Effect of Diazoxide Preconditioning on Cultured Rat Myocardium Microvascular Endothelial Cells against Apoptosis and Relation of PI3K/Akt Pathway

Abstract

Background: Anti-apoptotic mechanism for cell protection on reperfusion may provide a new method to reduce reperfusion injury. Aims: The aim of the present study is to explore the effect of mitochondrial ATP sensitive potassium channel (Mito-KATP) opener diazoxide (DZ) preconditioning on hypoxia/ reoxygen (H/R) injury of rat myocardium microvascular endothelial cells (MMECs) against apoptosis and relation of PI3K/Akt pathway. Study Design: Animal experimentation. Methods: The rat MMECs were cultivated, and H/R model was made to imitate ischemia-reperfusion injury. The cells were seeds in 96-wellplates (100μL/hole) or in 6cm diameter dishes (2 mL/dish) with the density of 1×106/mL and randomly divided into 4 groups (n=6 each): control group (Group N), hypoxia-regoxygen group (Group H/R), Diazoxide preconditioning+H/R group (Group DZ) and Diazoxide preconditioning +mitochondrial KATP blocker 5-hydroxydecanoate (5-HD) + H/R group (Group DZ+5-HD). The cells were exposed to 2h hypoxia followed by 2h reoxygenation. Diazoxide 100μmol/L and diazoxide 100μmol/L+ 5-HD100μmol/L were added to the culture medium 2h before hypoxia in DZ and DZ+5-HD groups respectively. Each group was observed the proliferation in MTT, apoptotic rate in Annexin V-FITC/PI double standard, cell structure of Hoechst staining, and the levels of PI3K, Akt and p53 mRNA by RT-qPCR. Results: Compared with Group N, apoptotic rate of Group H/R increased (p<0.01) and the vitality decreased significantly (p<0.05), and the expression of PI3K, Akt and p53 mRNA elevated in Group H/R (p<0.05). Compared with Group H/R, apoptotic rate and p53 mRNA level of Group DZ depressed significantly (p<0.01, p<0.05), while the vitality, PI3K and Akt mRNA levels increased (p<0.05). Compared with Group DZ, apoptotic rate and p53 mRNA level of Group DZ+5-HD increased significantly (p<0.01, p<0.05), but the vitality, PI3K and Akt mRNA levels decreased (p<0.05). Conclusion: Under the condition of H/R, mito-KATP opened by DZ may depend on PI3K/Akt pathway to regulate expression level of the downstream p53 mRNA to inhibit apoptosis and improve viability of MMECs at the same time.

Keywords

• Apoptosis
• diazoxide
• Mito KATP
• MMECs
• p53
• PI3K/Akt