| Background and aims: Endothelial cells have a robust capacity to recover vascular injury and maintain intimal layer integrity. Endothelial apoptosis is involved in the formation of atherosclerosis. In this study we investigated whether the anti-diabetic hormone exendin-4 could prevent oxidative stress–induced human umbilical vein endothelial cells (HUVECs) apoptosis and its underlying mechanisms.Methods: HUVECs were cultured in vitro and exposed to tert-butyl hydroperoxide (t-BHP) for 1～4 h at different concentrations range from 50 to 800μmol/L. Cell viability were determined by MTT assay,and cell apoptotic were evaluated by fluorescence microscopic analysis after Hoechst 33342/PI staining. HUVECs were treated with t-BHP (100μmol/L)for 0, 0.5, 1, 2, 4, 6 h respectively. Expression of phosphorylation of Akt, Bcl-2 and caspase-3 were detected by Western blot. HUVECs were cultured in vitro and exposed to t-BHP, or t-BHP plus Exendin-4 (2.5～25 nmol/L). Cell viability was determined by MTT assay and apoptosis was detected by fluorescence microscopic analysis after Hoechst 33342/PI staining. HUVECs were pre-incubated with exendin-4 (25 nmol/L) for 18 h, then phosphatidylinositol 3-kinase (PI3k) inhibitor, wortmannin (100 nmol/l) were added to the medium 30 min before t-BHP exposure. The protein expression of phosphorylation of Akt, Bcl-2 and caspase-3 were measured by Western blot.Results: The results showed that HUVECs viability decreased gradually in dose-dependent and time-dependent manner after exposure to t-BHP. When t-BHP were at 50～100μmol/L, cell apoptosis dominates (8.2%±0.59,37.3%±1.65,P < 0.01), but when t-BHP at higher concentrations, cell necrosis dominates (4.3%±0.57,15.5%±1.38,36.2%±2.19,P < 0.01). Caspase-3 were activated and reached the maximum at 4 h after t-BHP exposure. Similarly, Akt activity reached the maximum at 30 min, however, declined later on. Meanwhile, Bcl-2 had a significant decrease with time extended. After HUVECs pre-treatmented with exendin-4 (25 nmol/l), cell viability increased by 46.5% (P < 0.01), and the number of apoptosis decreased significantly (8.0%±1.45 vs 36.0%±1.25, P < 0.01), compared with the t-BHP treated group alone. Going a further step, we examined the underlying regulation. Pre-incubation of HUVECs with exendin-4 resulted a reduction of active caspase-3 (by 48.53%, P < 0.01), an enhancement of phosphorylated Akt (by 1.78 times, P < 0.01) and an increase in anti-apoptotic protein Bcl-2 expression (by 1.08 times, P < 0.01), compared with the t-BHP group. These results indicate that exendin-4 can protect t-BHP-induced HUVECs apoptosis. However, this protective effect were inhibited by the phosphatidylinositol-3 kinase (PI3k) inhibitor wortmannin.Conclusion: Our findings showed that oxidative stress induces caspase- dependent HUVEC apoptosis, which is partly mediated by the inactivation of Akt and the reduction of Bcl-2 expression.GLP-1 analogue exendin-4 has a protective effect against oxidative stress-induced endothelial cell apoptosis, which may associate to the involvement of survival pathway PI3k/Akt- Bcl-2. |
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