乌司他丁对大鼠肢体缺血再灌注急性肺损伤细胞自噬的影响

Effect of Ulinastatin on autophagy - associated proteins expression in acute lung injury induced by remote limb ischemia - reperfusion in rats

目的探讨乌司他丁对大鼠双下肢缺血再灌注导致急性肺损伤肺组织细胞自噬的保护效应。方法采用LIR模型，72只雄性sD大鼠随机数字表法随机分为假手术＋生理盐水组（s0＋Ns组），肢体缺m再灌注＋生理盐水组（I／R＋NS组），肢体缺咀再灌注＋乌司他丁组（I／R＋UTI组），每组再分4亚组，即肢体缺血3h再分别灌注0、2、4、8h，每亚组各6只。夹闭股动脉前10rain于大鼠左侧颈内静脉分别注射NS或UTI5×10^4U／kg各1mL，于再灌注各时点末采用免疫荧光法检测肺组织病理变化，利用反转录聚合酶链式反应法（RT—PCR）检测各组肺组织Beclinl和Atg5mRNA表达及蛋白印迹分析（Westernblot）技术检测微管相关蛋白轻链3（LC3）的表达。结果免疫荧光染色I／R＋NS组不同时点镜下可见肺组织结构紊乱，肺泡间隔增厚甚至实变，而sO＋Ns及I／R＋UTI两组上述病理现象减轻；采用2^-△△CT。法分析肺组织Beclinl和Atg5mRNA相对表达量I／R＋NS组4、8h时点明显增高（P〈0．01或P〈0．05），与SO＋NS组各相同时点比较均明显增高（P〈0．01或P〈0．05），I／R＋UTI组与I／R＋NS组比较4h时点降低最明显（P〈0．01）；Westernblot法检测LC3-Ⅱ蛋白含量及LC3-Ⅱ／GAPDH比值，I／R＋NS组4、8h时点明显增高（P〈0．01），与SO＋NS组各相同时点比较均明显增高（P〈0．01），I／R＋UTI组与I／R＋NS组各相同时点比较明显降低（P〈0．01）。结论乌司他丁可下调大鼠LIR—ALI肺组织细胞自噬的过表达，具有良好的保护作用。

Objective To investigate the protective effect of Ulinastatin （UTI） on expression of autophagy- associated proteins in lung tissues in acute lung injury induced by remote limb ischemia - reperfusion （ LIR -ALI ） in rats. Methods Seventy - two adult male Sprague - Dawly （ SD ） rats weighting 220 - 250 g were made LIR -ALI models and divided randomly into three groups （ 24 rats in each group） ： sham operation and normal saline （ SO ＋ NS） group, isehemia - reperfusion and normal saline （ I/R ＋ NS ） group and ischemia - reperfusion and UTI （ I/R ＋ UTI ） group, each group was divided into 4 subgroups （0, 2, 4, 8 h） respectively. The rats were anesthetized. Before 10 rain of ischemia NS/UTI 1 mL/5 ×10^4 U/kg was injected through left internal jugular vein, and at the end of 0, 2, 4, 8 h of reperfusion after 3 hrs of ischemia the tissues of lung were removed. The pathological changes of lung tissues were observed by immunofluorescence techniques; the expression of Beclinl protein and Atg5 mRNA in the lung tissues were detected by reverse transcription polymerase chain reaction （ RT - PCR） and analyzed by 2 ^△△CT method; microtubules associated protein light chain 3 （LC3） in the lung tissues were detected by Western blotting. Results The level of serum LDH in I/R groups were very higher than that in sham group （P 〈 0.01 ） showed that the rats models of LIR -ALI were established. Immunofluorescence demonstrated the injuries of lung tissues, the thickening of alveolar septum and partial consol in I/R ＋ NS groups, but the above - mentioned damage was not observed significantly in SO ＋ NS and I/R ＋ UTI groups. The expression of Beclinl protein, Atg5 mRNA, LC3 - II and the ratio of LC3 - Ⅱ/GAPDH in lung tissues were very higher at 4, 8 h in I/R ＋ NS group （P 〈 0.01 or P 〈 0.05 ） , and they were significantly higher at the same time compared with SO ＋ NS group （P 〈0.01 or P 〈0.05） ; but compared with I/R ＋ NS group, the expression of Beclinl protein, Atg5 mRNA at 4 h and LC3 - Ⅱ , the ratio of LC3 - Ⅱ/GAPDH at all the same time were significantly lower in I/R ＋ UTI group （P 〈 0.01 ）. Conclusion UTI has notable protective effects on LIR -ALI by inhibiting the expression of autophagy - associated proteins in lung tissues in rats.