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Microvascular dysfunction is a primary comorbidity in diabetes mellitus (DM). Advanced glycation end products (AGEs) are well recognized pathogenic mediators in DM patients which could impair endothelium. Nicorandil, a KATP channel opener, is considered protective of the cardiac microvasculature. However, whether nicorandil could protect cardiac microvascular endothelial cells (CMECs) against AGEs-induced cytotoxicity is not clear.
Human CMECs were exposed to different concentrations (50, 100, 200 μg/mL) of AGE-bovine serum albumin (AGE-BSA) for 24 hours to find work concentration. CMECs were treated with nicorandil at different concentration (10, 100, 1000 μmol) and AGE-BSA at 100 μmol for 24 hours. Wortmannin at 100 nmol was used to inhibit the PI3K/Akt signaling pathway and then suppress the upstream of autophagy. Chloroquine at 100 mmol was used to impede the downstream of autophagy. Viability and AGEs-induced cytotoxicity of CMECs were evaluated spectrophotometrically by a CCK-8 method. The proliferation and migration of CMECs were measured by scratch wound healing assays. Western blot was used to detect protein level.
AGE-BSA administration significantly reduced cellular viability, as identified with decreased optical density (OD) value at 450 nm evaluated by CCK8. Accordingly, AGE-BSA reduced migration measured by scratch wound healing assays in a dose-dependent manner (p<0.05). The dose of nicorandil at 100 μmol ameliorated the AGEs-induced cytotoxicity identified with increased OD value (Mean ± SEM, 1.503 ± 0.196 in the Nicorandil 100 μmol group vs. 1.178 ± 0.094 in the AGE-BSA 100 μg/mL group, p<0.05) evaluated by CCK8, and the scratch wound healing assays showed the same trend (p<0.05). However, nicorandil at 10 or 1000 μmol didn’t protect CMECs because of a lack of statistical significance. The inhibition of autophagy either by wortmannin or by chloroquine decreased the protective effects of nicorandil. Western blot revealed that phosphorylation of Akt was abrogated by wortmannin, which also decreased autophagy. The result of western blot demonstrated that autophagy was also suppressed by chloroquine, which was identified with increased type II of light chain 3 (LC3-II).
These results suggested that nicorandil could protect CMECs from AGE-induced cytotoxicity, and induction of autophagy via activating PI3K/Akt signaling pathway may be one of the mechanisms.