The Effects And Mechanisms Of Epoxyeicosatrienoic Acids On Endoplasmic Reticulum Stress In Endothelial Cells

Objective: To examine the effects of arachidonic cytochrome P450 epoxygenase metabolite epoxyeicosatrienoic acids (EETs) on endoplasmic reticulm (ER) stress in endothelial cells and potential molecular mechanisms.Methods: 1, Human umbilical vein endothelial cells (HUVECs) and 2h-11 cells were treated with 10μM tunicamycin (TM) to induce ER stress and then detected ER stress-related proteins, such as p-PERK, Grp78 and Xbp-1 using Western Blot.2, Exogenous EETs (8,9-EET, 11,12-EET and 14,15-EET) were added. ER stress related proteins such as p-PERK, Grp78 and Xbp-1 were detected using Western Blots and apoptosis of endothelial cells was assessed by flow cytometry. Cells viability was determined with cells counting assay-8.3, Related pathway inhibitors (Inhibitors of G protein, Gs protein, adenylate cyclase, protein kinase A, protein kinase G, PI3K, EGFR, MEK and PPAR-γ) were added. ER stress related proteins Grp78 was detected using Western Blots.Results: 1, TM treatment significantly increased the expression of ER stress related proteins p-PERK, Grp78 and Xbp-1 with maximum at 4h in 2h-11 cells and 8h in HUVECs (P<0.05).2, Exogenous EETs attenuate the expression of these proteins in 2h-11 cells and HUVECs (P<0.05). 11,12-EET attenuated the expression of p-PERK and Xbp-1 in a concentration-dependent manner in 2h-11 cells and 14,15-EET attenuated the expression of Xbp-1 in a concentration-dependent manner in HUVECs (P<0.05). Furthermore, EETs significantly inhibited the TM induced apoptosis of endothelial cells and increased cells viability (P<0.05).3, Antagonists of G protein, Gs protein, adenylate cyclase, protein kinase A (PKA) and protein kinase G (PKG) increased the expresssion of Grp78 in the presence of EETs (P<0.05) , however, inhibitors of MEK (PD98059), EGFR (AG1478), PI3K (LY294002) and PPAR-γ(GW9662) didn't increase the expresssion of Grp78 in endothelial cells in the presence of EETs (P>0.05).Conclusion: 1, EETs can inhibit TM-induced ER stress and apoptosis in endothelial cells, and increase cells viability.2, EETs inhibited ER stress via binding G protein coupling receptors and then activating PKA and PKG probably, but not through activating PI3K, EGFR, MEK and PPAR-γ.