爆震伤对大鼠肺组织凋亡影响研究

丛培芳， 柳云恩， 张玉彪， 佟昌慈， 史秀云， 刘 颖， 施 琳， 佟 周， 金红旭， 侯明晓

沈阳军区总医院 急诊医学部 全军重症(战)创伤救治中心实验室辽宁省重症创伤和器官保护重点实验室，辽宁 沈阳 110016

·爆震伤·

爆震伤对大鼠肺组织凋亡影响研究

丛培芳， 柳云恩， 张玉彪， 佟昌慈， 史秀云， 刘 颖， 施 琳， 佟 周， 金红旭， 侯明晓

沈阳军区总医院 急诊医学部 全军重症(战)创伤救治中心实验室辽宁省重症创伤和器官保护重点实验室，辽宁 沈阳 110016

目的 通过建立肺爆震伤大鼠模型，检测不同时间点大鼠肺组织中c-Jun氨基末端激酶(JNK)、P38、Bad及Bcl-xl的变化，探讨JNK/P38通路在肺爆震伤中的作用，旨在为肺爆震伤的损伤机制提供理论依据。方法 选取18只成年健康雄性SD大鼠，分别纳入对照组与爆震伤后24 h组、7 d组，每组各6只。TUNEL检测爆震伤后肺组织的凋亡情况；免疫荧光检测JNK与P38的表达情况；Western-blot检测JNK、P38、Bcl-xl与Bad的蛋白表达情况；Real Time PCR检测JNK、P38、Bcl-xl与Bad的mRNA表达情况。结果 TUNEL结果显示，与对照组比较，爆震伤后24 h组的肺组织细胞凋亡率明显升高；与24 h组比较，7 d组凋亡率降低，差异均有统计学意义(P<0.05)。免疫荧光结果显示，与对照组比较，JNK与P38在爆震伤后24 h表达升高；与24 h组比较，7 d组表达降低，差异均有统计学意义(P<0.05)。Western-blot与Real Time PCR结果显示，与对照组比较，JNK、P38及Bad在爆震伤后24 h蛋白与mRNA表达均升高；与24 h组比较，7 d组表达均降低，差异有统计学意义(P<0.05)。而Bcl-xl在爆震伤后24 h蛋白与mRNA表达低于对照组，7 d组表达高于24 h组，差异有统计学意义(P<0.05)。结论 肺爆震伤后的细胞凋亡反应受JNK/P38通路的调控，且与时间具有相关性。

爆震伤； 大鼠； c-Jun氨基末端激酶； P38

爆震伤是各种原因产生的冲击波，在击中机体后，释放出大量能量造成的各组织器官的损伤[1]。近年来，爆震伤所致受伤人数逐年上升，成为军事战争与日常生活中最常见的暴力致伤原因[2-3]。肺爆震伤是爆炸现场最常见的致命伤[4]，其过程中有多种发病机制参与，如活性氧、钙释放、谷氨酸毒性及线粒体功能障碍等[5-6]，这些机制导致的细胞凋亡反应是造成损伤的重要原因。c-Jun氨基末端激酶(c-Jun N-terminal kinase，JNK)与P38参与机体多种病理生理过程，其相关通路在应激反应所致的炎症与细胞凋亡中发挥重要作用[7]。因此，本研究通过建立肺爆震伤大鼠模型，检测不同时间点大鼠肺组织中JNK、P38、Bad及Bcl-xl的变化，探讨JNK/P38通路在肺爆震伤中的作用，旨在为肺爆震伤的损伤机制提供理论依据。现报道如下。

1 材料与方法

1.1 实验动物 选取18只成年健康雄性SD大鼠，体质量(200±10)g，由沈阳军区总医院动物实验中心提供。动物自由饮食、饮水(江苏省协同医药生物工程有限责任公司)，于清洁级动物房适应性喂养1周。18只大鼠分别纳入对照组与爆震伤后24 h组、7 d组，每组6只。

1.2 试剂与材料 TUNEL染色试剂盒(瑞士罗氏公司)；JNK、P38、Bad、Bcl-xl、GAPDH抗体及相应二抗(英国Abcam公司)；Western-blot一抗稀释液(中国碧云天生物技术研究所)；JNK、P38、Bad、Bcl-xl及β-actin 引物(中国Sangon公司)。

1.3 研究方法

1.3.1 动物模型建立 采用爆震冲击装置建立大鼠肺损伤模型。大鼠按体质量麻醉后置入保护管中，保护大鼠头、腹部，外露胸部。将保护管置于爆震伤装置上，下方压力达到0.12 MPa时，铝膜爆破，超压波显示压力为0.55 MPa，大鼠飞起高度为80～150 cm。对照组不致伤。

1.3.2 TUNEL 将肺组织进行固定、脱水、包埋、切片，然后根据试剂盒说明进行TUNEL染色，显微镜下观察肺组织凋亡情况。

1.3.3 免疫荧光 将切片后的肺组织进行一抗孵育过夜，然后加入荧光二抗，37℃孵育1 h。DAPI避光5 min，封片，观察。

1.3.4 Western-blot 分别于肺爆震伤后24 h、7 d时，取大鼠肺组织，提取蛋白后，将蛋白样品经SDS-聚丙烯酰胺凝胶电泳并转移至PVDF膜上，含5%脱脂奶粉中室温封闭1 h，一抗4℃孵育过夜。TBST漂洗4次，分别用相应二抗室温孵育2 h。TBST再次漂洗4次，ECL Western印迹试剂盒进行化学发光检测。

1.3.5 Real Time PCR Trizol试剂提取大鼠肺组织总RNA。浓度测定后，反转录合成cDNA。取cDNA产物，加入引物进行扩增，上机，计算。

2 结果

2.1 TUNEL检测爆震伤后肺组织的凋亡情况 与对照组比较，爆震伤后24 h的肺组织细胞凋亡率明显升高；与24 h组比较，7 d组凋亡率降低，差异均有统计学意义(P<0.05)。见图1。

2.2 免疫荧光检测JNK与P38的表达情况 与对照组比较，JNK与P38在爆震伤后24 h表达升高；与24 h组比较，7 d组表达降低，差异均有统计学意义(P<0.05)。见图2。

2.3 Western-blot检测JNK、P38、Bcl-xl与Bad的蛋白表达情况 与对照组比较，JNK、P38及Bad在爆震伤后24 h蛋白表达升高；与24 h组比较，7 d组表达降低，差异有统计学意义(P<0.05)。而Bcl-xl在爆震伤后24 h蛋白表达低于对照组，7 d组表达高于24 h组，差异有统计学意义(P<0.05)。见图3。

图1 TUNEL检测爆震伤后肺组织的凋亡情况(400倍)

图2 免疫荧光检测JNK与P38的表达情况(400倍;a.JNK表达情况；b.P38表达情况)

图3 Western-blot检测JNK、P38、Bcl-xl与Bad的蛋白表达情况(与对照组比较，①P<0.05；与24 h组比较，②P<0.05)

2.4 Real Time PCR 检测JNK、P38、Bcl-xl与Bad的mRNA表达情况 与对照组比较，JNK、P38及Bad在爆震伤后24 h mRNA表达升高；与24 h组比较，7 d组表达降低，差异有统计学意义(P<0.05)。而Bcl-xl在爆震伤后24 h mRNA表达低于对照组，7 d组表达高于24 h组，差异有统计学意义(P<0.05)。见图4。

3 讨论

丝裂原活化蛋白激酶家族参与细胞中各种重要信号转导途径的调节过程，其中，JNK与P38在细胞凋亡中发挥关键作用[8]。JNK与P38通路可被各种应激反应激活，参与并促进机体对应激的应答，活化下游凋亡因子，促使细胞发生凋亡[9-14]。有研究表明，JNK与P38可通过抑制抗凋亡蛋白Bcl-2的表达、促进促凋亡蛋白Bax的表达而导致凋亡发生[15-18]。Bcl-xl与Bad均为Bcl-2家族成员，分别属于其中的抗凋亡与促凋亡蛋白。Bcl-xl可维持线粒体膜的完整性，抑制细胞色素C的释放，减少凋亡的发生，而Bad则有相反作用[19-20]。本研究发现，发生肺爆震伤24 h后，肺组织发生凋亡，同时，JNK与P38处于高表达状态，导致其下游促凋亡蛋白Bad的表达升高，抗凋亡蛋白Bcl-xl的表达降低；而在肺爆震伤发生7 d时，JNK与P38的表达降低，使Bad的表达降低、Bcl-xl的表达升高。结果表明，肺爆震伤后的细胞凋亡反应受JNK/P38通路的调控，且与时间具有相关性。

图4 Real Time PCR检测JNK、P38、Bcl-xl与Bad的mRNA表达情况(与对照组比较，①P<0.05;与24 h组比较，②P<0.05)

综上所述，肺爆震伤后机体瞬间产生严重的应激反应，JNK/P38通路介导的细胞凋亡参与肺爆震伤的发生与发展，且与时间具有相关性。因此，JNK与P38对肺爆震伤的诊治与预后具有一定意义，以其为靶点或通过干预其信号传导与表达研发药物可能会成为肺爆震伤的有效治疗方法之一。

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Effect of blast injury on apoptosis of lung tissue in rats

CONG Pei-fang,LIU Yun-en,ZHANG Yu-biao,TONG Chang-ci,SHI Xiu-yun,LIU Ying,SHI Lin,TONG Zhou,JIN Hong-xu,HOU Ming-xiao

(Emergency Medicine Department of General Hospital of Shenyang Military Command, Laboratory of Rescue Center of Severe Wound and Trauma PLA, Severe Trauma and Organ Protection key Laboratory of Liaoning Province，Shenyang 110016, China)

Objective Through establishment of rats model with blast injury in lungs,to detect the changes of different time points in lung tissue of c-Jun amino terminal kinase(JNK),P38,Bad and Bcl-xl,to investigate the role of JNK/P38 pathway in blast injury in lungs,and to provide the theory basis for damage mechanism.Methods A total of 18 adult healthy female SD rats were selected and divided into the control group,the group of 24 hours after blast injury(group of 24 hours)and the group of 7 days after blast injury(group of 7 days),with 6 cases in each group.Apoptosis in lung was observed by TUNEL stain.Immunofluorescence was used to detected the expression of JNK and P38.The expressions of JNK,P38,Bcl-xl and Bad were detected by Western-blot.The expression of mRNA in these factors such as JNK,P38,Bcl-xl and Bad were detected by Real Time PCR.Results The result of TUNEL showed that,compared with the control group,the apoptosis rate of lung tissue in the group of 24 hours was significantly increased;compared with the group of 24 hours,the apoptosis rate in the group of 7 days was reduced,and the difference had statistical significance(P<0.05).The result of immunofluorescence showed that,compared with the control group,the expression of JNK and P38 increased after 24 hours of blast injury;compared with the group of 24 hours,the expression in 7 days reduced(P<0.05).The results of Western-blot and Real Time PCR showed that,compared with the control group,the expression of mRNA in JNK,P38 and Bad after 24 hours of injury were increased;compared with the group of 24 hours,the expression in 7 days reduced(P<0.05).The expression of Bcl-xl in the 24 hours protein and mRNA was lower than that in the control group after the blast injury,and the expression in 7 days was higher than that in the group of 24 hours,and the difference was statistically significant(P<0.05).Conclusion The cell apoptosis after lung burst was regulated by JNK/P38 pathway and was correlated with time.

Blast injury; Rats; C-Jun amino terminal kinase; P38

全军十二五面上项目(CSY12J002)；全军重大新药创制项目(2013ZX09J13109-02B)；全军十二五面上项目(CSY13J002)； 总后卫生部重大新上(ASM14L008)

丛培芳(1987-)，女，辽宁沈阳人，药师，硕士

侯明晓，E-mail：houmingxiao188@163.com

2095-5561(2017)04-0205-06 DOI∶10.16048/j.issn.2095-5561.2017.04.03

2017-07-17