| Objective:Alcoholic liver disease(alcoholic liver disease,ALD)is a liver disease caused by long-term heavy drinking with a histological spectrum ranging from hepatic steatosis,steatohepatitis,fibrosis,and ultimately to cirrhosis.Long-term heavy drinking may induce extensive necrosis of liver cells,and even acute liver failure.As is kown that the cause of the disease is clear,however,the mechanisms driving disease progression is incomplete understood.It is closely related to the toxic,oxidative stress,lipid peroxidation,immune response,endotoxin,cytokines of alcohol and its metabolites to liver.We established the model of ALD in rats by using intragastric administration and have concluded that the mRNA and protein expression of SIRT1 showed significant decreases and expression of SFRS10 decreased.Moreover,we have also elucidated that the expression of Lipin 1 and Lipin1-? enhanced while the expression of Lipin1-? decreased.Pihlajam?ki et al.discovered that the expression of SFRS10 decreased in both obese human liver and high-fat-fed mice and revealed that the amount expression of SFRS10 was related to its function of alternative splicing to express different isoforms of Lipin 1.Yin et al.reported that ethanol inhibited the expression of SIRT1 and simultaneously reduce the expression of SFRS10 in mice hepatocyte cell lines incubated with ethanol by concentration dependent manner.Therefore,the influence on the expression of SFRS10,Lipin 1 by the reduced SIRT1 is worthy of a further study.This study choose AML-12 cells of mice that could metabolizes ethanol.We detect the different expression of SIRT1,SFRS10,Lipin1-?,Lipin1-?,Lipin 1 mRNA by RT-qPCR and protein by Western blot following the application culturing the AML-12 cells by gradient alcohol and of si RNA interference technique;Lipid deposition in the cytoplasm was observed by oil red O staining;We use immunofluorescence staining to observe Lipin 1 distribution of cytoplasm and nucleus.The study aimed to illuminate SIRT1 influence on the expression of SFRS10,Lipin 1 at the cellular level.Methods: The new resuscitated AML-12 cells were plated and cultured at 5% CO2,37? in 12-well culture plates with HD-DMEM medium supplemented with 10% fetal bovine serum(FBS)and 1% penicillin and 1% sodium pyruvate,1‰HEPES.When the logarithmic phase cells fused completely to 60%-70% confluence,they were divided to 0mM group(normal control group),40 mM group,80 mM group,120 mM group according the ethanol concentration;When the expression of SIRT1 were interfered,they were divided to Blank group,SIRT1 siRNA negative control group,SIRT1 siRNA group and SIRT1 siRNA+Ethanol group.1 First,we cultured AML-12 cells stimulating with 40 mM,80mM,120 mM concentration ethanol and the control group was treated with the same volume sterile saline.After culturing for 24 h,the cells were collected for detecting the expression of SIRT1,SFRS10,Lipin 1,Lipin1-?,Lipin1-? mRNA by RT-qPCR and protein by Western blot;At the same time,the cells were fixed by 4% neutral formaldehyde stained by oil red O staining and immunofluorescence staining.2 The SIRT1 siRNA were used to transfect respectively to interfere SIRT1 expression.SIRT1 siRNA negative control sequence,SIRT1 siRNA were respectively transfected to AML-12 cells.Only add the same volume of liposomes as Blank control group.After cultured for 24 h,120mM concentration ethanol were added to SIRT1 siRNA +Ethanol group to continue to culture for 24 h,the cells were collected for detecting the expression of SIRT1,SFRS10,Lipin1-?,Lipin1-?,Lipin 1 mRNA by RT-qPCR and protein by Western blot.At the same time,the cells were fixed by 4% neutral formaldehyde stained by oil red O staining and immunofluorescence staining.Results:1 Changes of the expression of SIRT1,SFRS10,Lipin 1,Lipin1-? and Lipin1-? mRNA in AML-12 cells when stimulated by gradient ethanol 1.1 The changes of the expression of SIRT1,SFRS10,Lipin 1,Lipin1-? and Lipin1-? mRNA in AML-12 cells: As shown in Fig.1 and Table 2,with the increase of concentration of ethanol,the expression of SIRT1 decreased and the expression of SFRS10 decreased as well;The expression of Lipin 1 and Lipin1-? in the cytoplasm up-regulated gradually while the expression of Lipin1-? in the nucleus decreased gradually(F values are 35.64,16.59,100.81,78.92,28.41 respectively,P<0.01).As shown in Fig.2 and Table 3,the ratio of Lipin1-? to Lipin1-? up-regulated gradually with the increase of concentration of ethanol(P<0.01).1.2 The changes of protein expression of SIRT1,SFRS10,Lipin 1,Lipin1-? and Lipin1-? in AML-12 cells by Western blot: As shown in Fig.4 and Table 4,with the increase of concentration of ethanol,the expression of SIRT1 decreased and the expression of SFRS10 decreased as well;The expression of Lipin 1 and Lipin1-? in the cytoplasm up-regulated gradually while the expression of Lipin1-? in the nucleus decreased gradually(F values are 64.79,74.46,97.11,71.74,57.62 respectively,P<0.01).1.3 Oil red O staining to observe lipid deposition in the cytoplasm in AML-12 cells: As shown in Fig.5,nonsteatosis was observed in the AML-12 cells cytoplasm of normal control group.When cells stimulated by 40 mM concentration ethanol,there were a few of lipid droplets in cytoplasm.A quantity of lipid droplets distributed in the cytoplasm when stimulated by 120 mM concentration ethanol.1.4 Immunofluorescence staining to observe Lipin 1 distribution of cytoplasm and nucleus in AML-12 cells: As shown in Fig.6,in control group,Lipin 1 was present predominantly in the cytoplasm.Treatment with ethanol dramatically increased the intensity of Lipin 1 staining in the cytoplasm,suggesting an increase of Lipin 1 protein expression,while it decreased in the nucleus.2 Deletion SIRT1 gene to detect the expression of SIRT1,SFRS10,Lipin1-?,Lipin 1,Lipin1-? 2.1 Changes of mRNA expression of SIRT1,SFRS10,Lipin 1,Lipin1-? and Lipin1-? in AML-12 cells by RT-qPCR: As shown in Fig.7 and Table 5,kocking down SIRT1 gene,as SIRT1 siRNA group compared with Blank group,the expression of SIRT1 decreased by about 42% and the expression of SFRS10 decreased as well;The expression of Lipin 1 and Lipin1-? in the cytoplasm showed increased significantly while the expression of Lipin1-? in the nucleus decreased(all P<0.01).After knocking out SIRT1 gene and adding ethanol,the expression of SIRT1,SFRS10,Lipin1-? further decreased and the expression of Lipin 1,Lipin1-? showed more increased,the difference was statistically significant(P<0.05 or P<0.01).There were no statistical significance as SIRT1 siRNA negative control group compared with Blank group(all P>0.05).As shown in Fig.8 and Table 6,the ratio of Lipin1-? to Lipin1-? showed increased significantly by kocking down SIRT1 gene as SIRT1 siRNA group compared with Blank group,the difference was statistically significant(P<0.01);After knocking out SIRT1 gene and adding ethanol,the ratio of Lipin1-? to Lipin1-? showed more increased as SIRT1siRNA+Ethanol group compared with SIRT1 siRNA group,the difference was statistically significant(P<0.01).2.2 Changes of protein expression of SIRT1,SFRS10,Lipin1-?,Lipin 1,and Lipin1-? in AML-12 cells by Western blot: As shown in Fig.10 and Table 7,kocking down SIRT1 gene,as SIRT1 siRNA group compared with Blank group,the expression of SIRT1 decreased by about 45% and the expression of SFRS10 decreased as well;The expression of Lipin 1 and Lipin1-? in the cytoplasm showed increased significantly while the expression of Lipin1-? in the nucleus decreased(all P<0.01).After knocking out SIRT1 gene and adding ethanol,the expression of SIRT1,SFRS10,Lipin1-? further decreased and the expression of Lipin 1,Lipin1-? showed more increased,the difference was statistically significant(P<0.05 or P<0.01).There were no statistical significance as SIRT1 siRNA negative control group compared with Blank group(all P>0.05).2.3 Oil red O staining to observe lipid deposition in the cytoplasm in AML-12 cells: As shown in Fig.11,nonsteatosis was observed in the AML-12 cells cytoplasm of normal control group and negative control group.Deletion SIRT1 gene,there were lots of lipid droplets in cytoplasm.A quantity of lipid droplets distributed in AML-12 cells cytoplasm when stimulated by 120 mM concentration ethanol after deletion SIRT1 gene.2.4 Immunofluorescence staining to observe Lipin 1 distribution of cytoplasm and nucleus in AML-12 cells: As shown in Fig.12,in control group,Lipin 1 was present predominantly in the cytoplasm.Deletion SIRT1 gene,the expression of Lipin 1 protein in the cytoplasm increased and decreased it in nucleus.Treatment with ethanol dramatically increased the intensity of Lipin 1 staining in the cytoplasm after deletion SIRT1 gene.Conclusions:SIRT1 upstream regulates the expression of SFRS10,Lipin 1,which may partly be an important mechanism for AFLD. |
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