepG2 cellsCompared with the control,ethanol increased the concentration of ROS in BRL-3A and HepG2 cells.Compared with the ethanol-treated group,corosolic acid significantly decreased ethanol-increased ROS dose-dependently.6.Effect of corosolic acid on MAPK pathway of ethanol-incubated BRL-3A and HepG2 cellsCompared with the control,ethanol increased p-p38/p38 and p-JNK/JNK ratio in BRL-3A and HepG2 cells.Compared with the ethanol-treated group,corosolic acid significantly decreased p-p38/p38 and p-JNK/JNK ratio dose-dependently in the cells.It indicated that corosolic acid suppressed ethnaol-activated p38 and JNK MAPK pathway.7.Effect of corosolic acid on autophagy of ethanol-incubated BRL-3A and HepG2 cellsCompared with the control,ethanol suppressed protein expression of beclin-1 and down-regulated LC3II/LC3I ratio dose-dependently in BRL-3A and HepG2 cells.Compared with the ethanol-treated group,corosolic acid dose-dependently up-regulated the protein expression of beclin-1 and LC3II/LCI ratio in the cells.It indicated that corosolic acid up-regulated ethanol-suppressed autophagy.8.Effect of 3-MA?corosolic acid on viability of ethanol-incubated BRL-3A and HepG2 cellsIn the same condition of 100mM ethanol stimulation,3-MA significantly decreased corosolic acid-increased cell viability.It indicated that corosolic acid protected hepatocytes against ethanol-induced apoptosis through activating autophagy.9.Related mechanism of corosolic acid effect on autophagy in ethanol-incubated BRL-3 A and HepG2 cellsCompared with the control,ethanol down-regulated p-AMPK/AMPK ratio and beclin-1 expression.Compared with the ethanol-treated group,corosolic acid up-regulated p-AMPK/AMPK ratio and beclin-1 expression.The modulation effects of corosolic acid could be reversed by compound C,a inhibitor of AMPK pathway.10.The role of calcium in modulation of corosolic acid on autophagy in ethanol-incubated BRL-3 A and HepG2 cellsCompared with the control,ethanol increased concentration of intracellular calcium in the cells.Compared with the ethanol-treated group,corosolic acid decreased concentration of intracellular calcium dose-dependently.Compared with corosolic acid-treated group,neither BAPTA-AM or thapsigargin had obvious effect on protein expression of beclin-1 in the cells.Compared with corosolic acid-treated group,thapsigargin up-regulated concentration of intracellular calcium and ROS.Conclusion1.Corosolic acid suppressed ethanol-induced apoptosis in BRL-3A and HepG2 cells,through down-regulating ethanol-activated ROS/MAPK pathway and up-regulating ethanol-supressed autophagy.2.Corosolic acid up-regulated ethanol-suppressed autophagy through activating AMPK pathway in BRL-3A and HepG2 cells.Part ?:The effect of corosolic acid on the liver of ethanol-fed rat and the underlying mechanismObjectiveTo observe the effect of corosolic acid on the liver injury of ethanol-fed rats and explore the intrinsic mechanism.Methods1.Animal model and treatment30 male Sprague-Dawley rats were divided into 3 groups:(n=10 each):control group,ethanol-fed and therapeutic group.The control group was fed a standard diet for 12 weeks.The ethanol group was fed the control diet followed by intragastrical ethanol infusion:60%ethanol(Red Star Er Guo Tou Jiu,Beijing)diluted with water was given in an increasing dose every 4 weeks(1-4 weeks with 4.5g/kg/day,5-8 weeks with 6.5g/kg/day,9-12 weeks with 9 g/kg/day).The therapeutic group received both corosolic acid(4ml 20%twice a day,5-12 weeks)and ethanol as for the ethanol-fed group.Rats received oral gavages twice a day,12 hour apart.At the end of the experiment,rat livers were excised for further analysis.2.Histopathological measurement of the liver tissueHematoxylin eosin(HE)staining was used to detect pathological injury of the liver tissue.And oil red staining was used to observe lipid accumulation in the liver.3.Western-blot analysisProtein examples were extracted from the liver tissue.Then western-blot was used to detect the protein expression of ?-actin,bax,bcl-2,p38,phosphorylated-p38(p-p38),JNK,phosphorylated-JNK(p-JNK),beclin-1 and LC3?/LC ? in the liver tissue.Results1.Influence of corosolic acid on liver pathology of ethanol-fed SD ratsCompared with controls,ethanol-treated rats showed inflammation?hepatocyte swelling and increased lipid accumulation in the liver tissue.Compared with the ethanol-fed rats,corosolic acid decreased inflammation?hepatocyte swelling and lipid accumulation in the liver tissue.2.Influence of corosolic acid on apoptosis of liver tissue in ethanol-fed SD ratsCompared with controls,ethanol increased bax/bcl-2 ratio in the rat liver tissue.Compared with ethanol-treated group,corosolic acid decreased the bax/bal-2 ratio in the liver tissue.It indicated that corosolic acid alleviated ethanol-induced apoptosis in rat livers.3.Influence of corosolic acid on MAPK pathway of liver tissue in ethanol-fed SD ratsCompared with controls,ethanol increased p-p38/p38 and p-JNK/JNK ratio in the rat liver tissue.Compared with ethanol-treated group,corosolic acid decreased the p-p38/p38 and p-JNK/JNK ratio in the liver tissue.It indicated that corosolic acid inhibited ethanol-activated p38 and JNK MAPK pathway in rat livers.4.Influence of corosolic acid on autophagy in liver tissue in ethanol-fed SD rats and related mechanismCompared with controls,ethanol decreased p-AMPK/AMPK ratio,beclin-1 expression and LC3?/LC3I ratio in the rat liver tissue.Compared with ethanol-treated group,corosolic acid incresed p-AMPK/AMPK ratio,beclin-1 expression and LC3?/LC3I ratio in the liver tissue.It indicated that corosolic acid up-regulated ethanol-suppressed AMPK/autophagy pathway in rat livers.Conclusion1.Corosolic acid alleviated pathological injury and lipid accumulation in the liver of ethanol-gavaged rats.2.Corosolic acid suppressed hepatocyte apoptosis in the liver of ethanol-gavaged rats,through inhibited ethanol-activated MAPK pathway and restored ethanol-suppressed AMPK/autophagy pathway. |
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