摘要
肢体缺血再灌注(IR)所致多器官损伤,是一种由活性氧及炎症介质引发的全身性炎性损伤。一氧化碳(CO)作为气体信使分子,具有抗炎、抗氧化损伤的重要功效。本课题组先前的研究发现,肢体IR后脑、肝、肾组织中内源性CO的诱导酶(HO-1)表达上调,用工具药抑制HO-1的活性后,上述器官的损伤明显加重。该发现使我们想到,吸入适量外源性CO,可能对肢体IR所导致的多器官损伤有防治作用。
目的:观察吸入适量外源性CO对肢体IR所致多器官损伤的防治作用。
方法:健康清洁SD大鼠100只,随机分为假手术(S)组、假手术吸入CO(SC)组、IR组、IR吸入CO(RC)组,每组25只。各组再随机分为6h、24h、48 h、72h、10d五个观察时间点。通过夹闭大鼠双侧股动脉及橡皮筋绑扎双后肢根部4h,去血管夹及橡皮筋6-72 h、10d,复制大鼠肢体IR模型。S、SC组模拟手术过程,但不造成肢体缺血。S、IR组于手术后2h时开始持续吸入标准医用空气4h,SC、RC组于手术后2h时开始持续吸入含有CO(500 p.p.m)的医用空气4h。各组的6h、10d组于再灌注到6h、10d时取脑、肝、肾组织,6h组行HE染色,10d组流式细胞仪检测凋亡百分比及bax、bcl-2的表达变化。24、48、72h组分别再灌注到24、48、72h时采血行肝肾功能检查。
结果:1、SC与S组比较:各项指标无明显差异。2、IR组和SC组相比:(1) IR 6h组海马区细胞间隙变宽,胞体增大,胞核淡染、染色质边集,可见少数坏死、凋亡细胞;肝索结构紊乱,中央静脉变形,部分细胞胞浆中可见较大空泡,胞核肿胀、淡染,染色质边集,个别肝细胞胞浆深染,胞核变小、深染;肾组织中肾小球充血,嗜酸性增强,嵌顿细胞数明显增多,内皮细胞肿胀明显,毛细血管腔狭窄,肾小管上皮细胞肿胀,管腔变窄,腔内可见大量嗜酸性管型;(2) IR 24、48、72h组谷丙转氨酶值增高,但依次降低;IR 24h组血钾、肌酐、尿素氮值均增高,48h组仅尿素氮值增高;(3) IR 10d组三种组织凋亡百分比及bax蛋白FI值增高;但bcl-2蛋白FI值仅肾组织增高。3、RC和IR相比,RC中6h组脑(海马区)细胞间隙变窄,胞体无明显增大,胞核染色及位置趋近正常,未见核固缩;肝索结构较完整,肝小叶结构清晰,中央静脉形态正常,肝细胞大小及染色接近正常,部分肝细胞胞浆含有较多空泡,偶见胞质深染;肾小球细胞数少量增加,部分内皮细胞轻度肿胀,毛细血管及肾小管腔狭窄较轻,可见少量嗜酸性管型;24、48、72h组谷丙转氨酶较IR降低;24h组血钾、肌酐、尿素氮值均降低;48h组尿素氮值低于IR。10d组调亡百分比、bax蛋白FI值降低,bcl-2蛋白FI值增高。
结论:吸入外源性CO可减轻肢体缺血再灌注所致脑、肝、肾组织损伤。
Objective: To investigate the protective effection on the ischemia reperfusion (IR) on the double hind limbs injured brain, liver and kidney tissues through inhaled carbon monoxide (CO).
Methods: 100 SD rat were divided randomly into 4 groups, 25 SD rat in each group were divided further into 6h, 24h, 48h, 72h, 10d groups according to the time when obtained specimens. Of 4 groups, IR group: rats underwent 4-hour bilateral femoral artery occlusion of hind limb to produce ischemia followed by reperfusion; IR and inhaled CO group(RC): rats underwent 4-hour ischemia of hind limb followed by reperfusion, with inhaling CO after 2-hour ischemia for 4 hours; sham operation group(S): rats underwent sham operation which just expose bilateral femoral artery without IR; S and inhaled CO group(SC): rats underwent sham operation, and inhaled CO 2-hour later for 4 hours. 6h and 10d groups within which samples were taken after reperfusion for 6 hours or 10 days; the former followed by HE stain, and the later followed by flow cytometry. Within 24, 48, 72h groups, the blood samples were taken after reperfusion for 24, 48, 72 hours to detect the liver and renal function.
Results: 1:There is no difference between S and SC groups. 2 : Compared with that of group SC: In IR, pathological change of hippocampus, liver and kidney tissue in 6h group were significant; In 24, 48, 72 groups, plasma GPT were obviously increased, and were depressed by turns; in 24h group, plasma k+, Cr, BUN were increased; in 48h group, BUN were increased; in 10d group, the percentage of apoptosis, the FI of proteinum bax were increased; the FI of proteinum bcl-2 of kidney tissue were increased. 3 :Compared with that of group IR: In RC, pathological change in 6h group were slight; plasma GPT in 24, 48, 72h groups were decreased; in 24h group, plasma k+, Cr, BUN were decreased; in 48h group, plasma BUN were decreased; in 10d group, the percentage of apoptosis, the FI of proteinum bax of hippocampus, liver and kidney tissues were depressed,the FI of proteinum bcl-2 of these tissues were increased.
Conclusions: The ischemia reperfusion on the double hind limbs induced the injury of hippocampus, liver and kidney tissue. Inhaled CO can attenuate injury of these tissues.
引文
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