肢体缺血后处理对孕兔失血性休克急性肾损伤的保护及作用机制

Preventive effects and the mechanism of limb ischemic postconditioning on kidney against hemorrhagic shock in pregnant rabbits

目的:探讨肢体缺血后处理对孕兔失血性休克肾损伤的保护及可能的作用机制。方法:24只孕兔制成重度失血性休克模型,均游离双侧股动脉,随机分为假手术组(S组)、缺血再灌注组(IR组)、肢体缺血后处理组(LRIP组)(每组8只)。S组:游离双侧股动脉后无失血及输血;IR:孕兔股动脉放血使平均动脉压40 mmHg维持180 min后,1 h内匀速回输全部血液,使孕兔血压维持在MAP稳定在80 mmHg之上并平稳24 h;LRIP组:在急性失血性休克180 min后,给予夹闭双侧股动脉缺血30 s再灌注30 s共10次后(即缺血后处理),再输血复苏至24 h。分别采用全自动生化分析仪测定血尿素氮(BUN)、血肌酐(Cr)水平,酶联免疫吸附试验(ELISA)法测定不同时点(0 h、8 h、16 h、24 h)血清TNF-α、IL-10的含量,免疫组化法检测肾组织中核因子κB(NF-κB)表达;RTPCR法检测肾组织内TNF-α、IL-10及iNOS mRNA的表达;HE染色观察肾脏病理学改变。结果:(1)与I/R组相比,LRIP组不同时点Cr、BUN浓度降低,血清TNF-α含量降低、IL-10含量升高,肾组织NF-κB表达减少(P均<0.05);(2)与S组比较,肾内TNF-α、IL-10、iNOS mRNA在I/R组、LRIP组表达均上调(P<0.05或P<0.01),与I/R组相比,肾内TNF-α、iNOS mRNA表达在LRIP组下调,IL-10 mRNA表达上调(P<0.05);(3)S组肾脏未发生明显的病理改变,I/R组肾小管可见明显玻璃样改变和坏死,肾小管略扩张,肾小球和肾小管周围可见炎症细胞浸润,LRIP组肾脏组织结构正常,少量的炎症细胞浸润,肾小管上皮细胞相对完整,水肿减轻。结论:重复10次缺血30 s再灌注30 s的肢体后处理对肾脏有保护作用,其机制可能与抑制肾组织NF-κB转录、减少iNOS mRNA的表达、改善肾内炎症反应有关。

Objective： To investigate the protective effects of limb ischemic postconditioning on kidney injury induced by hemorrhagic shock in pregnant rabbits. Methods： The model of severe hemorrhagk shock was established in 24 pregnant rabbits. The rabbits were divided into three groups： Sham operation group（ S group）,ischemia reperfusion group（ IR）and limb remote ischaemic postconditioning group（ LRIP） randomly（ n = 8）. S group： only free bilateral femoral artery,no bleeding and transfusion; IR group： they were bleeding by femoral artery,and maintenance mean arterial pressure 40 mmHg for 180 min,then infused all the blood back within 1 hour and maintained MAP over 80 mmHg and stationary stability in 24 hours; LRIP group： they were given the occlusion of bilateral femoral artery,ischemia 30 seconds and reperfusion 30seconds for 10 times after acute hemorrhagic shock lasting 180 min,then infused blood to recovery lasting 24 h. automatic biochemical analyzer were used respectively to determine blood urea nitrogen（ BUN）,serum creatinine（ Cr）,enzymelinked immunosorbent assay（ ELISA） method at different time（ 0 h,8 h,16 h,24 h） to test the content of serum TNF alpha,IL-10,immunohistochemical method to detect nuclear factor kappa B in the kidney tissues（ NF-kB） expression; RTPCR method to detect renal tissue TNF alpha,IL-10 and iNOS mRNA expression; HE dyeing observation renal pathological changes. Results：（ 1） compared with I /R group,serum urea nitrogen,creatinine and TNF-α concentration were decreased,and IL-10 levels was increased in LRIP group at different point,NF-kB expression in kidney tissues decreased（ P〈0. 05）;（ 2） compared with S group,TNF-α mRNA,IL-10 mRNA,iNOS mRNA expression in kidney tissue were increased at I /R group and LRIP group（ P〈0. 05 or P〈0. 01）; compared with I /R group,TNF-α mRNA and iNOS mRNA expression decreased,IL-10 mRNA expression increased in renal tissue at LRIP group（ P〈0. 05）;（ 3） there were no obvious pathological changes in kidney at shame group,and there were obviously hyaline changes,renal tubular necrosis,renal tubules slightly expansion,the inflammatory cell infiltration around glomerular and renal tubular in the I /R group,there were normal structure in kidney tissue,a small amount of inflammatory cell infiltration,renal tubular epithelial cells relatively intact,edema decreased at LRIP group. Conclusion： ischemia 30 s and reperfusion 30 s repeated 10 times limb remote ischaemic postconditioning has a protective effect on kidney function. Its mechanism may be related to inhibit renal tissue NF-κB transcription,decreasing iNOS mRNA expression,improving renal inflammatory response.