CD38 Gene Silencing Protects H9c2 Cardiomyocytes From Hypoxia/Reoxygenation Injury Through Reducing Oxidative Stress

ObjectivesCD38 is a multifunctional enzyme that has both ADP-ribosyl cyclase and c ADPR hydrolase activities, being capable of cleaving NAD+ into cADPR and hydrolyzing c ADPR to ADPR. It is widely distributed in various tissues and has many biological functions. It has been reported that CD38 deficiency in mice significantly elevated the intracellular NAD+, which results in activation of SIRT1. SIRT1 as an important NAD+ dependent deacylation enzyme was reported to activate FOXO1 anti-oxidative stress signaling pathway. Our previous experimental results showed that mouse embryonic fibroblasts(MEFs) from CD38 knockout mice were significantly resistant to oxidative stress such as H2O2 injury and Hypoxia/Reoxygenation(H/R) compared with wild type MEFs, suggesting that CD38 deficiency may play a pivotal role in myocardial ischemia reperfusion injury. However, its role in myocardial ischemia and reperfusion injury has not been reported. In the present study, we explored the effects of CD38 gene silence on the H/R induced oxidative stress injury in vitro and the possible mechanisms. There is no doubt that our data should provide evidences for treating the cardiac ischemic diseases clinically. Methods1.Preparation of cell hypoxia re-oxygenation model: The hypoxic condition was generated using a hypoxia chamber and flushed with a mixture of gas(95% N2 and 5% CO2) for 4 hours. Followed by different hours of re-oxygenation, the cell activity was detected by CCK8 test.2.Detection of HR induced cell injury:After the H/R injury stimulation, the mitochondrial membrane potential(MMP) and apoptosis was detected by flow cytometry with JC-1 and Annexin-V/PI double stainingrespectively. The LDH release from the supernatant is also detected by multifunctional microplate reader.3.ROS detection:The ROS generation was detected by flow cytometry and fluorescence microscopy after fluorescence probe DCFH-DA staining.4.The effect of CD38 gene knockdown on FOXO1 nuclear translocation was detected by cellular immunofluorescence assay.5.The Real-time PCR was used to detect the interference efficiency of CD38 gene transcription in CD38 interference cell line and the mRNA expression levels of Catalase and SOD2 after hypoxia and re-oxygenation injury.6.Western bolt was used to detect the protein expression levels of Catalase and SOD2 after H/R stimulation. Results1.After 4 hours of hypoxia and different times of re-oxygenation, the activity of H9c2 cells showed a significant decrease, and presented in a time dependent manner. According to the result, 4 hours Hypoxia followed with 3 hours re-oxygenation was enough to mimic the acute myocardial ischemia reperfusion injury in vitro.2.After H/R stimulation, the cell activity and MMP were significantly decreased in control group, but attenuated in CD38 knockdown H9c2 cells. Consistent with the above result, the LDH release, and apoptosis were detected to be increased by H/R stimulation, but decreased in CD38 gene silenced cells.3.The generation of ROS was significantly induced by H/R stimulation while reduced in CD38 knockdown cells.4.CD38 gene silencing promote FOXO1 nuclear translocation in H9c2 cells.5.CD38 gene knockdown promotes the expressions of antioxidants Catalase and SOD2. ConclusionsIn summary, the results from our present study demonstrated that CD38 gene knockdown significantly protects heart from H/R induced injury in vitro, in which the protection is primarily associated with activating SIRT1/FOXO1 anti-oxidative stress signaling pathway and promoting the expression of antioxidant Catalase and SOD2.