Ophiopogonin D Increases SERCA2a Interaction With Phospholamban By Promoting CYP2J3 Upregulation

Radix Ophiopogonis is a traditional Chinese medicine.Shenmai injection consists of two ingredients which are Radix Ophiopogonis and red ginseng.The clinical application of shenmai injection is the treatment of heart disease.Ophiopogonin D（OPD）is one kind of component in Radix Ophiopogonis,which emerges the pharmacological activity in heart failure.The study demonstrated that OPD released severe symptoms in heart failure rats model.The mechanism involved indicates OPD enhances the activity of Ca²⁺-ATPasea2a（SERCA2a）and the level of calcium in endoplasmic reticulum.SERCA2a is a protein located in the endoplasmic reticulum membrane.The protein mentioned plays as a key role in heart failure.CYP2J3 is a member of Cytochrome P450 enzyme family.The distribution of CYP2J3 is mainly in the rattus heart.CYP2J3 plays an important role in the cardiovascular system.The study showed that CYP2J3enhanced the activity of myocardial cells in the CYP2J3 transgenic rat model.The aim of this research is to investigate whether CYP2J3 involved in myocardium protective action of OPD by activating the SERCA2a and PLB in cells.The results identify CYP2J3 as a critical intracellular target for OPD and the mechanism of CYP2J3-dependent regulation in intracellular calcium circle.Methods（1）H9c2 cells were treated with different doses of OPD,the cell viability was determined by cck-8 assay.（2）The effect of OPD on the expression of CYP2J3 in 293T cells was detected by recombinant plasmid assay.（3）The effect of OPD on expressions of SERCA2a and PLB phosphorylation levels were detected using the q uantitative qRT-PCR and western-blot assay.Effects of OPD on ER C a²⁺uptake was detected by Mag-Fluo-4/AM probe.（4）The effects of OPD on SERCA2a and PLB H9c2 cells were treated with different OPD and OPD′,the ER stress related gene expressions were detected with qRT-PCR and western-blot.Morphologies of endoplasmic reticul um were observed by endoplasmic reticulum fluorescent probe.The ef fect of OPD on OPD′-induced cell apoptosis was measured by flow cytometry.Results（1）The cell viability data showed no significant change in H9c2 cells after treated with 1-80μM OPD.（2）OPD qualitatively induced CYP2J3 protein and mRNA expression in H9c2 cells.OPD activatet PLB phosphorylation through two different pathways and improved SERCA2a activity in the two heart failure models in vitro.（3）OPD induced colocalization between SERCA2a and p-PLB,while SERCA2a and PLB protein showed decreased colocalization in H9c2 cells after OPD treatment.After transfection of siRNA,the suppression of CYP2J3 alleviated the physical binding between SERCA2a and p-PLB.（4）OPD promoted the translocation of PXR from cytoplasm to nucleus in a concentration-dependent manner.The expression of CYP2J3 decreased after transfection of PXR-siRNA into H9c2 cells.（5）OPD′triggered the endoplasmic reticulum stress in rat cardiomyocytes.OPD reversed the myocardial cytotoxicity caused by OPD’and reduced the expression of ATF-4 and CHOP,which are related to endoplasmic reticulum stress.The data shows that OPD-induced SERCA2a interaction and co-localization with PLB is mediated by CYP2J3.OPD promotes PXR translocation from cytoplasm to the nucleus.It is possible that PXR plays the role in regulating of CYP2J3.Our results identify CYP2J3 as an attractive target and OPD as a promising component for the HF therapy.Besides,The experimental results showed that OPD`induced myocar-dial toxicity may be associated with the endoplasmic reticulum stress.OPD may modulate the expression of CYP2J3 to relieve the endoplasmic reticulum stress triggered by OPD′.