Role Of PERK/eIF2α In Ox-LDL-induced Endothelial Cell Apoptosis And The Effect Of Simvastatin

Chapter I Role of PERK/eIF2a in ox-LDL-induced endothelial cell apoptosis and its mechanismObjective:To study on the effect of PERK/eIF2a in endothelial cell apoptosis induced by ox-LDL and its mechanism.Methods:(1) To investigate the effect of ox-LDL on PERK and p-eIF2a protein expression. PERK and p-eIF2a was determined by Western-blot. Endothelial cells were treated by ox-LDL (100μg/ml) for0,12,24or48hrs.(2) To study the effect of PERK gene silencing on ox-LDL induced endothelial cell apoptosis, caspase-3activity and CHOP mRNA level. Cells cultured in6well plates were randomly divided into6groups: control group, endothelial cells were incubated in DMEM medium containing1%calf serum for24hrs;100μg/ml ox-LDL group, endothelial cells were cultured in DMEM medium containing100μg/ml ox-LDL for24hours; non-targeting sh RNA group, after successful non-targeting shRNA transfection, cells were cultured in DMEM medium containing1%calf serum for24hrs; PERK sh RNA group, after successful PERK shRNA transfection, cells were cultured in DMEM medium containing1%calf serum for24hrs; Ox-LDL+non-targeting sh RNA group, after successful non-targeting shRNA transfection, cells were cultured in DMEM medium containing100μg/ml ox-LDL for24hrs; Ox-LDL+PERK sh RNA group, after successful PERK shRNA transfection, cells were cultured in DMEM medium containing100μp,g/ml ox-LDL for24hrs;(3) To study the effect of Salubrinal on ox-LDL induced endothelial cell apoptosis, caspase-3activity and CHOP mRNA level. Cells cultured in6well plates were randomly divided into4groups: control group, endothelial cells were incubated in DMEM medium containing1%calf serum for24hrs;100μg/ml ox-LDL group, endothelial cells were cultured in DMEM medium containing100μg/ml ox-LDL for24hrs;+Salubrinal group, cells were pretreated with40μM of Salubrinal (specific eIF2α phosphorylation inhibitor) for1h prior to ox-LDL exposure; Salubrinal group, endothelial cells were cultured in DMEM medium containing40μM of Salubrinal for24hrs.(4) To study the effect of PERK gene silencing on ox-LDL induced eIF2a. phosphorylation. Cells cultured in6well plates were randomly divided into4groups:control group, endothelial cells were incubated in DMEM medium containing1%calf serum for24hrs;100μg/ml ox-LDL group, endothelial cells were cultured in DMEM medium containing100μg/ml ox-LDL for24hrs; Ox-LDL+PERK sh RNA group, after successful PERK shRNA transfection, cells were cultured in DMEM medium containing100μg/ml ox-LDL for24hrs;+Salubrinal group, cells were pretreated with40μM of Salubrinal (specific eIF2a phosphorylation inhibitor) for1h prior to ox-LDL exposure.Results:(1) ox-LDL increased the expression of PERK and eIF2aphosphorylation in endothelial cells in time-dependent manner.(2) Incubation of endothelial cells with ox-LDL (100μg/ml) for24hrs significantly increased cell apoptosis concomitantly with the increase in caspase3activty and CHOP mRNA level. PERK gene silencing or pretreatment with Salubrinal (specific eIF2a. phosphorylation inhibitor) for1h, significantly inhibited ox-LDL-induced endothelial cell apoptosis, the increased caspase-3activity and CHOP mRNA level (P<0.01).(3) Ox-LDL (100μg/ml) increased eIF2a phosphorylation, which is inhibited by PERK gene silencing or pretreatment with Salubrinal (specific eIF2a phosphorylation inhibitor).Conclusions:Ox-LDL can trigger Endoplasmic Reticulum Stress. And ox-LDL induces endothelial cell apoptosis, which is related to inhibition of PERK/eIF2a CHOP/caspase-3pathway. Chapter Ⅱ The inhibitory effet of Simvastatin on ox-LDL-induced endothelial cell apoptosis and Endoplasmic Reticulum StressObjective:To study on the effect of Simvastatin on endothelial cell apoptosis induced by ox-LDL and its mechanism.Methods:Firstly, to study the effect of Simvastatin on ox-LDL induced endothelial cell apoptosis, caspase-3activity and CHOP mRNA level, endothelial cells were pre-treated with Simvastatin (0.1,0.5or2.5μM) for1h, and then cultured with ox-LDL (100μg/mL) for24hrs. To further examine the role of PERK/eIF2a CHOP/caspase-3pathway in these process induced by ox-LDL, endothelial cells were pre-treated with salubrinal (40μM) or DEVD-CHO (100μM) for1h, then exposed to ox-LDL (100μg/mL) for24hrs, the mRNA expression of CHOP, intracellular caspase-3activity and cell apoptosis were determined.Secondly, to study the effect of Simvastatin on ox-LDL induced the expression of PERK and eIF2a phosphorylation, endothelial cells were pre-treated with Simvastatin (0.1,0.5or2.5μM) for1h, and then cultured with ox-LDL (100μg/mL) for24hrs. To further examine the role of eIF2α, endothelial cells were pre-treated with salubrinal (40μM)-specific eIF2α phosphorylation inhibitor, then exposed to ox-LDL (100μg/mL) for24hrs. Expressions of PERK and eIF2α. phosphorylation were determined by western-blot. Results(1)Treatment with ox-LDL (100μg/ml) for24hrs caused a significant increase in the cell apoptosis, concomitantly with the increase in caspase3activty and CHOP mRNA level. Simvastatin (0.1,0.5or2.5μM) significantly attenuated the increase of above index induced by ox-LDL in a concentration-dependent manner, which was also attenuated by treatment with salubrinal (specific eIF2a phosphorylation inhibitor) or DEVD-CHO (specific caspase-3inhibitor)(2)Treatment with ox-LDL (100μg/ml) for24hrs significantly increased the expression of PERK and eIF2a phosphorylation. The results also showed that Simvastatin (0.1,0.5or2.5μM) concentration-dependently reduced PERK and eIF2α expression. Furthermore, pretreatment with salubrinal (specific eIF2α. phos-phorylation inhibitor) prevented ox-LDL-induced eIF2α phosphorylation, but had no effect on PERK expression.Conclusions Simvastatin attenuate ox-LDL-induced endothelial cell apoptosis, by inhibiting PERK/eIF2a/CHOP/caspase-3pathway.