Study On The Molecular Mechanism Of Ophiopogonin D Regulating CYP2J In Treating Heart Failure

Objective:Ophiopogonin D（OPD）is a major active ingredient in the Chinese medicine shenmai injection which commonly used in the treatment of cardiac diseases.OPD can protect cardiovascular,anti-inflammatory and inhibit tumor proliferation.CYP2J also plays a very important role in maintaining cardiovascular homeostasis.Previous studies by our research group have shown that OPD can reverse myocardial ischemia-reperfusion injury and myocardial hypertrophy caused by AngⅡin rats.The mechanism is related to the activation of the CYP2J3-EETs system by OPD.It also revealed for the first time the mechanism of OPD in the treatment of heart failure in H9c2 cells,that is,inducing the expression of CYP2J3,enhancing the interaction between SERCA2a and PLB,and improving calcium circulation.However,how OPD up-regulates the expression of CYP2J is still unclear,especially the molecular initiation event of OPD entering the cell is still unclear.The aim of this research is to investigate the mechanism of OPD alleviating heart failure in rats;and to explore whether its up-regulation of CYP2J expression is mediated by other receptors.To explore the mechanism that OPD can reduce the myocardial toxicity of OPD′.To supplement the myocardial protective effect and pharmacological activity of OPD.The experiment covers the whole framework of OPD entering the molecular initiation event of the cell,to the key event of the signal pathway to the beneficial outcome of myocardial protection,and provides a theoretical basis for the clinical application and further research of shenmai injection and Ophiopogon.Methods:（1）Rats were injected with ISO（5 mg/kg/d）for 7 consecutive days in subcutaneously to establish heart failure model,and then treated with OPD（20mg/kg/d）for 7 consecutive days;the serum NT-pro BNP,CTn-T,AST,CK,CK-MB,LDH and the content of MDA and SOD in myocardial tissue of rats were detected.The pathological changes of myocardial were detected by hematoxylin-eosin（HE）staining,the expression of apoptosis-related proteins and CYP2J3 were detected by Western-blot.（2）Different concentrations of OPD were used to treat AC-16 cells and H9c2 cells,and CCK-8 was used to detect the survival rate of the cells to further confirm the concentration of the cells.The expression changes of CYP2J were detected by Western-blot and Real Time PCR.The biological imaging of the Bn XPI probe is used to reflect the expression and activity changes of CYP2J2 in Ac-16 cell.（3）Detected the effect of OPD on the expression of CYP2J in the heart failure environment caused by ISO in vitro.（4）Detected the effect of OPD on the nuclear translocation of PXR.（5）Decrease the expression of PXR by helicid or si-PXR,Western-bolt detected the expression changes of CYP2J.（6）Dual luciferase reporter gene detection OPD mediates the binding of PXR to the CYP2J3 promoter region,and possible binding sequences.（7）H9c2 cells were used to treart by different doses of OPD and OPD′,CCK-8 was used to detect the survival rate of H9c2 cells and determines the concentration.（8）The effect of OPD and OPD′on the content of intracellular iron in H9c2 cells was detected by Fe²⁺detection kit and Ferro Orange fluorescent probe.（9）Detected the changes in the contents of MDA,LPO,GSH and GSH-Px in the cells during co-treatment.（10）The expression of ferroptosis-related proteins were detected by Western-blot.Results:（1）OPD can improve the cardiac function of isoproterenol-induced heart failure rat models.After OPD treatment,serum NT-pro BNP,CTn-T,AST,CK,CK-MB,and LDH levels all decreased;the ability to resist oxidative stress was improved;the damage of myocardial tissue was improved;the apoptosis of myocardial cells was improved and induced the expression of CYP2J3 in myocardial tissue.（2）The data of CCK-8 shows that OPD is not toxic to cells when the concentration is 0～20μM.（3）OPD（0.25μM,0.5μM,1μM）can significantly promoted the expression of CYP2J in cells and induced nuclear translocation of PXR;（4）When the expression of PXR was inhibited,the expression of CYP2J was also inhibited;（5）After H9c2 is administered by OPD,PXR binds to the promoter region of CYP2J3 and then regulates its expression.The sequence that PXR binds to CYP2J3 may be between-1500 bp and 0 bp.（6）OPD’triggered ferroptosis of cells which caused iron overload in H9c2 cells,increased the expression of ferroptosis related proteins TFR1,COX2,NOX1,ACSL4 and SLC7A11,and decreased the expression of GPX4 and FTH1.（7）OPD reversed the ferroptosis caused by OPD’and restored iron overload and the expression of iron death-related proteins.Conclusion:OPD can improve the cardiac function in rats with heart failure that caused by ISO and can induce changes in the expression of CYP2J3 in myocardial tissue.At the cellular level,OPD can induce the expression of CYP2J2 and CYP2J3,and the induction is mediated by PXR.In addition,it was found that the two isomers of OPD and OPD’have different pharmacological effects on H9c2 cells.OPD can protecting cardiomyocytes by reverse the ferroptosis induced by OPD′.