国际麻醉学与复苏杂志   2012, Issue (4): 5-5
    
肾缺血后处理对热休克蛋白表达的影响及作用
郭琼梅, 赵砚丽, 张东, 王振显, 岳立辉1()
1.河北省人民医院
Effect of ischemic postconditioning on expression of HSPs in rats after kidney ischemia reperfusion injury
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摘要：

目的 评价肾缺血后处理（ischemic postconditioning, IPo）对热休克蛋白（heat shock protein, HSP）70、HSP27和血红素加氧酶-1（heme oxygenase-1，HO-1，即HSP32）表达的影响及在减轻肾缺血再灌注（ischemia reperfusion, I/R）损伤中的作用。方法 健康雄性SD大鼠140只，体重250～280 g，采用随机数字表法随机分为4组(n=35)：假手术组（S组）仅开腹，游离双侧肾脏，分离双侧肾蒂不夹闭；I/R组，夹闭双侧肾蒂缺血45 min，恢复灌注；IPo组，夹闭双侧肾蒂45 min，再灌注10 s，缺血10 s，反复3次，恢复灌注；HSP抑制剂槲皮黄酮+IPo组（Q+IPo组）缺血前1 h腹腔注射槲皮黄酮100 mg/kg，余操作同IPo组。于再灌注即刻（T0）、1、3、6、12、24、48 h（T1～6）时取5只大鼠经心脏抽血后迅速处死取肾，采用RT-PCR和免疫组织化学法分别检测各时点肾组织HSP70、HSP27和HO-1 的mRNA和蛋白表达，测定T3时血清肌酐（creatinine, Cr）和尿素氮（urea nitrogen, BUN）浓度、肾组织丙二醛（methylene dioxyamphetamine, MDA）含量和超氧化物歧化酶（superoxide dismutase, SOD）活性、肾组织核因子 кB（nuclear factor-kappa B, NF-кB）表达和血清肿瘤坏死因子-α（tumor necrosis factor α, TNF-α）浓度，光镜下观察肾组织病理学结果。结果 S组HSP70、HSP27和HO-1的mRNA有微量表达，蛋白几乎无表达，其余组在T0时开始表达，逐渐升高，T3时达高峰，随后逐渐下降。与S组比较，其余组各时点HSP70、HSP27和HO-1 的mRNA和蛋白表达上调（P﹤0.05），IPo组较I/R组T2~5时HSP70、HSP27和HO-1 的mRNA和蛋白表达上调（P﹤0.05），Q+IPo组较IPo组T2~5时HSP70、HSP27和HO-1 的mRNA和蛋白表达下调（P﹤0.05）。T3时血清Cr、BUN和TNF-α浓度I/R组分别为（102±5）µmol/L、（25.7±3.9）mmol/L、（2.29±0.18）ng/ml，IPo组分别为（64±5）µmol/L、（11.3±3.0）mmol/L、（1.76±0.13）ng/ml，Q+IPo组分别为（101±6）µmol/L、（26.5±4.5）mmol/L、（2.31±0.17 ）ng/ml，均高于S组（46±6）µmol/L、（5.1±1.9）mmol/L和（1.13±0.14）ng/ml（P﹤0.05），IPo组三者浓度较I/R组降低（P﹤0.05），Q+IPo组较IPo组升高（P﹤0.05）。T3时MDA含量I/R组（2.20±0.23 ）nmol/mgprot、IPo组（1.35±0.13 ）nmol/mgprot和Q+IPo组（2.25±0.16）nmol/mgprot较S组（1.02±0.19）nmol/mgprot升高（P﹤0.05），SOD活性I/R组（104±6）U/mgprot、IPo组（124±4）U/mgprot和Q+IPo组（106±5）U/mgprot较S组（147±6）U/mgprot降低（P﹤0.05），IPo组与I/R组比较MDA含量降低和SOD活性升高（P﹤0.05），Q+IPo组与IPo组比较MDA含量升高和SOD活性降低（P﹤0.05），肾组织NF-кB表达I/R组、IPo组和Q+IPo组较S组增高，IPo组较I/R组表达降低，Q+IPo组较IPo组表达增高（P﹤0.05）。I/R组与Q+IPo组相比，各指标差异无统计学意义（P>0.05）。与S组比较，其余三组有程度不等的肾组织病理学损伤，IPo组损伤较I/R组减轻，Q+IPo组损伤程度与I/R组相似。结论 IPo上调了HSP70、HSP27和HO-1的表达；HSP高表达参与了肾IPo减轻肾I/R损伤的过程
关键词： 缺血后处理； 再灌注损伤；肾；热休克蛋白
Abstract:

Obiective To investigate the effect of ischemic postconditioning on expression of heat shock protein (HSP)70, HSP27 and HO-1 (heme oxygenase-1) and the role of which in reduction renal ischemia-reperfusion (I/R) injury in rats. Methods One hundred forty healthy male SD rats weighing 250-280 g were randomly divided into 4 groups (n=35 each) : group I sham operation group (group S); group II I/R; group III IPo; group IV quercetin+IPo group (group Q+IPo). The rats were anesthetized with intraperitoneal (IP) chloral hydrate 300 mg/kg. Bilateral kidneys were exposed and their pedicels were clamped for 45 min followed by reperfusion in group II-IV. In group III and IV 3 cycles of 10 s reperfusion followed by 10 s ischemia were applied immediately after 45 min kidney ischemia. In group IV quercetin (a inhibitor of HSP) 100 mg/kg was given IP at 30 min before ischemia. Five rats were sacrificed at 0, 1, 3, 6, 12, 24, and 48 h (T0-6) of reperfusion and the kidneys were immediatedly removed for determination the expression of HSP70mRNA, HSP27mRNA, HO-1 mRNA, HSP70, HSP27 and HO-1. At T3 before the rats were sacrificed blood samples were obtained for determination of serum creatinine(Cr), urea nitrogen (BUN) and tumor necrosis factor α (TNF-α) concentrations and kidneys were removed for determination of the expression of nuclear factor-kappa B (NF-кB), the methylene dioxyamphetamine (MDA) content and superoxide dismutase (SOD) activity, and observation of histopathology with light microscope. Results The expression of HSP70mRNA, HSP27mRNA, HO-1 mRNA, HSP70, HSP27 and HO-1 was traced in group S, and in the other three groups the expression of HSP70mRNA、HO-1mRNA and HSP70、HO-1 increased at T1 and meet peakpoint at T3, then begain to decreased. Compared with group S, the expression of HSP70mRNA, HSP27mRNA, HO-1mRNA, HSP70, hsp27 and HO-1 was up-regulated at T0-6 in other 3 groups (P<0.05), Compared with group I/R, the expression of HSP70mRNA, HSP27mRNA, HO-1mRNA, HSP70, hsp27 and HO-1 was up-regulated at T2-5 in group IPo (P<0.05), Compared with group IPo, the expression of HSP70mRNA, HSP27mRNA, HO-1mRNA, HSP70, hsp27 and HO-1 was down-regulated at T2-5 in group Q+IPo (P<0.05). There were not significant defferent between group I/R and group Q+IPo (P>0.05). Serum Cr, BUN and TNF-α concentrations at T3 in group I/R (102±5) µmol/L, (25.7±3.9) mmol/L, (2.29±0.18) ng/ml, in group IPo (64±5) µmol/L, (11.3±3.0) mmol/L, (1.76±0.13) ng/ml, and in grou Q+IPo(101±6) µmol/L, (26.5±4.5) mmol/L, (2.31±0.17) ng/ml were increased than those in group S (46±6) µmol/L, (5.1±1.9) mmol/L and (1.13±0.14) ng/ml(P﹤0.05), and the serum Cr, BUN and TNF-α concentrations in group IPo were decreased than those in group I/R, and those in group Q+IPo were increased than those in group IPo (P﹤0.05). There were not significant defferent between group I/R and group Q+IPo (P>0.05). The MDA content (2.20±0.23) nmol/mgprot in group I/R, (1.35±0.13) nmol/mgprot in group IPo and (2.25±0.16) nmol/mgprot in group Q+IPo was in creased than that in group S (P﹤0.05), the activity of SOD (104±6) U/mgprot in group I/R, (124±4) U/mgprot in group IPo and (106±5) U/mgprot in group Q+IPo was decreased than that in group S (P﹤0.05); the MDA content was decreased and SOD activity was increased in group IPo compared with group I/R, and the MDA content was increased and SOD activity was decreased in group Q+IPo compared with group IPo (P﹤0.05). The expression of NF-кB in group was up-regulated at T3 in group I/R, IPo and Q+IPo compared with group S, that in group IPo was down-regulated compared with group I/R, and that in Q+IPo was up-regulated compared with group IPo (P﹤0.05). There were not significant defferent between group I/R and group Q+IPo (P>0.05). Microscope examination showed that the injury to kidney was attenuated in group IPo compared with group I/R, the injury to kidney was not significant defferent between group I/R and group Q+IPo. Conclusion IPo increased the expression of HSP70, HSP27, and HO-1 and the expression of HSP is involved in the reduction of renal I/R injury by IPo in rats.
Key words: Ischemic postconditioning; Ischemic reperfusion injury; Renal; Heat shock protein