Effects Of Medium Chain Fatty Acids On Further Liver Injury And Lipid Metabolic Disorders In Human Liver Cells With Steatosis

With the improvement of people's living standards and physical inactivity,resulting in a significant increase in the incidence of obesity and related metabolic diseases,which damage the body's health.In recent years,looking for a healthy and effective way to lose weight has always been concerned.However,interactions of dietary fat(as the main cause precipitation of lipid)and body especially in the intrinsic molecular mechanisms are still unclear.The dissertation is armed at the establishment of cellular steatosis model,exploration of medium-chain fatty acids the impact on hepatic steatosis cells in apoptosis,oxidative stress,immune response and lipid metabolism in a more systematic study by using a combination of biological techniques.An experimental cellular model of NAFLD was established by MTT cell viability assay,LDH method to detect cell necrosis,Hoechst 33342 staining for apoptosis assays,oil red O staining and triglycerides quantitative assay,scanning electron microscopy cell injury assay,which had significant lipid accumulation in the absence of overt cytotoxicity.The mechanisms that promote disease progression from NAFLD to further liver injury are investigated by combining a variety of analytical methods,including apoptotic cells were quantified by FCM(staining with Annexin V-FITC/PI)and were observed under fluorescence microscope(staining with Hoechst 33342/PI),determination of antioxidant defense and oxidative stress by ELISA assay,2,DE and MS analysis of different proteins and inflammatory cytokines was measured by Western blot analysis,ELISA and real-time PCR analysis.We also further examined the effects of MCFA on lipid deposition in the liver and protein expression of lipid-sensing genes in the cellular model of steatosis by MTT cell viability assay,oil red O staining and triglycerides quantitative assay,and mRNA and protein expression of lipid-sensing genes was measured by Western blot analysis,ELISA and real-time RT-PCR analysis.The main research conclusions in this dissertation are summarized as follows:In Chapter 1,a background of fatty acid biological characteristics and the research progress of non-alcoholic fatty liver disease are summarized,and further made a prospect for molecular mechanism of NAFLD.Fatty acid biological characteristics including metabolism of fatty acids characteristics,physiological functions,research progress of security and potential limitations.Pathogenesis research of NAFLD including causes of the disease,model of building,as well as lipid metabolism related signaling pathways.In Chapter 2,we explored the conditions for the establishment of non-alcoholic fatty liver disease model,which has significant lipid accumulation in the absence of overt cytotoxicity.MTT and LDH studies the effect of mixed fatty acids(oleic acid and palmitic acid,2:1)and a single fatty acid(oleic acid)on proliferation and apoptosis in L02 and HepG2 cells,and the results indicated that fatty acids cytotoxicity have a concentration and exposure time-dependent manner.There was no effect on cell viability in any given time after treatment at a concentration of less than 400 ?m,and the L02 cells more sensitive to the toxic effects of fatty acids than HepG2 cells.Hoechst staining results further shown that apoptosis was positively correlated with concentration of fatty acids,apoptosis was not caused at concentrations less than 400 ?M,and cytotoxicity can be caused at 800 ?M compared with the control group.The intracellular triglyceride content was determined by Oil Red O staining and phosphoglycerol oxidase method,and the results show that mixed fatty acid is more suitable for the establishment of fatty liver steatosis model than single fatty acid.A cellular steatosis model,which is suitable to experimentally investigate the impact of fat accumulation in the liver,was established in L02 human liver cells with a mixture of free fatty acids(oleate/palmitate,2:1)at 200?M for 24 h incubation.In Chapter 3,we studied the effect dietary fatty acids on apoptosis and oxidative stress in the human liver steatosis,in order to explore the mechanisms that promote disease progression from nonalcoholic fatty liver disease to further liver injury.Flow cytometric analysis showed that medium-chain fatty acid(MCFA)markedly decreased the percentage of late apoptotic and necrotic cells compared with long-chain fatty acid(LCFA),and MCFA inhibited the activities of Caspase-3 and Caspase-9.Proteomic analysis further showed that LCFA inhibited the expression of antioxidant enzymes,and increased the expression of proteins associated with oxidative stress,we also had similar results by measure activities of intracellular SOD,MDA,ROS and GSH-PX.It was found that LCFA(palmitate),not MCFA induced apoptosis and oxidative stress in the hepatic cells with steatosis.In conclusion,selection of dietary fats has potential to translate therapeutically through a personalized nutrition approach.In Chapter 4,we studied the effect dietary fatty acids on immune response in the human liver steatosis,in order to explore the mechanisms that promote disease progression from nonalcoholic fatty liver disease to further liver injury.The production of NO and activity of iNOS,indicating that both MCFA groups exerted no effect on the production of NO and iNOS after 12 h exposure,but C16:0 treatment tended to increase the level of NO and activity of iNOS when compared to NR,although no significant difference.Western blot analysis found that the levels of inflammatory markers(IL-6,IL-1-? and TNF-?)were substantially reduced by MCFA compared with LCFA,and C16:0 treatment had a markedly positive effect.These results indicated the specificity of FA-induced apoptosis in human liver cells with steatosis by other ways,and reasonable selection of dietary fats has potential to translate therapeutically by ameliorating disease progression in patients with NAFLD.In Chapter 5,Accumulation of lipids in the liver can lead to cell dysfunction and steatosis,an important factor in pathogenesis causing non-alcoholic fatty liver disease.The mechanisms related to lipid deposition in the liver,however,remain poorly understood.This study investigate the effects of medium-chain fatty acid(MCFA)on the lipolysis and expression of adipogenic genes in human liver cells with steatosis,is aimed to find a substitute for dietary fat.Oil Red O staining and phosphoglycerol oxidase method showed that:compared with LCFA(oleic acid),MCFA can significantly reduce lipid droplets accumulated within L02 fatty cells,and act similar effect as gemfibrozil drug.The results of RT-PCR and WB shown that:MCFA can down-regulation expression of liver X receptor-a,sterol regulatory element binding protein-1,acetyl-CoA carboxylase,fatty acid synthase,CD 36 and lipoprotein lipase in this cellular model,and have positive effects on adipose triglyceride lipase and hormone-sensitive lipase.These results suggest that MCFA is capable of reducing lipid accumulation by down-regulating expression of adipogenic genes in human liver cells.In Chapter 6,medium-chain fatty acids impact on response of hepatic steatosis cells to apoptosis,oxidative stress,lipid metabolism and immune response are summarized,and the further research aspects are prospected.In summary,the molecular mechanisms of interaction between dietary fat and NAFLD cells still need further exploration.This study including the establishment of NAFLD cell model,as well as cellular activity and apoptosis,oxidative stress,lipid deposition and the immune response after explore to MCFA.This study further explored the mechanisms in fatty lipid metabolism and immune regulation at cell and molecular level.This research can provide guide significance for obesity to reasonable choice of the type of dietary fat,and provide a scientific basis of prevention and treatment of NAFLD.