PON2 Mediates Cardioprotection By Against Cytotoxicity Of Ox-LDL Through The JNK And Oxidative Stress Pathways

Objective:Paraoxonase 2(PON2)is one of the paraoxonase family members and it is widely expressed in human tissues.It has antiatherogenic and antioxidative stress effects.At present,a lot of studies have focused on the effects of PON2,but little research has been studied on the intracellular pathways.This study aimed to evaluate the intracellular mechanisms of PON2against cytotoxicity of ox-LDL in neonatal rat ventricular cells.Methods:The SD neonatal rats born within 1-3 days of each gender were selected,which were provided by Experimental Animal Center of Hebei Medical University.After anesthesia,neonatal rats were removed from the heart for cell culture.The recombinant adenovirus with PON2 interference gene was used to transfect the cardiomyocytes of neonatal rat to make the PON2 protein low expression.The adenovirus carriers,ox-LDL,PON2 protein and JNK inhibitor SP6000125 were used for pre-treatment.The myocardial cells of neonatal rats were grouped into:control group,ox-LDL group,ox-LDL+PON2 protein group,ox-LDL+SP600125 group,control AD group,control AD+ox-LDLgroup,PON2 siRNA group,ox-LDL+PON2 siRNA group,The cell viability was measured by MTT assay.The cell apoptosis was detected by caspase 3/7 activity assay kit.The phosphorylation level of JNK and the protein expression of Nox2/gp91~phoxhox and PON2 were detected by western blot.Results:1.Compared with the control group,the survival rate of the ox-LDL group significantly decreased[(0.400±0.150)vs(0.654±0.153),P<0.05];compared with the ox-LDL group,the survival rate of the ox-LDL+PON2protein group and the ox-LDL+SP600125 group significantly increased[(0.560±0.200),(0.550±0.180)vs(0.400±0.150),P<0.05],compared with the control AD group,the survival rate of the ox-LDL+PON2 siRNA group significantly decreased[(0.230±0.110)vs(0.371±0.151),P<0.05].Compared with the control group,the activity of caspase 3/7 significantly increased in ox-LDL group[(11962.33±643.41)vs(6620.5±342.01),P<0.05];compared with ox-LDL group,the activity of caspase 3/7 significantly decreased in ox-LDL+PON2 protein group and ox-LDL+SP600125 group[(7462.00±402.75),(7521.80±499.80)vs(11962.33±643.41),P<0.05],compared with the control AD group,the activity of caspase 3/7 significantly increased in ox-LDL+PON2 siRNA group[(13562.5±574.42)vs(11816.33±640.21),P<0.05].It was confirmed that PON2 can reduce the apoptosis of cardiomyocyte induced by ox-LDL and reduce the damage of cardiomyocyte.2.Compared with the control group,the protein expression of Nox2/gp91~phoxhox significantly increased in the ox-LDL group[(1.400±0.140)vs(0.540±0.230),P<0.05];compared with the ox-LDL group,the protein expression of Nox2/gp91~phoxhox in ox-LDL+PON2 protein group significantly decreased[(0.868±0.226)vs(1.400±0.140),P<0.05];compared with the control AD group,the protein expression of Nox2/gp91~phoxhox in ox-LDL+PON2siRNA group significantly increased[(1.740±0.140)vs(1.396±0.140),P<0.05].It was confirmed that PON2 can inhibit oxidative stress induced by ox-LDL.3.After the transfection of neonatal rat cardiomyocytes with an adenovirus which was carrying the PON2 interference gene,the protein expression of PON2 significantly reduced,which was indicated that the low expression model of PON2 was successfully established.Compared with the control group,the phosphorylation level of JNK significantly increased in the ox-LDL group[(0.370±0.020)vs(0.150±0.020),P<0.05];compared with the ox-LDL group,the phosphorylation level of JNK decreased in the ox-LDL+PON2 group[(0.204±0.096)vs(0.370±0.020),P<0.05];compared with the control AD group,the phosphorylation level of JNK significantly increased in ox-LDL+PON2 siRNA group[(0.620±0.060)vs(0.3689±0.024),P<0.05].It was confirmed that PON2 inhibits the activation of JNK signaling pathway induced by ox-LDL.Conclusion:1.PON2 can reduce the apoptosis and the damage of cardiomyocyte induced by ox-LDL.2.PON2 can alleviate ox-LDL damage to cardiomyocytes by inhibiting oxidative stress pathway.3.PON2 can alleviate ox-LDL damage to cardiomyocytes by inhibiting JNK pathway.