高遷移率族蛋白-1、糖基化終產(chǎn)物受體及Toll樣受體-4通路對(duì)爆震傷致大鼠急性肺損傷影響研究

佟昌慈， 柳云恩， 張玉彪， 施 琳， 叢培芳， 劉 穎， 史秀云， 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(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 110016

·爆震傷·

高遷移率族蛋白-1、糖基化終產(chǎn)物受體及Toll樣受體-4通路對(duì)爆震傷致大鼠急性肺損傷影響研究

佟昌慈， 柳云恩， 張玉彪， 施 琳， 叢培芳， 劉 穎， 史秀云， 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(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 110016

目的 探討高遷移率族蛋白(HMGB)-1、糖基化終產(chǎn)物受體(RAGE)及Toll樣受體(TLR)-4通路對(duì)爆震傷致大鼠急性肺損傷的調(diào)控機(jī)制。方法 選取40只SD大鼠，10只納入對(duì)照組；其余30只在建立爆震傷致大鼠急性肺損傷模型后，分別納入6 h組、12 h組、24 h組。ELISA檢測(cè)大鼠血清炎癥因子HMGB-1、白細(xì)胞介素(IL)-1α、IL-8；Real Time PCR、Western Blot及免疫熒光檢測(cè)大鼠肺組織炎癥因子HMGB-1、IL-1α、IL-8及通路相關(guān)蛋白R(shí)AGE、TLR-4、p38絲裂原活化蛋白激酶(MAPK)、兩面神激酶(JAK)-2、信號(hào)傳導(dǎo)及轉(zhuǎn)錄激活因子(STAT)-3。結(jié)果 ELISA結(jié)果顯示，與對(duì)照組比較，12 h組、24 h組HMGB-1與IL-1α表達(dá)增高，12 h組表達(dá)量最高；6 h組、12 h組IL-8表達(dá)增高，12 h組表達(dá)量最高(P<0.05)。Real Time PCR、Western Blot及免疫熒光結(jié)果顯示，與對(duì)照組比較，12 h組、24 h組HMGB-1與IL-1α表達(dá)增高，12 h組表達(dá)量最高；6 h組、12 h組IL-8表達(dá)增高，12 h組表達(dá)量最高；12 h組RAGE表達(dá)增高；12 h組、24 h組TLR-4、p38 MAPK表達(dá)增高，12 h組表達(dá)量最高；6 h組、12 h組、24 h組JAK-2、STAT-3表達(dá)增高，12 h組表達(dá)量最高(P<0.05)。結(jié)論 爆震傷致大鼠急性肺損傷誘導(dǎo)HMGB-1釋放，結(jié)合RAGE與TLR-4受體，激活p38 MAPK與JAK/STAT通路，促進(jìn)炎癥因子IL-1α、IL-8的作用，調(diào)節(jié)炎癥反應(yīng)。

爆震傷； 急性肺損傷； 大鼠； 高遷移率族蛋白； 糖基化終產(chǎn)物受體； Toll樣受體

隨著現(xiàn)代局部軍事沖突的增加，高能武器的大量使用，恐怖事件、瓦斯與地鐵內(nèi)爆炸等事故的發(fā)生率上升，爆震傷已經(jīng)成為青壯年死亡的重要原因，造成了嚴(yán)重的生命損害，消耗了大量的醫(yī)療資源。爆震傷是一種復(fù)雜的物理損傷，沖擊波直接或間接作用機(jī)體并釋放出能量造成損傷，常累及多個(gè)重要器官，具有傷情復(fù)雜、死亡率較高、臨床傷情變化較快、休克率高等特點(diǎn)[1-3]。因此，對(duì)爆震傷的合理救治不僅是當(dāng)前醫(yī)學(xué)研究的熱點(diǎn)，更是社會(huì)安全穩(wěn)定發(fā)展的迫切需要。肺是爆震傷的主要累及器官，也是導(dǎo)致傷員死亡最常見的致命傷之一。爆炸形成的沖擊波作用于機(jī)體，引起胸廓變形，胸腔內(nèi)壓增加，并通過肺實(shí)質(zhì)傳播，導(dǎo)致即刻或遲發(fā)性的大出血與肺泡、肺毛細(xì)血管破裂，出現(xiàn)肺部出血、肺組織挫傷及肺水腫等[3-5]。隨著病情的惡化，會(huì)發(fā)展為急性肺損傷(acute lung injury，ALI)、急性呼吸窘迫綜合征(acute respiratory distress syndrome，ARDS)及多器官功能障礙綜合征(multiple organ dysfunction syndrome，MODS)[6-7]。有研究表明，ALI常伴隨炎癥反應(yīng)、活性氧釋放、谷氨酸毒性及線粒體功能障礙等[8-9]。目前，肺爆震傷患者只能接受常規(guī)抗炎與機(jī)械輔助治療[10]，無針對(duì)性治療與保護(hù)類藥物，死亡率居高不下。因此，研究爆震傷致ALI的發(fā)病機(jī)制與救治方法具有重要意義。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物 選取沈陽軍區(qū)總醫(yī)院實(shí)驗(yàn)動(dòng)物科的健康清潔級(jí)SD大鼠40只，體質(zhì)量(200±22)g，隨機(jī)分成4組(對(duì)照組，6 h組、12 h組、24 h組)，每組各10只。清潔級(jí)動(dòng)物房常規(guī)飼養(yǎng)，適應(yīng)性喂養(yǎng)3 d。

1.2 實(shí)驗(yàn)試劑 ELISA、Trizol reagent、SYBR Green PCR試劑盒、逆轉(zhuǎn)錄試劑盒均購自日本TAKALA公司，蛋白提取試劑盒、BCA蛋白定量試劑盒均購自南京Vazyme公司，ECL顯色試劑盒購自美國(guó)BIO-RAD公司，二抗、高遷移率族蛋白(high mobility group box protein，HMGB)-1抗體、糖基化終產(chǎn)物受體(receptor for advanced glycation end-products，RAGE)抗體、Toll樣受體(toll-like family of receptors，TLR)-4抗體、兩面神激酶(Janus kinase，JAK)-2抗體、信號(hào)傳導(dǎo)及轉(zhuǎn)錄激活因子(signal transducers and activators of transcription，STAT)-3抗體均購自英國(guó)Abcam公司，白細(xì)胞介素(interleukin，IL)-1α抗體、IL-8抗體、p38絲裂原活化蛋白激酶(mitogen-activated protein kinases，MAPK)抗體、β-actin抗體均購自美國(guó)Santa公司。

1.3 動(dòng)物模型建立 采用自主設(shè)計(jì)研發(fā)的高仿真爆震傷模擬裝置，建立爆震傷致大鼠ALI模型。裝置設(shè)計(jì)如下：下方為空氣壓縮裝置，長(zhǎng)度約100.0 cm，周徑約30.0 cm，將16層厚度約0.8 μm的鋁薄置于中間層。通過空氣壓縮裝置壓縮空氣，當(dāng)達(dá)到一定壓力時(shí)，鋁膜破裂產(chǎn)生沖擊波。大鼠稱質(zhì)量后進(jìn)行麻醉，將麻醉后的大鼠置于保護(hù)罩內(nèi)，保護(hù)大鼠其他部位，只顯露胸部，隨后將大鼠固定于裝置的網(wǎng)狀部位。通電后記錄鋁膜爆破的時(shí)間、下方空氣壓縮裝置內(nèi)壓力及上方壓力傳感器所記錄的超壓波壓力。

1.4 ELISA 取各組大鼠血液，分離血清后，通過ELISA試劑盒檢測(cè)HMGB-1、IL-1α、IL-8的濃度。具體步驟見試劑盒說明書。

1.5 Real Time PCR Trizol reagent提取大鼠肺組織總RNA。取4.0 μg總RNA，65℃變性5 min，逆轉(zhuǎn)錄酶42℃逆轉(zhuǎn)錄30 min，合成cDNA。采用SYBR熒光染料，Smart Cycler System軟件進(jìn)行分析。反應(yīng)體系如下：cDNA 2.0 μl，ddH2O 6.4 μl，上下游引物各0.8 μl，SYBR Premix Ex TaqTMⅡ 10.0 μl。擴(kuò)增條件：56℃退火30 s，72℃延伸30 s，45個(gè)循環(huán)。根據(jù)GenBank HMGB-1、IL-1α、IL-8、RAGE、TLR-4、p38 MAPK、JAK-2、STAT-3及β-actin cDNA序列，Primer 5引物軟件設(shè)計(jì)特異性引物(表1)。

1.6 Western Blot 取大鼠肺組織，加入蛋白裂解液，勻漿機(jī)制成勻漿，12 000 r/min離心5 min，吸取上清蛋白液，BCA蛋白定量試劑盒與酶標(biāo)儀測(cè)定蛋白濃度。實(shí)驗(yàn)蛋白樣品配平后，加入相應(yīng)比例的6×Loading Buffer，煮沸變性5 min，SDS-PAGE電泳后，轉(zhuǎn)膜，5%脫脂奶粉PBST緩沖液室溫封閉2 h，PBST洗膜3次。加入一抗HMGB-1、IL-1α、IL-8、RAGE、TLR-4、p38 MAPK、JAK-2、STAT-3及β-actin 4℃孵育過夜。PBST洗膜3次，每次10 min，辣根過氧化物酶標(biāo)記二抗，室溫孵育1.5 h。洗膜3次，ECL顯影。1.7 免疫熒光 石蠟切片脫蠟與水化，3% H2O2室溫孵育10 min以消除內(nèi)源性過氧化物酶的活性。浸泡3次，每次5 min?？乖迯?fù)：高壓鍋中加入0.01 mol/L檸檬酸鈉緩沖液，放入組織芯片，加熱，保壓100 s后，冷卻至室溫。PBS浸泡2次，每次5 min。正常山羊血清封閉，室溫孵育30 min，倒去血清，滴加一抗工作液，4℃過夜。PBS浸泡3次，每次5 min，滴加適量熒光二抗，室溫孵育1 h，PBS浸泡3次，每次5 min。滴加適量的DAPI，室溫孵育10 min，PBS浸泡3次，每次5 min，封片鏡檢。

2 結(jié)果

2.1 ELISA檢測(cè)大鼠血清炎癥因子HMGB-1、IL-1α、IL-8 與對(duì)照組比較，12 h組、24 h組HMGB-1與IL-1α表達(dá)增高，12 h組表達(dá)量最高；6 h組、12 h組IL-8表達(dá)增高，12 h組表達(dá)量最高(P<0.05)。見表2。

表2 ELISA檢測(cè)大鼠血清炎癥因子

注：與對(duì)照組比較，①P<0.05

2.2 Real Time PCR、Western Blot及免疫熒光檢測(cè)大鼠肺組織炎癥因子HMGB-1、IL-1α、IL-8 與對(duì)照組比較，12 h組、24 h組HMGB-1與IL-1α表達(dá)增高，12 h組表達(dá)量最高；6 h組、12 h組IL-8表達(dá)增高，12 h組表達(dá)量最高(P<0.05)。見圖1、2。

2.3 Real Time PCR、Western Blot及免疫熒光檢測(cè)通路相關(guān)蛋白 RAGE、TLR-4、p38 MAPK、JAK-2、STAT-3與對(duì)照組比較，12 h組RAGE表達(dá)增高；12 h組、24 h組TLR-4、p38 MAPK表達(dá)增高，12 h組表達(dá)量最高；6 h組、12 h組、24 h組JAK-2、STAT-3表達(dá)增高，12 h組表達(dá)量最高(P<0.05)。見圖3、4。

3 討論

爆震傷致ALI患者病情嚴(yán)重、救治困難，易早期死亡[11]。肺組織的原發(fā)性損傷是由爆炸時(shí)瞬間產(chǎn)生的沖擊波與高能碎片沖擊所致，之后，肺組織細(xì)胞的損傷壞死誘導(dǎo)內(nèi)源性炎癥介質(zhì)的表達(dá)與釋放，引起繼發(fā)性損傷[12]。

HMGB-1是一種非組蛋白染色體結(jié)合蛋白，當(dāng)機(jī)體處于穩(wěn)態(tài)、無外界刺激時(shí)，HMGB-1主要存在于細(xì)胞核中[13-15]；當(dāng)外界信號(hào)刺激細(xì)胞時(shí)，細(xì)胞應(yīng)激，HMGB-1賴氨酸殘基被乙酰化后，釋放到細(xì)胞外，誘導(dǎo)局部組織或全身性炎癥反應(yīng)，細(xì)胞破損或壞死亦可導(dǎo)致HMGB-1的釋放增加[16-17]。HMGB-1可刺激巨噬細(xì)胞、中性粒細(xì)胞及單核細(xì)胞，使腫瘤壞死因子、IL-1α、IL -1β、IL -6、IL-8、巨噬細(xì)胞炎性蛋白-1α等分泌量增加[16，18-22]。HMGB-1激活炎癥反應(yīng)主要通過結(jié)合晚期RAGE實(shí)現(xiàn)[23]。RAGE是廣泛存在于不同細(xì)胞表面的免疫球蛋白超家族跨膜蛋白，在正常組織細(xì)胞中的表達(dá)水平很低，其配體聚集時(shí)可誘導(dǎo)其表達(dá)增加[24-25]。HMGB-1與RAGE受體結(jié)合后，激活JAK/STAT信號(hào)轉(zhuǎn)導(dǎo)通路，活化NF-κB，促進(jìn)炎癥因子表達(dá)，反饋調(diào)節(jié)HMGB-1。另外，HMGB-1可激活p38 MAPK通路，調(diào)節(jié)炎癥反應(yīng)[26-27]。本研究結(jié)果中，爆震傷致大鼠ALI誘導(dǎo)了HMGB-1的釋放，促進(jìn)了RAGE、p38 MAPK、JAK-2、STAT-3的表達(dá)。這說明，爆震傷致ALI引起的細(xì)胞應(yīng)激與損傷壞死可促進(jìn)HMGB-1釋放，增加HMGB-1與RAGE受體結(jié)合，激活p38 MAPK與JAK/STAT通路，促進(jìn)IL-1、IL-8等炎癥因子釋放，誘導(dǎo)炎癥反應(yīng)。

圖1 Real Time PCR與Western Blot檢測(cè)大鼠肺組織炎癥因子HMGB-1、IL-1α、IL-8(與對(duì)照組比較，①P<0.05)

圖2 免疫熒光檢測(cè)大鼠肺組織炎癥因子HMGB-1(400倍)

圖3 Real Time PCR與Western Blot檢測(cè)通路相關(guān)蛋白R(shí)AGE、TLR-4、p38 MAPK、JAK-2、STAT-3(與對(duì)照組比較，①P<0.05)

圖4 免疫熒光檢測(cè)通路相關(guān)蛋白R(shí)AGE、TLR-4(400倍)

研究發(fā)現(xiàn)，通過阻斷抗體或基因沉默敲除RAGE并不能完全抑制HMGB-1誘導(dǎo)的炎癥反應(yīng)，Toll樣受體家族中的TLR-2與TLR-4受體也可與HMGB-1結(jié)合，活化NF-κB，誘導(dǎo)炎癥反應(yīng)發(fā)生[28-29]。HMGB-1介導(dǎo)的TLR-4可激活I(lǐng)KK-α與IKK-β，而RAGE僅可激活I(lǐng)KK-β[30-31]。TLR-4還可激活p38 MAPK通路[32-33]。本研究結(jié)果中，爆震傷致大鼠ALI誘導(dǎo)了HMGB-1的釋放，促進(jìn)了TLR-4、p38 MAPK的表達(dá)。這說明，爆震傷致ALI引起的細(xì)胞應(yīng)激與損傷壞死可促進(jìn)HMGB-1釋放，除結(jié)合RAGE受體外，還可結(jié)合TLR-4受體并激活p38 MAPK通路，促進(jìn)IL-1、IL-8等炎癥因子釋放，誘導(dǎo)炎癥反應(yīng)。

綜上所述，爆震傷致大鼠急性肺損傷誘導(dǎo)HMGB-1釋放，結(jié)合RAGE與TLR-4受體，激活p38 MAPK與JAK/STAT通路，促進(jìn)炎癥因子IL-1α、IL-8作用，調(diào)節(jié)炎癥反應(yīng)。

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Effects of HMGB-1,RAGE and TLR-4 pathway on acute lung injury induced by blast in rats

TONG Chang-ci,LIU Yun-en,ZHANG Yu-biao,SHI Lin,CONG Pei-fang,LIU Ying,SHI Xiu-yun,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 To investigate the mechanism of high mobility group protein-1(HMGB-1),receptor for advanced glycation end products(RAGE)and Toll-like receptor-4(TLR-4)pathway on acute lung injury induced by blast in rats.Methods There were 40 SD rats were selected,10 rats were divided into the control group;the other 30 rats were divided into the 6 hours group,12 hours group and 24 hours group after the establishment of rat model with acute lung injury induced by blast.The levels of serum HMGB-1,IL-1α and IL-8 were detected by ELISA.The expression of HMGB-1,IL-1α,IL-8,RAGE,TLR-4,p38 MAPK,JAK-2 and STAT-3 in lung tissue were detected by Western Blot,Real Time PCR and immunofluorescence.Results The result of ELISA showed that,compared with the control group,the expression of HMGB-1 and IL-1α in 12 hours group and 24 hours group both increased,and the level reached the highest in 12 hours group;the expression of IL-8 in 6 hours group and 12 hours group increased,and the level reached the higher in 12 hours group(P<0.05).The results of Western Blot,Real Time PCR and immunofluorescence showed that,compared with the control group,the expression of HMGB-1 and IL-1αin 12 hours group and 24 hours group increased,and the level reached the highest in 12 hours group;the expression of IL-8 in 6 hours and 12 hours increased,and the level reached the highest in 12 hours group;the expression of RAGE in 12 hours group increased;the expression of TLR-4 and p38 MAPK in 12 hours group and 24 hours group increased,and the level reached the highest in 12 hours group;the expression of JAK-2 and STAT-3 in 6 hours,12 hours and 24 hours groups increased,and the level reached the highest in 12 hours group(P<0.05).Conclusion The acute lung injury induced by blast injury in rats can promote the release of inflammatory factors and promote the inflammatory response by promoting the release of HMGB-1,binding to RAGE and TLR-4,activating p38 MAPK and JAK/STAT pathway.

Blast injury; Acute lung injury; Rats; High mobility group protein; Receptor for advanced glycation end products; Toll-like receptor

佟昌慈(1988-)，女，遼寧撫順人，技士，碩士

侯明曉，E-mail：houmingxiao188@163.com

2095-5561(2017)04-0198-07 DOI∶10.16048/j.issn.2095-5561.2017.04.02

2017-07-17