title: Ophiopogonin D maintains Ca2+ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/EETs and suppressing ER stress.
abstract: OBJECTIVE
  CYP2J3 in myocardium metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs), which have diverse biological activities in rat heart. In this study we examined whether CYP2J3 was involved in cardioprotective effects of ophiopogonin D (OPD), a steroidal glycoside isolated from Chinese herb Radix ophiopogonis.
  
  
  METHODS
  Rat cardiomyoblast cell line (H9c2 cells) was tested. Intracellular Ca(2+) concentrations ([Ca(2+)]i) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with qRT-PCR and Western blot analyses. Cell apoptosis was quantified with Hoechst 33258 staining and TUNEL assay. The level of 14,15-DHET, a stable metabolite of 14,15-EET, was assessed with ELISA.
  
  
  RESULTS
  Angiotensin II (10(-6) mol/L) significantly decreased the expression of calcium-regulating molecules (SERCA2a, PLB, RyR2 and FKBP12.6), and elevated [Ca(2+)]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 stress-mediated 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 Ca(2+) homeostasis, ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous 14,15-EET also prevented angiotensin II-induced abnormalities in Ca(2+) homeostasis, whereas transfection with CYP2J3 siRNA diminished the effects of OPD on Ca(2+) homeostasis. Furthermore, the intracellular Ca(2+) chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells.
  
  
  CONCLUSIONS
  OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca(2+) homeostasis and ER stress.
authors.affiliation: Department of Pharmacology, Anhui Medical University, Hefei 230032, China.|Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Wen-ting
authors.initials: WT
authors.lastname: You
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.|Department of Pharmacology, Guangxi Medical University, Nanning 530021, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Zeng-chun
authors.initials: ZC
authors.lastname: Ma
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Qian-de
authors.initials: QD
authors.lastname: Liang
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Cheng-rong
authors.initials: CR
authors.lastname: Xiao
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Xiang-lin
authors.initials: XL
authors.lastname: Tang
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Hong-ling
authors.initials: HL
authors.lastname: Tan
authors.affiliation: Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China.
authors.forename: Bo-li
authors.initials: BL
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Yu-guang
authors.initials: YG
authors.lastname: Wang
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Gao
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002316:Cardiotonic Agents / Q000494:pharmacology*; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D006706:Homeostasis / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D012503:Saponins / Q000494:pharmacology*; D013150:Spirostans / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D002316:Cardiotonic Agents; D012503:Saponins; D013150:Spirostans; C046996:ophiopogonin D; D003577:Cytochrome P-450 Enzyme System; C106098:Cyp2j3 protein, rat; D002118:Calcium
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