缺血预处理对大鼠缺血再灌注肾损伤诱发凋亡的保护机制

摘要

Objective To investigate the protective effect of ischemia preconditioning(IPC)on apoptosis in-duced by renal ischemia - reperfusion(IR)and relations to the changing expressions of Bcl - 2,Bax in rat kidney. Methods Ischemia models were induced by clipping bilateral renal pedicles for 30 min by using the artery clamp;IPC group was induced by clipping bilateral renal pedicles for 15 min,4 days later IR was performed again by clipping bila-teral renal pedicle for 30 min. Rats were randomly divided into 5 groups with 5 animals in each group:control group(C group),sham - operation group(S group),IR group,IPC group(IPC ﹢ IR group),sham IPC group(S ﹢ IR group),all groups were randomly divided into 9 sub groups(0 h,3 h,6 h,12 h,24 h,48 h,3 d,5 d,7 d)except C group according to the time points after reperfusion. Occurrence of apoptosis was detected by terminal deoxynuleotidyl transferase media-ted dUTP nick end and labeling(TUNEL)method;the mRNA expression and protein levels of Bax and Bcl - 2 were de-tected by reverse transcriptase - polymerase chain reaction and quantitave immunohistochemisty. Results (1)Com-pared with S group and S ﹢ IR group,serum creatinine,blood urea nitrogen,kidney pathological damage scores in IR group gradually increased after IR,and peak point was 24 h after reperfusion;among all the subgroups there was a sig-nificant difference(all P ﹤ 0. 01). The expression of Bax,Bcl - 2 mRNA raised sharply in IR group after reperfusion, peaking at 6 h,24 h of reperfusion respectively,2. 66 ± 0. 12,2. 70 ± 0. 10,and among all the subgroups there was a sig-nificant difference(all P ﹤ 0. 01);the expression of Bax,Bcl - 2 protein had significant difference(all P ﹤ 0. 05). TUNEL immunofluorescence staining showed C group and S group had no obvious apoptosis cells in renal tubular epi-thelium;epithelial cell apoptosis after IR gradually increased in IR group,peaking at 24 h of reperfusion[(25. 07 ± 2. 29)% ].(2)Compared with IR group and S ﹢ IR group,pathological injury was significantly decreased in IPC ﹢ IR group;the expression of Bax,Bcl - 2 mRNA and protein,apoptosis cells were significantly decreased in IPC ﹢ IR group (all P ﹤ 0. 05). Conclusions Bax,Bcl - 2 are closely associated with kidney injury induced by IR. IPC may regulate acute kidney injuries by regulating Bax/ Bcl - 2.%目的：探讨缺血预处理（IPC）对大鼠缺血再灌注（IR）肾损伤引起肾小管上皮细胞凋亡的保护作用及其与 Bcl-2、Bax 表达变化的关系。方法采用夹闭小鼠双侧肾蒂30 min 的方法建立小鼠 IR 肾损伤模型；微型动脉夹夹闭小鼠双侧肾蒂15 min 的方法进行预缺血，4 d 后再进行 IR。实验共分为5个大组：对照组（C组），假手术组（S 组），IR 组，IPC 组（IPC ﹢ IR 组），IPC 对照组（S ﹢ IR 组）；除 C 组外，其他4组根据再灌注的时间分亚组（再灌注后0 h，3 h，6 h，12 h，24 h，48 h，3 d，5 d，7 d）；反转录聚合酶链反应（RT-PCR）方法检测小鼠肾组织 Bax、Bcl-2 mRNA 表达水平；免疫组织化学方法检测小鼠肾组织 Bax、Bcl-2的分布及蛋白质表达水平。原位末端转移酶标记（TUNEL）法免疫荧光染色检测肾小管上皮细胞凋亡。结果1.与 S 组、S ﹢ IR 组比较，IR组 IR 后血肌酐、血尿素氮、肾脏病理损害评分逐渐加重，再灌注24 h 达到峰值，各亚组之间差异均有统计学意义（P 均﹤0.01）；肾组织 Bax、Bcl-2 mRNA 表达急剧上升，分别于6 h、24 h 到达峰值，相对表达水平分别为2.66±0.12、2.70±0.10，各亚组间比较差异均有统计学意义（P 均﹤0.01）；肾组织 Bax、Bcl-2蛋白质的表达水平，各亚组间比较差异均有统计学意义（P 均﹤0.05），TUNEL 免疫荧光染色 C 组、S 组小鼠肾小管上皮未见明显的凋亡细胞；IR 组在 IR 后肾小管上皮细胞凋亡数逐渐增多，再灌注后24 h 达到峰值[（25.07±2.29）%]。2. IPC ﹢ IR 组肾脏病理损害明显减轻，Bax、Bcl-2 mRNA 及蛋白质表达水平、细胞凋亡明显低于 IR 组及 S ﹢ IR 组（P 均﹤0.05）。结论 Bax/ Bcl-2与 IR 肾损伤密切相关，IPC 可能通过调控 Bax/ Bcl-2参与急性肾损伤的调控。