Protections And Mechanisms Of Liver-specific Deletion Of CD38 Gene On Non-alcoholic Fatty Liver In Mice

Background and Aims:Non-alcoholic fatty liver disease(NAFLD)is a clinicopathological syndrome characterized by excessive lipid accumulation in hepatocytes due to factors other than alcohol and other well-defined liver damage factors.It is an acquired metabolic stress liver injury associated with insulin resistance and genetic susceptibility.Due to changes in lifestyle and diets,the incidence of NAFLD has been gradually increasing in recent years.If not controlled,NAFLD will be developed into irreversible nonalcoholic fatty liver hepatitis and cirrhosis,and eventually developed into liver cancer.CD38,the main NADase in mammalian cells,can regulate intracellular NAD+level which is closely related to metabolic diseases.However,the roles of CD38 in NAFLD are still unclear.The aim of this study was to explore the effects and mechanisms of CD38 on NAFLD induced by high fat diet.Obviously,our study will provide a new insight in early prevention and treatment of NAFLD.Methods:1.Preparation and analysis of non-alcoholic fatty liver model induced by high fat diet in vitro: Liver-specific CD38 knockout(CD38-LKO)and control(CD38fl/fl)male mice with 8-week age were divided into four groups: 1)WT-ND group: wild type(CD38fl/fl)mice were fed with normal diet(ND);2)KO-ND group: CD38-LKO(KO)mice were fed with normal diet;3)WT-HFD group: CD38fl/fl mice were fed with high fat diet(HFD);4)KO-HFD group:CD38-LKO(KO)mice were fed with high fat diet,and the mice were fed with ND or HFD for 16 weeks.The mouse weights were monitored weekly and the glucose tolerance test and insulin tolerance test were performed for all mice before they were sacrificed.Then the liver tissue from the same part of each mouse was fixed in 10% formaldehyde.The paraffin sections and frozen sections were stained with HE and oil red respectively to analyze the accumulation of lipid droplets in the liver.The intracellular triglyceride(TG) content,the malondialdehyde(MDA)content and the superoxide dismutase(SOD)activity were examined in liver tissue.The RNA and total protein were simultaneously extracted from liver tissue and the expressions of genes involved in oxidative stress and fatty acid oxidation were detected in liver tissue by Q-pcr and Western blot.2.Preparation and analysis of oleic acid(OA)-induced hepaticsteatosis models in mouse primary hepatocytes: 1)The primary hepatocytes were isolated from C57BL/6 and CD38-LKO mice at 6-8 weeks of age by in vitro perfusion method;2)The expressions of CD38 were examined in primary hepatocytes treated with different concentrations of oleic acid;3)The contents of intracellular TG and MDA were detected,and the relative SOD activity and the mitochondrial membrane potential were also examined;4)The RNA and total protein were extracted from the primary hepatocytes,and the expressions of genes involved in oxidative stress and fatty acid oxidation such as CAT,SOD2,CPT-1 and ACOX1 were analyzed.3.Preparation and analysis of oleic acid(OA)-induced hepaticsteatosis models in the hepatocyte cell lines with overexpression of CD38 in vivo: 1)CD38over-expressed stable cell lines were established by transfecting flag CD38 plasmids in HEP1-6 cells;2)The control cells and over-expressing CD38 cells were stimulated with oleic acid for 24 hours,and the lipid accumulation was examined by Oil Red staining.The contents of TG and MDA were detected with the corresponding kits;3)The expressions of genes involved in oxidative stress and fatty acid oxidation such as CAT,SOD2,PPARα and CPT-1 were examined.Results:1.The expression of CD38 was up-regulated in liver tissues from wild type mice fed with HFD compared with ND feeding.In addition,its expressions were also significantly increased in HEP1-6 cells and primary hepatocytes with stimulation of oleic acid in a dose-dependent manner.2.The HFD-induced accumulation of the intracellular lipids was significantly attenuated in liver of CD38 conventional knockout mice.The insulin resistance was markedly improved in the livers of the liver-specific CD38 knockout mice.The results from HE staining,Oil red staining and the intracellular triglyceride Kit showed that the lipid accumulation in liver was reduced in CD38-LKD mice compared with wild type mice.Moreover,oxidative stress was improved in the null mice and the expressions of sirt1 and its target genes PPARα and SOD2 were increased in liver tissues from CD38-LKO mice fed with HFD.3.Deletion of CD38 gene in hepatocytes significantly reduced the oleic acid-induced the elevation of triglyceride in hepatocytes,and improved the oxidative stress injury through reducing the content of MDA,increasing the SOD activity and mitochondrial membrane potential.In addition,CD38 deficiency in hepatocytes promoted the expressions of Sirt1 and the genes involved in oxidative stress and fatty acid oxidation such as CAT,SOD2,CPT-1 and ACOX1.4.Overexpression of CD38 in HEP1-6 cell lines significantly promoted the oleic acid-induced lipid accumulation,and the increases of the intracellular TG and MDA contents.In contrast,the expressions of Sirt1 and the genes involved in fatty acid oxidation and anti-oxidative stress such as PPARα,CPT-1,SOD2 and CAT were significantly decreased and the expression of NOX2 protein was increased in the CD38 overexpressing cells.Conclusions:1.The expressions of CD38 were up-regulated in liver from mice under HFD and in hepatocytes treated with oleic acid.2.Deletion of CD38 gene in vivo and in vitro improved lipid accumulation and oxidative stress injury with treatment of HFD or stimulation of oleic acid,while overexpression of CD38 in hepatocytes aggravated the effects in vivo.3.Liver-specific deletion of CD38 gene alleviated the HFD or oleic acid-induced the lipid accumulation and oxidative stress injury through activating Sirt1/PPARα/SOD2 signaling pathway.