c-Jun氨基末端激酶-小干扰RNA预处理对小鼠呼吸机相关性肺损伤的影响

Effect of c - Jun N - terminal kinase - small interfering RNA pretreatment on ventilator - induced lung injury in mice

目的观察c-Jun氨基末端激酶（JNK）靶向小干扰RNA（JNK-siRNA）预处理对小鼠呼吸机相关性肺损伤（VILI）的影响。方法实验用C57BL／6J小鼠50只，采用随机数字表法分为5组（n=10）：常规潮气量组（NVT组）、大潮气量组（HVT组）、HVT＋载体组（HVT＋Vehicle组）、HVT＋阴性对照组（HVT＋siRNANegative组）和HVT＋JNK-siRNA组（HVT＋siRNAJNK）。小鼠机械通气4h后抽取动脉血进行血气分析，记录动脉血氧分压（PaO：）。实验结束后留取小鼠左肺，分别检测肺组织湿／干重比（W／D），光镜下观察肺组织病理改变并测定肺泡损伤率（IAR），电镜下观察肺组织超微结构改变，反转录-聚合酶链反应（RT-PCR）和Westernblot分别检测JNKmRNA及磷酸化JNK（p-JNK）蛋白表达水平，原位末端细胞凋亡检测法（TUNEL法）检测肺组织细胞凋亡指数（AI）。结果与NVT组比较，HV，组肺组织中JNKmRNA（0．93±0．15比n41-t-n09）及p-JNK蛋白（2．78±0．78比1．05±0．21）表达水平均增加（P〈0．01），PaO2[（61±5）mmHg比（77±6）mmHg]降低（P〈0．01），W／D（5．45±0．83比2．82±0．73）、IAR[（44．44±5．77）％比（5．33±1．93）％]和AI[（46．15±2．57）％比（2．73±0．76）％]均升高（P〈0．01）；肺组织形态学结构及超微结构均呈明显损伤性变化。与HVT＋Vehicle组和HVT＋siRNANegative组比较，HVT＋siRNAJNK组肺组织中JNKmRNA（0．53±0．10比0．88±0．16、0．88±0．11）及p-JNK蛋白（1．32±0．46比2．82±0．76、2．96±0．54）表达水平均降低（P〈0．01），PaO2[（87±9）mmHg比（59±7）mmHg、（63±6）mmHg]升高（P〈0．01），W／D（2．88±0．45比5．42±0．56、5．23±0．66）、IAR[（20．52±2．88）％比（39．84±3．06）％、（38．62±4．34）％]和AI[（13．72±1．66）％比（45．36±2．48）％、（44．43±2．48）％]均降低（P〈0．01），肺组织形态学结构及超微结构异常改变均有不同程度的减轻。结论JNK-siRNA预处理可减轻小鼠呼吸机相关性肺损伤，其机制可能与其抑制JNK信号途径介导的细胞凋亡有关。

Objective To observe the effect of small interfering RNA （siRNA） of e -Jun N -ter- minal kinase （JNK） pretreatmcnt on ventilator - induced lung injury （VILI） in mice. Methods Fifty C57BL/6J mice were randomly allocated into five groups： normal tidal volume group （NVT group） , high tidal volume group （ HVT group）, HVT ＋ vehicle group （ HVT ＋ vehicle group）, HVT ＋ negative control siRNA group （ HVT ＋ siRNANegative group） and HVT ＋ JNK siRNA group （ HVT ＋ siRNAJNK group） , 10 mice per group. After 4 h of mechanical ventilation, arterial blood samples were obtained for blood gas a- nalysis and partial pressure of oxygen （ PaO2 ） was recorded. Mice were euthanized after experiment fin- ished, and left lung tissue was excised. Wet lung weight and dry lung weight （W/D） and total lung water content （TLW） were determined. Pathological changes of lung tissue were observed and the injured alveo- lus rate （IAR） counted by light microscope, and changes of uhrastructure of lung tissue were observed by transmission electron microscope. The expression levels of JNK mRNA and phosphorylated JNK （p -JNK） protein were detected by reverse transcription - polymerase chain reaction （ RT - PCR） and Western blot- ting. Apoptosis index （AI） of lung tissue was determined by terminal - deoxynucleoitidyl transferase medi- ated nick end labeling （TUNEL） method. Results Compared to NVT group, the expression levels of JNK mRNA （0. 93 ± 0. 15 vs. 0. 41 ± 0. 09） and p - JNK protein （2.78 ± 0.78 vs. 1.05 ± 0. 21 ） were signifi- cantly increased （P 〈0.01） in, PaO2[ （61 ±5） mmHg vs. （77±6） mmHg] was significantly decreased （P〈0.01）, W/D （5.45±0.83 vs. 2.82 ±0.73）, IAR [（44.44±5.77）% vs. （5.33±1.93）%]and AI[ （46. 15 ±2. 57） % vs. （2. 73 ±0. 76）% ] were all notably increased （P 〈0. 01 ）, and morpho- logical and uhrastruetural demage in lung tissue was notably aggravated in HVT group. Compared to HVT ＋ vehicle group and HVT ＋ siRNANegative group, the expression levels ofJNK mRNA （0. 53±0. 10 vs. 0. 88 ± 0. 16 ; 0. 88 ± 0. 11 ） and p - JNK protein （ 1.32 ±0. 46 vs. 2. 82 ± 0. 76 ; 2. 96 ±0. 54 ） were significantly reduced in HVr group （P〈0. 01）, Pa02[ （87 ±9） mmHg vs. （59 ±7） mmHg; （63 ±6） mmHg] was significantly increased （P〈 0.01）, W/D （2.88 ± 0.45 vs. 5.42 ± 0.56; 5.23 ± 0.66）, IAR [（20.52±2.88）% vs. （39.84 ±3.06）%; （38.62 ±4.34）%; and AI [（13.72 ± 1.66）% vs. （45.36 ± 2.48 ） % ; （44.43 ± 2.48 ） % ] were all decreased （ P 〈 0. 01 ）, and morphological and ultra- structural demage in the lung tissue were alleviated in HVT ＋ siRNAJNK group. Conclusion JNK - siRNA pretreatment can alleviate VILI in mice, and the mechanism may be associated with inhibition of pneumo- cyte apoptosis mediated by JNK signal pathway.