异氟醚预处理对大鼠肾缺血再灌注时细胞凋亡的影响

Effect of isoflurane preconditioning on cell apoptosis during renal ischemia-reperfusion in rats

目的评价异氟醚预处理对大鼠肾缺血再灌注时细胞凋亡的影响。方法SD雄性大鼠30只，体重300—320g，12～14周龄，采用随机数字表法将其分为3组（n=10）：假手术组（S组）、肾缺血再灌注组（I／R组）和异氟醚预处理＋肾缺血再灌注组（Iso＋I／R组）。采用右肾切除、左肾蒂夹闭30min时恢复灌注的方法建立肾缺血再灌注损伤模型。Iso＋I／R组在密闭麻醉箱中吸入1．5％异氟醚（O：2L／rain）1h，洗脱30min后制备模型。其余两组吸人O2 2L／min1h。于再灌注2h时采集腹主动脉血样3ml，测定血清肌酐（cr）、尿素氮（BUN）、胱抑素C（CysC）浓度；取左肾组织，采用TUNEL法检测细胞凋亡情况，计算凋亡指数（AI）；采用实时荧光定量PCR法检测p53mRNA、BaxmRNA和Bcl-2mRNA表达水平，采用Westernblot法检测Bax、Bcl-2、caspase-3表达水平，计算Bcl-2和Bax表达的比值。结果与S组比较，I／R组血清Cr、BUN、CysC浓度升高，Bcl-2和Bax及其mRNA表达上调，Bcl-2／Bax比值降低，caspase-3表达上调，AI增加（P〈0．05）；与I／R组比较，Iso＋I／R组血清Cr、BUN、CysC浓度降低，Bcl-2mRNA、BaxmRNA和Bax表达下调，Bcl-2／Bax比值升高，caspase-3表达下调，AI降低（P〈0．05）；各组间p53mRNA表达比较差异无统计学意义（P〉O．05）。结论异氟醚预处理减轻大鼠肾缺血再灌注损伤的机制与其调节Bax和Bcl-2平衡、抑制肾细胞凋亡有关。

Objective To evaluate the effect of isoflurane preconditioning on cell apoptosis during renal ischemia-repeffusion （I/R） in rats. Methods Thirty male Sprague-Dawley rats, aged 12-14 weeks, weighing 300-320 g, were randomly divided into 3 groups （n = 10 each） using a random number table： sham operation group （group S） ; I/R group; isoflurane preconditioning plus I/R group （group Iso＋I/R）. The rats were anesthetized with intraperitoneal pentobarbital sodium 30 mg/kg. To establish the model of renal I/R injury, the right kidney was removed, and the left renal pedicle was occluded for 30 rain with atraumatic mini-clamp for 30 min, followed by 2 h repeffusion. In Iso＋I/R group, 1.5% isoflurane was inhaled for 1 h, followed by 30 rain of washout before I/R. In S and I/R groups only oxygen 2 L/min was inhaled for 1 h. Arterial blood samples were taken at 2 h of reperfusion to determine the concentrations of serum creatinine （Cr）, blood urea nitrogen （BUN） and eystatin C （CysC）. The animals were then sacrificed, and left kidneys were sampled for determination of the cell apoptosis （by TUNEL） , expression of p53, Bax and Bcl-2 mRNA （using real-time fluorescent quantitative PCR）, and expression of Bax, Bcl-2, and caspase-3 （by Western blot analysis ）. The Bcl-2/Bax ratio was calculated. Results Compared with group S, the serum Cr, BUN, and CysC concentrations were significantly increased, the Bcl-2/Bax ratio was decreased, Bcl-2 mRNA, Bax mRNA, Bcl-2, Bax and caspase-3 expression was up regulated, and AI was increased in group I/R. Compared with group I/R, the serum Cr, BUN, and CysC concentrations were significantly decreased, the Bcl-2/Bax ratio was increased, Bcl-2 mRNA, Bax mRNA, Bax and caspase-3 expression was down-regulated, and AI was decreased in group Iso＋I/R. There was no significant difference in p53 mRNA expression among the three groups. Conclusion Regulating the balance between Bel-2 and Bax and inhibiting apoptosis in kidney ceils are involved in the mechanism by which isoflurane preconditioning reduces renal I/R injury in rats.