Exendin-4Protected HUVECs From Tunicamycin-induced Apoptosis Via Affecting Endoplasmic Reticulum Stress-related Signal Pathway

Aims: The unusual increase of apoptosis of endothelial cells is a significant factor foratherosclerosis. In this study we adopted tunicamycin,which was a commonly usedsubstance to study ERs,to investigate the mechanism of HUVECs apoptosis inducedby tunicamycin,and to study whether the agonist of GLP-1receptor， Exendin-4could influence HUVECs apoptosis induced by tunicamycin and its underlyingmechanisms.Methods: Primary HUVECs were induced by tunicamycin at concentrations rangingfrom0to20μg/ml for16-24h.MTT assay was used to measure the cell viability,andfluorescence microscopic analysis with Hoechst/PI staining was used to evaluate theapoptotic rate. Western blot was used to detect the expression of IRE1а、P-IRE1а、JNK、P-JNK and caspase-3after HUVECs were exposed to10μg/ml tunicamycin for0、8、14、18h. MTT assay and fluorescence microscopic analysis with Hoechst/PIstaining were used to evaluate cell viability and the apoptotic rate of HUVECsinduced by tunicamycin at10μg/ml,with pretreatment of Exendin-4(5～150nmol/L).Expression of IRE1а、P-IRE1а、JNK、P-JNK and caspase-3was detected by westernblot after HUVECs were exposed to10μg/ml tunicamycin for18h,with pretreatmentof Exendin-4（50nmol/L）or1mmol/L tauroursodeoxycholic acid（TUDCA）.Results: The viabilities of HUVECs were gradually reduced in dose-dependent andtime-dependent manners after exposure to tunicamycin at different concentrations.The apoptosis of HUVECs increased gradually,along with the increase oftunicamycin’s concentration. Treatment of HUVECs with10μg/ml or15μg/mltunicamycin produced the maximal apoptotic response.The necrosis of HUVECs increased,when the cells were treated with tunicamycin at concentration≥15μg/ml.The ratio of P-IRE1а/IRE1а、P-JNK/JNK and active caspase-3/procaspase-3gradually increased when HUVECs were exposured to tunicamycin(10μg/ml) for0、8、14、18h. The cell viability gradually increased with pretreatment of Exendin-4(5～150nmol/L),compared with tunicamycin-alone group. Fluorescence microscopicanalysis with Hoechst/PI staining showed that the apoptotic rate of HUVECsdecreased40%in Exendin-4-pretreatment group than that of tunicamycin-alone group.Pretreatment of HUVECs with Exendin-4resulted in a23.25%decrease of theexpression of p-IRE1а than that of tunicamycin-alone group,and resulted in29.25%and22.2%decreases of p-JNK and active caspase-3,respectively(P<0.01).The effectof Exendin-4was similar to that of TUDCA. Compared with tunicamycin-alone group,the ratios of p-IRE1а/IRE1а、p-JNK/JNK and active caspase-3/procaspase-3were allreduced both in Exendin-4-pretreatment group and TUDCA-pretreatment group.Conclusion: Tunicamycin could induce apoptosis of primary HUVECs viaIRE1а/JNK/caspase-3signal pathway,and Exendin-4could protect HUVECs fromtunicamycin-induced apoptosis through inhibiting this pathway.