Ophiopogonin D upregulates CYP2J3/EETs to activate PI3K/Akt-eNOS pathway and rescue H2O2-induced H9c2 cell injury / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To explore the protective effect and mechanism of ophiopogonin D (OP-D) on oxidative stress injury of H9c2 cells induced by H2O2. METHODS An oxidative damage model of H9c2 cells was established by H2O2 induction. The cells were divided into control group (cultured in serum-free medium for 28 h), H2O2 injury model group (treated with H2O2400μmol·L-1 for 3 h), OP-D 5, 10 and 20 μmol · L-1 pretreatment groups (treated with H2O2400 μmol · L-1 for 3 h after OP-D pretreat?ment for 24 h), and an inhibitor of CYP2J3, 6-(2-proparglyloxyphenyl) hexanoic acid (PPOH) group (OP-D 20μmol·L-1+PPOH 10μmol·L-1, PPOH was added to the cells 1 h before OP-D treatment). Cell activity was measured by MTT method, levels of dihydroxyeicosatrienoic acid (11,12-DHET and 14,15-DHET respectively) were detected by enzyme-linked immunosorbent assay (ELISA), while levels of malondialdehyde (MDA), nitric oxide (NO) and activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) were detected by assay kits. Flow cytometry (FCM) was used to detect reactive oxygen species (ROS) and apoptosis. Western blotting was used to detect the expressions of CYP2J3, Akt phos?phorylation (p-Akt) protein and endothelial nitric oxide synthase phosphorylation (p-eNOS) protein in cells, and the possible mechanism by which OP-D reduces oxidative stress was further verified with PPOH. RESULTS H2O2400μmol · L-1 significantly inhibited H9c2 cell viability (P<0.01), and OP-D signifi?cantly increased the cell survival rate after H2O2 injury (P<0.01). Different concentrations of OP-D increased the level of 11,12- DHET and 14,15-DHET (P<0.05, P<0.01). OP-D increased the level of NO in cells after H2O2-induced injury (P<0.05, P<0.01), enhanced the activity of SOD (P<0.05), and decreased the level of MDA and LDH (P<0.05, P<0.01). OP-D significantly reduced oxidative stress and apoptosis after H2O2 injury (P<0.05, P<0.01). OP-D pretreatment increased the protein and mRNA expression of CYP2J3 (P<0.05, P<0.01) and the phosphorylation of PI3K/Akt-eNOS pathway after H2O2 injury (P<0.05, P<0.01). After PPOH was given in advance, the protective effect of OP-D was inhibited (P<0.05, P<0.01). CONCLUSION OP-D can reduce H2O2-induced H9c2 cell damage, which may be related to the activation of PI3K pathway and the phosphorylation of its downstream factors Akt and eNOS by inducing CYP2J3 expression and increasing the contents of 11,12-DHET and 14,15-DHET.