爆震傷對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)傷救治中心實驗室遼寧省重癥創(chuàng)傷和器官保護(hù)重點實驗室，遼寧 沈陽 110016

·爆震傷·

爆震傷對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)傷救治中心實驗室遼寧省重癥創(chuàng)傷和器官保護(hù)重點實驗室，遼寧 沈陽 110016

目的 通過建立肺爆震傷大鼠模型，檢測不同時間點大鼠肺組織中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，分別納入對照組與爆震傷后24 h組、7 d組，每組各6只。TUNEL檢測爆震傷后肺組織的凋亡情況；免疫熒光檢測JNK與P38的表達(dá)情況；Western-blot檢測JNK、P38、Bcl-xl與Bad的蛋白表達(dá)情況；Real Time PCR檢測JNK、P38、Bcl-xl與Bad的mRNA表達(dá)情況。結(jié)果 TUNEL結(jié)果顯示，與對照組比較，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)計學(xué)意義(P<0.05)。免疫熒光結(jié)果顯示，與對照組比較，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)計學(xué)意義(P<0.05)。Western-blot與Real Time PCR結(jié)果顯示，與對照組比較，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)計學(xué)意義(P<0.05)。而Bcl-xl在爆震傷后24 h蛋白與mRNA表達(dá)低于對照組，7 d組表達(dá)高于24 h組，差異有統(tǒng)計學(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，爆震傷所致受傷人數(shù)逐年上升，成為軍事戰(zhàn)爭與日常生活中最常見的暴力致傷原因[2-3]。肺爆震傷是爆炸現(xiàn)場最常見的致命傷[4]，其過程中有多種發(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，本研究通過建立肺爆震傷大鼠模型，檢測不同時間點大鼠肺組織中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)報道如下。

1 材料與方法

1.1 實驗動物 選取18只成年健康雄性SD大鼠，體質(zhì)量(200±10)g，由沈陽軍區(qū)總醫(yī)院動物實驗中心提供。動物自由飲食、飲水(江蘇省協(xié)同醫(yī)藥生物工程有限責(zé)任公司)，于清潔級動物房適應(yīng)性喂養(yǎng)1周。18只大鼠分別納入對照組與爆震傷后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Abcam公司)；Western-blot一抗稀釋液(中國碧云天生物技術(shù)研究所)；JNK、P38、Bad、Bcl-xl及β-actin 引物(中國Sangon公司)。

1.3 研究方法

1.3.1 動物模型建立 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，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，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。

1.3.5 Real Time PCR Trizol試劑提取大鼠肺組織總RNA。濃度測定后，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)，計算。

2 結(jié)果

2.1 TUNEL檢測爆震傷后肺組織的凋亡情況 與對照組比較，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)計學(xué)意義(P<0.05)。見圖1。

2.2 免疫熒光檢測JNK與P38的表達(dá)情況 與對照組比較，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)計學(xué)意義(P<0.05)。見圖2。

2.3 Western-blot檢測JNK、P38、Bcl-xl與Bad的蛋白表達(dá)情況 與對照組比較，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)計學(xué)意義(P<0.05)。而Bcl-xl在爆震傷后24 h蛋白表達(dá)低于對照組，7 d組表達(dá)高于24 h組，差異有統(tǒng)計學(xué)意義(P<0.05)。見圖3。

圖1 TUNEL檢測爆震傷后肺組織的凋亡情況(400倍)

圖2 免疫熒光檢測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JNK、P38、Bcl-xl與Bad的蛋白表達(dá)情況(與對照組比較，①P<0.05；與24 h組比較，②P<0.05)

2.4 Real Time PCR 檢測JNK、P38、Bcl-xl與Bad的mRNA表達(dá)情況 與對照組比較，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)計學(xué)意義(P<0.05)。而Bcl-xl在爆震傷后24 h mRNA表達(dá)低于對照組，7 d組表達(dá)高于24 h組，差異有統(tǒng)計學(xué)意義(P<0.05)。見圖4。

3 討論

絲裂原活化蛋白激酶家族參與細(xì)胞中各種重要信號轉(zhuǎn)導(dǎo)途徑的調(diào)節(jié)過程，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Bad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，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，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，且與時間具有相關(guān)性。

圖4 Real Time PCR檢測JNK、P38、Bcl-xl與Bad的mRNA表達(dá)情況(與對照組比較，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，且與時間具有相關(guān)性。因此，JNK與P38對肺爆震傷的診治與預(yù)后具有一定意義，以其為靶點或通過干預(yù)其信號傳導(dǎo)與表達(dá)研發(fā)藥物可能會成為肺爆震傷的有效治療方法之一。

[1] Born CT.Blast trauma:the fourth weapon of mass destruction[J].SJS,2005,94(4):279-285.

[2] Mirza FH,Parhyar HA,Tirmizi SZ.Rising threat of terrorist bomb blasts in karachi-a 5-year study[J].J Forensic Leg Med,2013,20(6):747-751.

[3] Zafar H,Jawad A,Shamim MS,et al.Terrorist bombings:medical response in a developing country[J].JPMA,2011,61(6):561-566.

[4] Wolf SJ,Bebarta VS,Bonnett CJ,et al.Blast injuries[J].Lancet,2009,374(9687):405-415.

[5] Mazzeo AT,Beat A,Singh A,et al.The role of mitochondrial transition pore,and its modulation,in traumatic brain injury and delayed neurodegeneration after TBI[J].Exp Neurol,2009,218(2):363-370.

[6] Finnie JW.Neuroinflammation:beneficial and detrimental effects after traumatic brain injury[J].Inflammopharmacology,2013,21(4):309-320.

[7] Kim JY,Huh KH,Park YJ,et al.Molecular mechanisms of cell death of mycophenolic acid-treated primary isolated rat islets:implication of mitogen-activated protein kinase activation[J].Transplant Proc,2008,40(8):2575-2577.

[8] Morrison DK.Map kinase pathways[J].Cold Spring Harb Perspect Biol,2012,4(11):a011254.

[9] Verma G,Datta M.The critical role of jnk in the er-mitochondrial crosstalk during apoptotic cell death[J].J Cell Physiol,2012,227(5):1791-1795.

[10] Shaukat Z,Liu D,Hussain R,et al.The role of jnk signalling in responses to oxidative DNA damage[J].Curr Drug Targets,2016,17(2):154-163.

[11] Sui X,Kong N,Ye L,et al.P38 and jnk mapk pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents[J].Cancer Lett,2014,344(2):174-179.

[12] Li YH,Zhang CH,Qiu J,et al.Antidepressant-like effects of chaihu-shugan-san via SAPK/JNK signal transduction in rat models of depression[J].Pharmacogn Mag,2014,10(39):271-277.

[13] Weston CR,Davis RJ.The JNK signal transduction pathway[J].Curr Opin Cell Biol,2007,19(2):142-149.

[14] Mishra DR,Chaudhary S,Krishna BM,et al.Identification of critical elements for regulation of inorganic pyrophosphatase(ppa1)in mcf7 breast cancer cells[J].PloS one,2015,10(4):e0124864.

[15] Pang YW,Sun YQ,Sun WJ,et al.Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos[J].J Pineal Res,2016,60(2):155-166.

[16] Dinh CT,Goncalves S,Bas E,et al.Molecular regulation of auditory hair cell death and approaches to protect sensory receptor cells and/or stimulate repair following acoustic trauma[J].Front Cell Neurosci,2015,9:96.

[17] Kuwana T,Mackey MR,Perkins G,et al.Bid,bax,and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane[J].Cell,2002,111(3):331-342.

[18] Mangano EN,Litteljohn D,So R,et al.Interferon-gamma plays a role in paraquat-induced neurodegeneration involving oxidative and proinflammatory pathways[J].Neurobiol Aging,2012,33(7):1411-1426.

[19] Jeong HS,Choi HY,Choi TW,et al.Differential regulation of the antiapoptotic action of b-cell lymphoma 2(bcl-2)and b-cell lymphoma extra long(bcl-xl)by c-jun n-terminal protein kinase(jnk)1-involved pathway in neuroglioma cells[J].Biol Pharm Bull,2008,31(9):1686-1690.

[20] Chu R,Upreti M,Ding WX,et al.Regulation of bax by c-jun nh2-terminal kinase and bcl-xl in vinblastine-induced apoptosis[J].Biochem Pharmacol,2009,78(3):241-248.

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

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

叢培芳(1987-)，女，遼寧沈陽人，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