Mechanism Of FoxO3a On Apoptosis Induced By Palmitate In Liver Cells

BackgroundNonalcoholic fatty liver disease (NAFLD) is genetic-environment-metabolic stress-related liver disease, includes a wide spectrum of liver diseases, ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD has been described as the hepatic component of the metabolic syndrome (MS). Incidence increases year by year and age of onset become increasingly younger; the prognosis is no longer well, it very likely develop into cirrhosis, liver cancer or related death. Pathogenesis of NAFLD is complex and unclear, the current working model explaining the pathogenesis of NAFLD is the "two-hit" hypothesis, which can not explain all the problems in the development and progression of NAFLD.Recent studies also show that about 10 to 20% of NAFLD patients has not insulin resistance (IR), about 50% of NAFLD is not fully consistent with the diagnostic criteria of MS. We speculate that there may be more hits or unknown initiating factors in the development of NAFLD. Liver cell would death and/or apoptosis after it injuries by different causes. It is well know that the Fas expression and apoptosis in liver cells is increased in NAFLD. Unsaturated fatty acids (especially polyunsaturated fatty acids) have a protective effect on the liver, while the saturated fatty acids have toxic effect. Palmitate is one of the most saturated fatty acid in blood and liver. In recent years, FoxO proteins(Forkhead box-containing protein, class O) can regulate cell fate by modulating the expression of genes involved in cell cycle transitions, apoptosis, DNA repair, oxidative stress and longevity, as well as metabolism in the area of immune regulation and tumor formation.FoxO3a is kind of FoxO protein, it locates in the nucleus and induces the transcription of genes encoding pro-apoptotic proteins such as FasL, Bim, p130 and Cdknlb/p27, thereby inducing cell apoptosis. Once phosphorylated by kinase, it is shifted out the nucleus and bind to 14-3-3 protein, which can protect the cell from apoptosis. Therefore, we speculated that palmitate accumulation in the liver cells and FoxO3a in the Apoptosis may be an important pathogenesis of NAFLD.According to those researches, and our recent research related to the pathogenesis of fatty liver, in this study, following three sections were studied on the molecular mechanism of apoptosis. It is expected to reveal the pathogenesis of NAFLD, interfere the disease progression and provide the theoretical basis of prevention of complications.Methods一,A fat over accumulation profile and apoptosis in liver cells induced by palmitate.1. Model of hepatocellular steatosis:Detection of intracellular TG content and oil red O stain identified cellular lipid droplet accumulation in the HepG2.2.15 cell and L02 cell induced by palmitate.2.Group:the cells were cultured in palmitate of 100μmol/L, 200μmol/L,400μmol/L and 600μmol/L for 24 hours or palmitate of 400μmol/L for 6h,12h,24h and 48h.3. We examined the changes cell viability, apoptosis rate and the activity of Caspase3 in the HepG2.2.15 and L02 cell by MTT, Propidium Iodide (PI) fluorescent staining and flow cytometry and Caspase3 Detection Kit.4. Bim mRNA,p27kip1 mRNA of cells were measured by RT-PCR; the total FoxO3a, p-FoxO3a (Thr32) protein were measured by Western Blot.二,The alteration of apoptosis induced by palmitate in HepG2.2.15 cells after transfection of FoxO3a siRNAl.1. the most efficient siRNA was selected to the further study.①Prime synthesis:according to human FoxO3a gene sequence, Three siRNAs (PI, P2, P3) and a random negative control (siRNA-NC) were designed and synthesized to silence FoxO3a.②Concentration of transfection:The negative control siRNAs labelled with fluorescence(5'FAM)were transfected into cells.Then fluorescent microscope was employed to determine the concentration at which siRNAs were transfected most effectively.③RT-PCR:The FoxO3a mRNA was measured after HepG2.2.15 cell transfected with the siRNAs respectly.④Western Blot:The FoxO3a protein was measured.2. HepG2.2.15 cells transfected by FoxO3a small interfering RNA1 (siRNA1) cultured by palmitate.HepG2.2.15 cells were cultured in five groups:(1) Mock group:cells were cultured in DMEM medium containing LipofectamineTM 2000 for 6 hours, then cultured for 48 hours.(2) P1 groups:cells were transfected with FoxO3a siRNAl for 54 hours.(3) P1+PA groups:cells were transfected with FoxO3a siRNAl for 48 hours,then cultured in 400μmol/L palmitate medium for 6 hours.(4) NC groups:cells were transfcctcd with negative siRNA for 54 hours.(5) NC+PA groups:cells were transfcctcd with negative siRNA for 48 hours, then cultured in 400μmol/L palmitate medium for 6 hours.三,The relation between FOXO3a expression in peripheral blood mononuclear cells and the severity of patients with nonalcoholic fatty liver disease1.42 NAFLD patients among whom 22 were nonalcoholic steatohepatitis(NASH) and 20 were Simple steatosis(SS), and 20 healthy person as control were enrolled in the study.2. RT-PCR:RT-PCR measured FoxO3a mRNA, Bim mRNA and p27kipl mRNA in peripheral blood mononuclear cells (PBMC).3. Multiple linear regression analysis:The relationships between FoxO3a mRNA and clinical indicators and biochemical parameters were analyzed by multiple linear regression analysis. Results1. Lipid droplet and TG content in cells increased gradually with time after cultured with palmitate.2.A concentration-dependent relation between palmitate and cells injury was observed. The cells apoptosis rate and Caspase3 activity were increased significantly, while the cellular viability was decreased.3.A significant time-dependent relation between palmitate and cells injury was observed.The cells apoptosis rate and Caspase3 activity were increased significantly, while the cellular viability was decreased.4. Bim mRNA, p27kipl mRNA expression increased gradually with time-dependent of palmitate cultured. The total FoxO3a protein of cells had no significant change, p-Fox03a protein gradually decreased with time prolonged. FoxO3a protein expression of HepG2.2.15 cell was higher than the L02 cells.5. siRNAs in 50 nmol/L had stronger fluorescence. siRNA1 was the most potent silencer.6. There were significant differences in expressions of FoxO3a mRNA after HepG2.2.15 cell transfected with siRNA1 and siRNA2 (P< 0.05), the expressions of FoxO3a mRNA decreased gradually in PI and P2 group with the time. There were no difference in P3 group(P>0.05); P1 (siRNA1) was the most potent silencer. The expressions of FoxO3a protein were gradually suppressed by siRNA1.7. The nucleus had less green fluorescent, cytoplasm had more in P1 group; on the contrary in P1+PA group.8. The apoptosis rate, Caspase3 activity remained unchanged among the NC group, P1 group and mock group (P>0.05). Apoptosis, Caspase3 activity decreased in P1+PA group than the NC+PA group (P<0.05).9. The expression of Bim mRNA,p27kip1 mRNA remained unchanged among the NC group, P1 group and mock group (P>0.05); P1+PA group had less than the NC+PA group(P<0.05).10. The total FoxO3a protein decreased in P1+PA group and P1 group, while the other groups had no different.p-FoxO3a expression at protein levels had no different among the NC group and mock group,P1 group. The NC+ PA group was the lowest; P1+PA group had more than in P1 group.11.Clinical research:In addition to age and height, there were significant differences in BMI, WHR, FFA, HDL-C, TC, TG, LDL-C, GPT between NAFLD and the health people (P<0.05 or P<0.001). BMI, WHR, FFA, HDL-C, TC, LDL-C, GPT was higher in NASH than SS group(P<0.05 or P<0.001); FOXO3a mRNA and Bim mRNA, p27kip1 mRNA gradually increased in Health people, nonalcoholic fatty liver and nonalcoholic steatohepatitis (P<0.05 or P< 0.001), the linear correlation between FOXO3a mRNA and Bim mRNA. In multiple linear regression analysis, WHR, FFA, triglyceride, BMI, LDL-C associated with FOXO3a mRNA, waist-hip ratio had most effection (multiple correlation coefficient R=0.953, coefficient of determination R2= 0.908). after diet restraint, weight control and taking lipid-lowering drugs et al, WHR, blood lipids and other improved(P<0.05 or P<0.001), and FOXO3a mRNA expression decreased (t=23.25, P<0.05).Conclusions1.HepG2.2.15 cell and L02 cell could be induced to injury and apoptosis by palmitate and had a significant concentration- and time-dependent.2. Bim mRNA, p27kipl mRNA were increased, then apoptosis occurred.3. HepG2.2.15 cells had higher expression of FoxO3a protein, and FoxO3a activation was increased by palmitate.4. siRNA1 was the most effective silencer.5. Apoptosis didn't induce by palmitate in HepG2.2.15 cells transfected FoxO3a siRNA1, Lipoapoptosis reduced through the decreased Caspase3 activity, inhibition of Bim, p27kipl expression. Palmitate promoted FoxO3a dephosphorylation (deactivation) and entered the nucleus.6. NAFLD had higher level of blood lipids and FOXO3a mRNA in peripheral blood mononuclear cells, FoxO3amRNA and BimmRNA had the linear relationship. The level of blood lipids affected FoxO3amRNA expression.