Mechanism Of The Cardioprotective Effects Of Ophiopogonin D Via CYP2J3/EETs System

Objective: Cytochrome P450 2J3（CYP2J3） is highly expressed in the myocardium,kidney and liver system, and this isozyme metabolizes arachidonic acid（AA） into four different epoxyeicosatrienoic acid（EET） regioisomers（5,6-, 8,9-, 11,12-, and 14,15-EET）. Accumulating evidence suggests that EETs have crucial and diverse protective roles within the cardiovascular system. Specically, EETs have been reported to possess anti-arrhythmic, anti-inlammatory, anti-apoptotic, anti-oxidant, angiogenesis, and the role of diastolic blood vessels. Ophiopogonin D（OPD）, a steroidal glycoside isolated from Radix ophiopogonis（a tuber of Ophiopogon japonicus Ker Gawl, Liliaceae）, is a traditional Chinese herbal medicine that has been used to treat in cardiovascular diseases. Our previous study found out that OPD increased the expression of CYP2J3 m RNA. In this study, we further confirmed wether OPD could increase CYP2J3 m RNA and protein expression, and the content of its metabolites EETs. We also examined the effects of OPD on Ca²⁺homeostasis and endoplasmic reticulum stress-mediate apoptosis in H9c2, and whether CYP2J3 was involved in cardioprotective effects of OPD.Methods: Rat cardiomyoblast cell line（H9c2 cells） was tested. MTS was used to measure cell viability. The levels of 14,15-DHET, a stable metabolite of 14,15-EET,was assessed with ELISA. Intracellular Ca²⁺concentrations([Ca²⁺]i) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with q RT-PCR and Western blot analyses. Cell apoptosis was quantified with Hochest 33258 staining and TUNEL assay.Results: Angiotensin II（10-6mol/L） significantly decreased the expression of calciumregulating molecules（SERCA2a, PLB, Ry R2 and FKBP12.6）, and elevated [Ca²⁺]i in H9c2 cells. Furthermore, angiotensin II markedly increased the expression of ER stress signaling molecules（GRP78, CHOP, p-JNK and cleaved caspase-12） and ER stressmediated apoptosis. OPD（100, 250 and 500 nmol/L） dose-dependently increased CYP2J3 expression and 14,15-DHET levels in normal H9c2 cells. Pretreatment of H9c2 cells with OPD suppressed angiotensin II-induced abnormalities in Ca²⁺homeostasis,ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous14,15-EET also prevented angiotensin II-induced abnormalities in Ca²⁺homeostasis,whereas transfection with CYP2J3 si RNA diminished the effects of OPD on Ca²⁺homeostasis. Furthermore, the intracellular Ca²⁺chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells.Conclusion: OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca²⁺homeostasis and ER stress.