缺血后处理对大鼠脑缺血—再灌注中细胞凋亡的作用及其机制研究

Effects of ischemic postconditioning on apoptosis in cerebral ischemia-reperfusion injury in rats and its mechanisms

摘要:

目的 观察缺血后处理（IP）对大鼠脑缺血－再灌注（I/R）中细胞凋亡的影响，并探寻其机制。方法 开颅永久性阻断大脑中动脉+临时夹闭双侧颈总动脉法制作模型。将大鼠随机分为空白对照组（Control组）12只、假手术组（Sham组）、I/R组、IP组各48只、I/R+caspase-3抑制剂组（I/R+Z-DEVD-FMK组）、I/R+溶剂组（I/R+DMSO组）各12只。通过免疫荧光及Western blot法检测细胞凋亡数、细胞色素c（cyt-c）释放及caspase-3活性。结果 IP组较I/R组TUNEL阳性细胞数减少（P<0.05）。Sham组、IP组和I/R组均有细胞呈cyt-c/TUNEL双阳性，但cyt-c阳性不全伴有TUNEL阳性。I/R组与IP组cyt-c呈双峰样释放（再灌注3 h和48 h），在48 h时IP组较I/R组降低（P<0.05）。I/R组caspase-3活性在再灌注3 h时开始升高，12 h和24 h时最高。各相应时点IP组较I/R组的caspase-3活性降低（P<0.01和P<0.05）。I/R+Z-DEVD-FMK组cyt-c后期释放量小于I/R+DMSO组（P<0.01），但完整细胞数多于 I/R+DMSO组（P<0.01）。结论 IP可以抑制凋亡；cyt-c参与凋亡，并与caspase-3形成相互作用的反馈回路。IP对该反馈回路具有调节作用。

Abstract:

Objective To study the effects of ischemic postconditioning (IP) on apoptosis in cerebral ischemia-reperfusion (I/R) injury in rats, and possible mechanisms. Methods Focal cerebral ischemia rat model was developed by cauterizing the middle cerebral artery permanently and occluding the bilateral common carotid arteries temporarily. Rats were assigned randomly into following groups: Control, Sham-operated, I/R, IP, I/R+Z-DEVD-FMK and I/R+DMSO. Apoptosis, cytochrome c (cyt-c) and caspase-3 expression were detected by immunofluorescence staining and Western blot.Results The number of TUNEL-positive cells was lower in the IP group than in the I/R group (P<0.05). Some cells were cyt-c/TUNEL double-positive in the Sham-operated, IP, and I/R groups. But not all cyt-c positive cells were TUNEL positive. Cyt-c was released in a double hump-type in the I/R and IP groups (3 hrs and 48 hrs after reperfusion). The amount of released cyt-c in the IP group was much lower 48 hrs after reperfusion than in the I/R group (P<0.05). The activity of caspase-3 increased 3 hrs after reperfusion, and peaked at 12 hrs and 24 hrs in the I/R group. The activity of caspase-3 in the IP group was lower than in the I/R group at each time point (P<0.05). The release of cyt-c in the I/R+Z-DEVD-FMK group was much less than in the I/R+DMSO group (P<0.01). The number of intact cell in ischemic penumbra in the I/R+Z-DEVD-FMK group was more than in the I/R+DMSO group (P<0.01).Conclusions IP can inhibit apoptosis of ischemic neurons after I/R. Cyt-c is involved in apoptosis of cerebral I/R injury in rats. There might be a feedback loop between cyt-c and caspase-3 in a focal cerebral ischemia rat model. IP plays a role in this loop.