Study On The Mechanism Of Hepatocyte Steatosis And Apoptosis Induced By OALNs

Nonalcoholic fatty liver disease(NAFLD)is the most common liver disease.Unbalanced dietary fatty acid intake is one of the main causes of NAFLD,which can cause steatosis in the liver and further induce lipotoxicity(cellular stress,damage and even death).Oleic acid induced lipid accumulation in hepatocytes had been demonstrated to be an efficient cell model for the study of NAFLD.However,the modeling effects of oleic acid are quite different in many animal experiments because fatty acids are insoluble in water and the drug delivery systems chosen by various researchers are different.Therefore,we hope to prepare a more stable oleic acid lipid nanoparticles(OALNs)that can be easily delivered in vivo and in vitro experiments,observe the effects it causes on hepatocytes and explore the mechanism of toxic effects of oleic acid overload.Establish a stable pathogenic model for further studies of NAFLD.Experimental method:1.Preparation method of OALNs.The oil phase was composed of oleic acid and egg yolk lecithin,which was dispersed into the water phase(including glycerin and Tween-80)by stirring.OALNs was prepared by high-speed homogenization and high-pressure homogenization.2.Effects of OALNs on steatosis and lipid metabolism genes in hepatocytes.The degree of cellular steatosis was observed by oil red O staining.The expression of lipid metabolism genes and proteins was detected by q RT-PCR and Western blot.3.Study on the mechanism of toxic effects of OALNs on hepatocytes.The hepatocyte viability was detected by CCK-8.Morphological observation,DAPI staining and flow cytometry were used to determine the major cell death modes,and the hepatocytes shape and structure were observed by transmission electron microscopy.DCFH-DA probe was used to detect the level of intracellular ROS.Transcriptome sequencing was used to analyze cellular gene expression changes.Cellular immunofluorescence staining was performed to observe protein localization,and Western blot was used to detect intracellular protein expression.4.Animal experiments to verify the mechanism of steatosis and toxic effects of OALNs on mice liver.The KM mice were injected intraperitoneally with OALNs.The changes of serum AST and ALT were detected.H&E staining,TUNEL staining and immunohistochemical staining was performed to detect pathological changes.Western blot was performed to detect liver protein expression in mice.Experimental results:1.Stable OALNs was prepared in this study.After 3 months of storage,the particle size,the Zeta and the PDI of OALNs had no significant change,which confirms the good stability of the OALNs.2.OALNs induced hepatocytes steatosis and upregulated the expression of lipid metabolism factors.OALNs can significantly induce hepatocyte steatosis.Treatment with OALNs increased the m RNA and protein expressions of SREBP-1c,FASN,and PPARγin a dose-dependent manner in HL-7702 cells.3.OALNs induced apoptosis via the ROS/DDIT3/BCL2/BAX/Caspase pathway.The survival rate of hepatocytes decreased with increasing dosing concentrations of OALNs.DAPI staining showed nuclear fragmentation and chromatin condensation.Flow cytometry results showed that the ratio of apoptotic cells increased.ROS gradually accumulated in cells with drug concentration.DDIT3 and TRIB3 were detected by transcriptome sequencing,which were the differentially expressed genes related to apoptosis.The m RNA and protein level of DDIT3 were increased,and BAX was transferred to mitochondria.The results of transmission electron microscopy and JC-1detection suggested that mitochondria were damaged.The expression of BCL2 protein was down-regulated,and the expressions of BAX,Cleaved Caspase-3/9 and Cleaved PARP proteins were all up-regulate.4.The mechanism of OALNs-induced hepatic steatosis and apoptosis was verified in vivo experiments.Compared with the normal mice,the livers of the OALNs mice showed fat vacuolation and increased triglyceride content,and AST and ALT were significantly increased.The apoptosis rate in the liver tissue of the mice in the administration group increased.The nuclear localization of DDIT3 increased drastically in the liver of mice.The expression levels of DDIT3,BCL2,BAX and Cleaved Caspase-3 were consistent with in vitro experiments.Conclusion:This study used OALNs to establish a NAFLD model in vitro and in vivo,which is characterized by highly pronounced steatosis and/or apoptosis.OALNs caused excessive oleic acid uptake by hepatocytes,resulting in hepatic steatosis,which further induced apoptosis via the ROS/DDIT3/BCL2/BAX/Caspase pathway.