叢培芳， 柳云恩， 張玉彪， 佟昌慈， 史秀云， 劉 穎， 施 琳， 佟 周， 金紅旭， 侯明曉

沈陽(yáng)軍區(qū)總醫(yī)院 急診醫(yī)學(xué)部 全軍重癥(戰(zhàn))創(chuàng)傷救治中心實(shí)驗(yàn)室遼寧省重癥創(chuàng)傷和器官保護(hù)重點(diǎn)實(shí)驗(yàn)室，遼寧 沈陽(yáng) 110016

·爆震傷·

爆震傷對(duì)大鼠肺組織凋亡影響研究

叢培芳， 柳云恩， 張玉彪， 佟昌慈， 史秀云， 劉 穎， 施 琳， 佟 周， 金紅旭， 侯明曉

沈陽(yáng)軍區(qū)總醫(yī)院 急診醫(yī)學(xué)部 全軍重癥(戰(zhàn))創(chuàng)傷救治中心實(shí)驗(yàn)室遼寧省重癥創(chuàng)傷和器官保護(hù)重點(diǎn)實(shí)驗(yàn)室，遼寧 沈陽(yáng) 110016

目的 通過(guò)建立肺爆震傷大鼠模型，檢測(cè)不同時(shí)間點(diǎn)大鼠肺組織中c-Jun氨基末端激酶(JNK)、P38、Bad及Bcl-xl的變化，探討JNK/P38通路在肺爆震傷中的作用，旨在為肺爆震傷的損傷機(jī)制提供理論依據(jù)。方法 選取18只成年健康雄性SD大鼠，分別納入對(duì)照組與爆震傷后24 h組、7 d組，每組各6只。TUNEL檢測(cè)爆震傷后肺組織的凋亡情況；免疫熒光檢測(cè)JNK與P38的表達(dá)情況；Western-blot檢測(cè)JNK、P38、Bcl-xl與Bad的蛋白表達(dá)情況；Real Time PCR檢測(cè)JNK、P38、Bcl-xl與Bad的mRNA表達(dá)情況。結(jié)果 TUNEL結(jié)果顯示，與對(duì)照組比較，爆震傷后24 h組的肺組織細(xì)胞凋亡率明顯升高；與24 h組比較，7 d組凋亡率降低，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。免疫熒光結(jié)果顯示，與對(duì)照組比較，JNK與P38在爆震傷后24 h表達(dá)升高；與24 h組比較，7 d組表達(dá)降低，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。Western-blot與Real Time PCR結(jié)果顯示，與對(duì)照組比較，JNK、P38及Bad在爆震傷后24 h蛋白與mRNA表達(dá)均升高；與24 h組比較，7 d組表達(dá)均降低，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。而B(niǎo)cl-xl在爆震傷后24 h蛋白與mRNA表達(dá)低于對(duì)照組，7 d組表達(dá)高于24 h組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論 肺爆震傷后的細(xì)胞凋亡反應(yīng)受JNK/P38通路的調(diào)控，且與時(shí)間具有相關(guān)性。

爆震傷； 大鼠； c-Jun氨基末端激酶； P38

爆震傷是各種原因產(chǎn)生的沖擊波，在擊中機(jī)體后，釋放出大量能量造成的各組織器官的損傷[1]。近年來(lái)，爆震傷所致受傷人數(shù)逐年上升，成為軍事戰(zhàn)爭(zhēng)與日常生活中最常見(jiàn)的暴力致傷原因[2-3]。肺爆震傷是爆炸現(xiàn)場(chǎng)最常見(jiàn)的致命傷[4]，其過(guò)程中有多種發(fā)病機(jī)制參與，如活性氧、鈣釋放、谷氨酸毒性及線粒體功能障礙等[5-6]，這些機(jī)制導(dǎo)致的細(xì)胞凋亡反應(yīng)是造成損傷的重要原因。c-Jun氨基末端激酶(c-Jun N-terminal kinase，JNK)與P38參與機(jī)體多種病理生理過(guò)程，其相關(guān)通路在應(yīng)激反應(yīng)所致的炎癥與細(xì)胞凋亡中發(fā)揮重要作用[7]。因此，本研究通過(guò)建立肺爆震傷大鼠模型，檢測(cè)不同時(shí)間點(diǎn)大鼠肺組織中JNK、P38、Bad及Bcl-xl的變化，探討JNK/P38通路在肺爆震傷中的作用，旨在為肺爆震傷的損傷機(jī)制提供理論依據(jù)?，F(xiàn)報(bào)道如下。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物 選取18只成年健康雄性SD大鼠，體質(zhì)量(200±10)g，由沈陽(yáng)軍區(qū)總醫(yī)院動(dòng)物實(shí)驗(yàn)中心提供。動(dòng)物自由飲食、飲水(江蘇省協(xié)同醫(yī)藥生物工程有限責(zé)任公司)，于清潔級(jí)動(dòng)物房適應(yīng)性喂養(yǎng)1周。18只大鼠分別納入對(duì)照組與爆震傷后24 h組、7 d組，每組6只。

1.2 試劑與材料 TUNEL染色試劑盒(瑞士羅氏公司)；JNK、P38、Bad、Bcl-xl、GAPDH抗體及相應(yīng)二抗(英國(guó)Abcam公司)；Western-blot一抗稀釋液(中國(guó)碧云天生物技術(shù)研究所)；JNK、P38、Bad、Bcl-xl及β-actin 引物(中國(guó)Sangon公司)。

1.3 研究方法

1.3.1 動(dòng)物模型建立 采用爆震沖擊裝置建立大鼠肺損傷模型。大鼠按體質(zhì)量麻醉后置入保護(hù)管中，保護(hù)大鼠頭、腹部，外露胸部。將保護(hù)管置于爆震傷裝置上，下方壓力達(dá)到0.12 MPa時(shí)，鋁膜爆破，超壓波顯示壓力為0.55 MPa，大鼠飛起高度為80～150 cm。對(duì)照組不致傷。

1.3.2 TUNEL 將肺組織進(jìn)行固定、脫水、包埋、切片，然后根據(jù)試劑盒說(shuō)明進(jìn)行TUNEL染色，顯微鏡下觀察肺組織凋亡情況。

1.3.3 免疫熒光 將切片后的肺組織進(jìn)行一抗孵育過(guò)夜，然后加入熒光二抗，37℃孵育1 h。DAPI避光5 min，封片，觀察。

1.3.4 Western-blot 分別于肺爆震傷后24 h、7 d時(shí)，取大鼠肺組織，提取蛋白后，將蛋白樣品經(jīng)SDS-聚丙烯酰胺凝膠電泳并轉(zhuǎn)移至PVDF膜上，含5%脫脂奶粉中室溫封閉1 h，一抗4℃孵育過(guò)夜。TBST漂洗4次，分別用相應(yīng)二抗室溫孵育2 h。TBST再次漂洗4次，ECL Western印跡試劑盒進(jìn)行化學(xué)發(fā)光檢測(cè)。

1.3.5 Real Time PCR Trizol試劑提取大鼠肺組織總RNA。濃度測(cè)定后，反轉(zhuǎn)錄合成cDNA。取cDNA產(chǎn)物，加入引物進(jìn)行擴(kuò)增，上機(jī)，計(jì)算。

2 結(jié)果

2.1 TUNEL檢測(cè)爆震傷后肺組織的凋亡情況 與對(duì)照組比較，爆震傷后24 h的肺組織細(xì)胞凋亡率明顯升高；與24 h組比較，7 d組凋亡率降低，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)圖1。

2.2 免疫熒光檢測(cè)JNK與P38的表達(dá)情況 與對(duì)照組比較，JNK與P38在爆震傷后24 h表達(dá)升高；與24 h組比較，7 d組表達(dá)降低，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)圖2。

2.3 Western-blot檢測(cè)JNK、P38、Bcl-xl與Bad的蛋白表達(dá)情況 與對(duì)照組比較，JNK、P38及Bad在爆震傷后24 h蛋白表達(dá)升高；與24 h組比較，7 d組表達(dá)降低，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。而B(niǎo)cl-xl在爆震傷后24 h蛋白表達(dá)低于對(duì)照組，7 d組表達(dá)高于24 h組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)圖3。

圖1 TUNEL檢測(cè)爆震傷后肺組織的凋亡情況(400倍)

圖2 免疫熒光檢測(cè)JNK與P38的表達(dá)情況(400倍;a.JNK表達(dá)情況；b.P38表達(dá)情況)

圖3 Western-blot檢測(cè)JNK、P38、Bcl-xl與Bad的蛋白表達(dá)情況(與對(duì)照組比較，①P<0.05；與24 h組比較，②P<0.05)

2.4 Real Time PCR 檢測(cè)JNK、P38、Bcl-xl與Bad的mRNA表達(dá)情況 與對(duì)照組比較，JNK、P38及Bad在爆震傷后24 h mRNA表達(dá)升高；與24 h組比較，7 d組表達(dá)降低，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。而B(niǎo)cl-xl在爆震傷后24 h mRNA表達(dá)低于對(duì)照組，7 d組表達(dá)高于24 h組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)圖4。

3 討論

絲裂原活化蛋白激酶家族參與細(xì)胞中各種重要信號(hào)轉(zhuǎn)導(dǎo)途徑的調(diào)節(jié)過(guò)程，其中，JNK與P38在細(xì)胞凋亡中發(fā)揮關(guān)鍵作用[8]。JNK與P38通路可被各種應(yīng)激反應(yīng)激活，參與并促進(jìn)機(jī)體對(duì)應(yīng)激的應(yīng)答，活化下游凋亡因子，促使細(xì)胞發(fā)生凋亡[9-14]。有研究表明，JNK與P38可通過(guò)抑制抗凋亡蛋白Bcl-2的表達(dá)、促進(jìn)促凋亡蛋白Bax的表達(dá)而導(dǎo)致凋亡發(fā)生[15-18]。Bcl-xl與Bad均為Bcl-2家族成員，分別屬于其中的抗凋亡與促凋亡蛋白。Bcl-xl可維持線粒體膜的完整性，抑制細(xì)胞色素C的釋放，減少凋亡的發(fā)生，而B(niǎo)ad則有相反作用[19-20]。本研究發(fā)現(xiàn)，發(fā)生肺爆震傷24 h后，肺組織發(fā)生凋亡，同時(shí)，JNK與P38處于高表達(dá)狀態(tài)，導(dǎo)致其下游促凋亡蛋白Bad的表達(dá)升高，抗凋亡蛋白Bcl-xl的表達(dá)降低；而在肺爆震傷發(fā)生7 d時(shí)，JNK與P38的表達(dá)降低，使Bad的表達(dá)降低、Bcl-xl的表達(dá)升高。結(jié)果表明，肺爆震傷后的細(xì)胞凋亡反應(yīng)受JNK/P38通路的調(diào)控，且與時(shí)間具有相關(guān)性。

圖4 Real Time PCR檢測(cè)JNK、P38、Bcl-xl與Bad的mRNA表達(dá)情況(與對(duì)照組比較，①P<0.05;與24 h組比較，②P<0.05)

綜上所述，肺爆震傷后機(jī)體瞬間產(chǎn)生嚴(yán)重的應(yīng)激反應(yīng)，JNK/P38通路介導(dǎo)的細(xì)胞凋亡參與肺爆震傷的發(fā)生與發(fā)展，且與時(shí)間具有相關(guān)性。因此，JNK與P38對(duì)肺爆震傷的診治與預(yù)后具有一定意義，以其為靶點(diǎn)或通過(guò)干預(yù)其信號(hào)傳導(dǎo)與表達(dá)研發(fā)藥物可能會(huì)成為肺爆震傷的有效治療方法之一。

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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

全軍十二五面上項(xiàng)目(CSY12J002)；全軍重大新藥創(chuàng)制項(xiàng)目(2013ZX09J13109-02B)；全軍十二五面上項(xiàng)目(CSY13J002)； 總后衛(wèi)生部重大新上(ASM14L008)

叢培芳(1987-)，女，遼寧沈陽(yáng)人，藥師，碩士

侯明曉，E-mail：houmingxiao188@163.com

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

2017-07-17