Protection And Its Mechanisms Of CD38 Deficiency On Lipid Overload-induced Cardiac Oxidative Stress In Mice

Background and Aims:In recent decades,with the development of global economy,the number of patients suffered from obesity is increasing.And the occurrence of obesity has adverse impacts on our body,and increases the incidence of cardiovascular diseases and the oxidative stress injury in heart tissue.It’s been reported that CD38 knockout can effectively suppress the obesity induced by high fat diet（HFD）,and our previous studies have suggested that CD38 knockout can protect the heart from ischemia-reperfusion injury through activating Sirt1/FOXOs-mediated antioxidant stress pathways.However,the roles of CD38 in lipid overload-induced heart injury were not evaluated.In the present study,we investigated the preventive effects and mechanisms of CD38 deficiency on mice heart injury induced by HFD.And our study could provide new targets and experimental basis for the prevention and treatment of cardiac damage induced by obesity.Methods:1.Preparation and analysis of the model of cardiac lipid overload induced by high fat diet:the 8-10 weeks C57BL/6 mice were randomly divided into two groups:（1）Wild Type mice group;（2）the CD38^-/-mice group,the number of mice in each group is 5 or 6.Then,the mice were feeded by high fat diet for 12 weeks.Finally,the metabonomics analysis of mice hearts was conducted to determine the protective effects of CD38 knockout on injured heart induced by lipid overload.2.Preparation and analysis of the model of cardiac lipid overload induced by oleic acid in cardiomyocytes:In order to determine CD38 gene knockout effects on myocardial cells,normal and CD38 knockdown of H9C2 cardiomyocytes were treated under the condition with or without oleic acid（0.5 m M）for 24h.Oil red staining was used to examine the lipid accumulation.The intracellular TG content was detected by Triglyceride assay kit.The CCK-8 kit was used to examine the cell viability.The enzyme activity of SOD and LDH were detected by special kit,and the content of intracellular ROS was examined.3.Mechanism analysis:To determine the protective mechanisms of CD38knockout on injured heart induced by lipid overload,we examined the protein and mRNA level of Sirt3,FOXO3,FASN,as well as genes involved in oxidative stress and apoptosis were detected by Western Blot and Q-PCR in H9C2 cell according to the results of metabonomics of heart tissue in mice.Results:1.The principal components and cluster analysis showed that there are differences in 271 compounds between WT mice and CD38^-/-mice after HFD,87 of those metabolites were significant differences,among which 60 compounds were significantly increased and 27 metabolites were decreased obviously in the heart of CD38^-/-mice.2.We found the level of NAD⁺,NMN（the NAD⁺precursor）,the metabolites of CD38 including niacinamide and ADPR were significantly increased in CD38deficiency mice comparing with those in WT mice under HFD.More importantly,the level of reduced GSH was increased,but oxidized glutathione GSSG content was lower in heart tissues of CD38 deficient mice compared to WT counterparts.Therefore,the ratio of GSH/GSSG representing the oxidative stress was increased in CD38 deficient heart.3.The results showed that the activity of SOD and the viability of myocardial cells were significantly increased in CD38 knockdown cells,but the activity of LDH and the level of ROS were significantly decreased after the OA treatment.4.Our results showed that the content of long chain fatty acids and polyunsaturated fatty acid in the heart of CD38^-/-mice was lower than the WT group under HFD.Moreover,the lipid accumulation,the content of TG and the expression of FASN were obviously decreased in CD38 knockdown myocardial cells after the OA treatment.5.We observed that the level of total protein acetylation in was significantly upregulated after the OA treatment.But compared with the control group,the level of protein acetylation was significantly decreased,while the levels of protein and mRNA of Sirt3,SOD2 and FOXO3a were significantly upregulated in CD38 knockdown cells.6.We found the expressions of NOX2,NOX4 and Bax were significantly decreased in CD38 knockdown cells compared with the control group,while the expression of Bcl2 was significantly increased,so the ratio of Bcl2/Bax was significantly increased with or without OA treatment.Conclusion:1.CD38 knockout alleviated the cardiac oxidative stress injury and apoptosis induced by lipid overload.2.CD38 knockout reduced the cardiac lipid synthesis in vivo and in vitro.3.CD38 deficiency protected heart from lipid overload-induced oxidative stress via activating Sirt3/FOXO3 pathway.