二氮嗪预处理对培养的大鼠心肌微血管内皮细胞抗细胞凋亡作用及其与 PI3K/Akt 通路的关系。
Effect of Diazoxide Preconditioning on Cultured Rat Myocardium Microvascular Endothelial Cells against Apoptosis and Relation of PI3K/Akt Pathway.
机构信息
Department of Anesthesiology, Affiliated Hospital of Nantong University, Nantong, China.
Department of Pharmacy, Affiliated Hospital of Nantong University, Nantong, China.
出版信息
Balkan Med J. 2014 Mar;31(1):83-7. doi: 10.5152/balkanmedj.2013.8458. Epub 2014 Mar 1.
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.
背景
细胞保护的抗细胞凋亡机制可能为减轻再灌注损伤提供新的方法。
目的
本研究旨在探讨线粒体三磷酸腺苷敏感性钾通道（mito-KATP）开放剂二氮嗪（DZ）预处理对大鼠心肌微血管内皮细胞（MMEC）缺氧/复氧（H/R）损伤的影响及其与 PI3K/Akt 通路的关系。
研究设计
动物实验。
方法
培养大鼠 MMEC，制作 H/R 模型模拟缺血再灌注损伤。将细胞以 100μL/孔（1×106/mL）的密度接种于 96 孔板或 6cm 直径培养皿（2mL/皿）中，随机分为 4 组（每组 6 只）：对照组（N 组）、缺氧-复氧组（H/R 组）、DZ 预处理+H/R 组（DZ 组）和 DZ+mito-KATP 阻断剂 5-羟癸酸（5-HD）+H/R 组（DZ+5-HD 组）。细胞先缺氧 2h，再复氧 2h。DZ 组和 DZ+5-HD 组分别在缺氧前 2h 加入培养液中二氮嗪 100μmol/L 和二氮嗪 100μmol/L+5-HD100μmol/L。各组分别采用 MTT 观察细胞增殖、Annexin V-FITC/PI 双标准检测细胞凋亡率、Hoechst 染色观察细胞形态结构、RT-qPCR 检测 PI3K、Akt 和 p53mRNA 水平。
结果
与 N 组比较，H/R 组细胞凋亡率增加（P<0.01），活力明显降低（P<0.05），PI3K、Akt 和 p53mRNA 表达上调（P<0.05）；与 H/R 组比较，DZ 组细胞凋亡率和 p53mRNA 水平明显降低（P<0.01、P<0.05），活力、PI3K 和 AktmRNA 水平升高（P<0.05）；与 DZ 组比较，DZ+5-HD 组细胞凋亡率和 p53mRNA 水平明显升高（P<0.01、P<0.05），但活力、PI3K 和 AktmRNA 水平降低（P<0.05）。
结论
在 H/R 条件下，DZ 开放 mito-KATP 可能通过 PI3K/Akt 通路调节下游 p53mRNA 的表达水平，抑制细胞凋亡，同时提高 MMEC 的活力。
Zotero 插件