王大龍，冷玉芳

(1.山東省勝利油田中心醫(yī)院麻醉科，東營(yíng) 257000;2.蘭州大學(xué)第一醫(yī)院麻醉科，蘭州 730000)

二硫代氨基吡咯烷對(duì)大鼠急性肺損傷保護(hù)作用

王大龍1，冷玉芳2

(1.山東省勝利油田中心醫(yī)院麻醉科，東營(yíng) 257000;2.蘭州大學(xué)第一醫(yī)院麻醉科，蘭州 730000)

目的 探討二硫代氨基吡咯烷(PDTC)對(duì)內(nèi)毒素(LPS)誘導(dǎo)急性肺損傷(ALI)大鼠肺組織的保護(hù)作用及其機(jī)制。方法將72只Wistar大鼠隨機(jī)分為對(duì)照組(C組),LPS組(L組),PDTC+LPS組(P組)。各組分為1,3,6,12 h亞組,每組6只。L組和P組制作內(nèi)毒素性肺損傷模型。P組腹腔注射LPS前30 min尾靜脈注射PDTC,120 mg·kg-1,L組和C組給予等體積0.9%氯化鈉溶液。觀察大鼠肺組織病理變化,肺組織濕/干質(zhì)量比(W/D)變化,RT-PCR法檢測(cè)大鼠肺組織細(xì)胞間黏附分子-1 mRNA(ICAM-1mRNA)的表達(dá),放射免疫法檢測(cè)大鼠血清腫瘤壞死因子α(TNF-α)、白細(xì)胞介素6(IL-6)含量變化。結(jié)果病理切片顯示,與C組比較,L組肺組織結(jié)構(gòu)破壞嚴(yán)重,W/D明顯增大,ICAM-1mRNA在各時(shí)間點(diǎn)表達(dá)明顯增強(qiáng)(P＜0.05),血清TNF-α、IL-6含量在1,3,6 h增高(P＜0.05)。與L組比較,P組肺組織結(jié)構(gòu)破壞明顯減輕,W/D減小(P＜0.05),ICAM-1mRNA表達(dá)明顯減少(P＜0.05),血清TNF-α、IL-6含量1,3,6 h降低(P＜0.05或P＜0.01)。結(jié)論P(yáng)DTC能明顯抑制ICAM-1mRNA表達(dá),降低TNF-α和IL-6含量,PDTC可能對(duì)ALI大鼠有保護(hù)作用。

二硫代氨基吡咯烷；核因子-κB；內(nèi)毒素；損傷,肺,急性

急性肺損傷(acute lung injury,ALI)/急性呼吸窘迫綜合征(acute respiratory distress syndrome,ARDS)是由嚴(yán)重感染、創(chuàng)傷和休克等多種病因所致的彌漫性肺實(shí)質(zhì)損傷,其在分子水平上表現(xiàn)為眾多促炎性細(xì)胞因子、粘附分子的過(guò)度表達(dá),伴有多種炎癥遞質(zhì)的產(chǎn)生。炎癥遞質(zhì)的釋放導(dǎo)致肺組織廣泛損害,并累及諸多肺外器官[1]。研究證明,抗氧化藥二硫代氨基吡咯烷(pyrrolidine dithiocarbamate,PDTC)能夠通過(guò)抑制重要的核因子-κB(nuclear factor-kappa B,NF-κB)活化,減少致炎細(xì)胞因子表達(dá)與合成釋放[2-3]。筆者在本實(shí)驗(yàn)中建立大鼠內(nèi)毒素(lipopolysaccharides,LPS)性肺損傷模型,研究PDTC對(duì)LPS誘導(dǎo)ALI大鼠肺組織細(xì)胞間黏附分子1(intercellular adhesion molecule-1mRNA, ICAM-1mRNA)和血清腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、白細(xì)胞介素6(interleukin-6,IL-6)含量的影響,以探討PDTC肺損傷的保護(hù)作用及其機(jī)制。

1 材料與方法

1.1 動(dòng)物與試劑 雄性Wistar大鼠(由蘭州大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供)72只,LPS(EColi055:B5)和PDTC購(gòu)自sigma公司(批號(hào):P8765,純度＞99%),核酸提取試劑購(gòu)自百泰克,RNA試劑盒購(gòu)自Fermentas公司。

1.2 造模與給藥方法 72只Wistar大鼠,體質(zhì)量200～250 g,隨機(jī)分為3組:對(duì)照組(C組),LPS組(L組),PDTC+LPS組(P組)。根據(jù)腹腔注射LPS到取肺組織的時(shí)間分為1,3,6,12 h亞組,每組6只。L組和P組腹腔注射LPS8 mg·kg-1制作內(nèi)毒素性肺損傷模型,C組腹腔注射同體積0.9%氯化鈉溶液。P組在腹腔注射LPS前30 min,尾靜脈注射PDTC120 mg·kg-1(稀釋到60 mg·mL-1),L組和C組給予同體積0.9%氯化鈉溶液。按分組規(guī)定的時(shí)間點(diǎn),10%水合氯醛350 mg·kg-1腹腔注射麻醉,開胸心臟放血處死,采集標(biāo)本。

1.3 觀察指標(biāo)

1.3.1 肺組織病理檢查 取右肺上葉,4%多聚甲醛固定,石蠟包埋,蘇木精-伊紅(hematoxylin-eosinstaining,HE)染色后光鏡下觀察肺組織形態(tài)學(xué)變化。

1.3.2 肺組織濕/干重比值(wet/dry ratio of lung,W/D)監(jiān)測(cè) 取左肺,濾紙拭去表面血液,稱濕重后置于80℃烤箱烘烤48 h,使肺組織達(dá)恒重后稱干重,計(jì)算W/D值。

1.3.3 ICAM-1mRNA監(jiān)測(cè) 用PCR專用器械迅速取右肺下葉,經(jīng)預(yù)冷的焦碳酸二乙酯(diethypyrocarbonate,DEPC)水沖洗表面的血液后立即置于液氮中速凍,-80℃冰箱保存,待測(cè)逆轉(zhuǎn)錄PCR(reverse transcription-PCR,RT-PCR)。用RNA提取試劑Trizol提取肺組織總RNA,反轉(zhuǎn)錄合成cDNA第一條鏈。取反轉(zhuǎn)錄產(chǎn)物2 μL進(jìn)行PCR反應(yīng)(94℃變性5 min,59℃復(fù)性30 s,72℃延伸1 min,35個(gè)循環(huán))。擴(kuò)增組織中ICAM-1的引物為:5′-AGGTATCCATCCATCCCACA-3′和5′-AGTGTCTCATTCCCACGGAG-3′,片段長(zhǎng)度為388 bp;擴(kuò)增內(nèi)對(duì)照β-actin的引物為:β-actin引物5′-GTGGGCCGCTGTAGGCACCAA-3′和5′-CTCTTTGATGTCGCACGATTTC-3′,片段長(zhǎng)度為540 bp。由上海賽百盛有限公司合成。PCR擴(kuò)增產(chǎn)物用1%瓊脂糖凝膠電泳,溴化乙錠染色顯示,凝膠圖像分析系統(tǒng)分析,以ICAM-1mRNA/β-actinmRNA電泳帶的熒光強(qiáng)度比值作為ICAM-1mRNA的表達(dá)量指標(biāo)。

1.3.4 TNF-α、IL-6含量監(jiān)測(cè) 取血液37℃水浴0.5 h后離心(2 500×g,10 min),取上層血清-70℃冰箱保存?zhèn)溆?。放免法測(cè)定TNF-α、IL-6含量。

1.4 統(tǒng)計(jì)學(xué)方法 用SPSS 18.0版統(tǒng)計(jì)軟件分析,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(±s)表示,組內(nèi)及組間比較采用t檢驗(yàn),以P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 兩組形態(tài)學(xué)改變比較 光鏡下可見C組肺泡結(jié)構(gòu)完整,肺泡壁無(wú)水腫,無(wú)炎癥細(xì)胞浸潤(rùn);L組基本看不到完整肺泡結(jié)構(gòu),肺間質(zhì)及肺泡毛細(xì)血管明顯擴(kuò)張、片狀出血,支氣管壁、肺泡間隔及毛細(xì)血管周圍有大量炎癥細(xì)胞浸潤(rùn);P組肺泡結(jié)構(gòu)輕微破壞,可見代償增大的肺泡,無(wú)明顯的水腫、淤血,炎癥細(xì)胞浸潤(rùn)較L組少。

2.2 兩組W/D值比較 與C組比較,P組和L組W/D值在模型建立后1,3,6,12 h均明顯升高(P＜0.05);與L組比較,P組W/D值在相同時(shí)間點(diǎn)均明顯降低(P＜0.05),見表1。

2.3 兩組肺組織ICAM-1mRNA表達(dá) 與C組比較,L組和P組模型建立后相同時(shí)間點(diǎn)ICAM-1mRNA表達(dá)量均顯著增加(P＜0.05),12 h達(dá)到高峰;與L組相比, P組在各時(shí)間點(diǎn)ICAM-1mRNA表達(dá)量顯著降低(P＜0.05)。見表2。

2.4 兩組血清TNF-α、IL-6含量 與C組相比,L組和P組血清TNF-α水平在1,3,6 h均顯著升高(P＜0.01),1 h達(dá)高峰,隨著時(shí)間延長(zhǎng)而降低;與L組比較,P組在1,3,6 h TNF-α表達(dá)量均顯著低于L組(P＜0.05或P＜0.01)。與C組比較,L組和P組血清IL-6水平在1,3,6 h均顯著升高(P＜0.01),3 h達(dá)高峰;與L組比較,P組1,3 h IL-6表達(dá)量均顯著低于L組(P＜0.05),見表3。

表1 3組大鼠肺組織W/D比值Tab.1 W/D ratio in lung tissue of three groups of rats ±s

表1 3組大鼠肺組織W/D比值Tab.1 W/D ratio in lung tissue of three groups of rats ±s

與C組比較,*1P＜0.05;與L組比較,*2P＜0.05Compared with group C,*1P＜0.05;Compared with group L,*2P＜0.05

組別例數(shù)1 h3 h6 h12 h C組64.27±0.164.26±0.114.25±0.204.26±0.23 L組64.61±0.08*14.83±0.12*15.22±0.14*15.75±0.27*1P組64.36±0.10*1*24.56±0.24*1*24.83±0.16*1*25.24±0.38*1*2

表2 3組大鼠肺組織ICAM-mRNA表達(dá)量Tab.2 ICAM-mRNA expression in lung tissue of three groups of rats ±s

表2 3組大鼠肺組織ICAM-mRNA表達(dá)量Tab.2 ICAM-mRNA expression in lung tissue of three groups of rats ±s

與C組比較,*1P＜0.05;與L組比較,*2P＜0.05Compared with group C,*1P＜0.05;Compared with group L,*2P＜0.05

組別例數(shù)1 h3 h6 h12 h C組60.400±0.0180.410±0.0260.390±0.0190.410±0.020 L組60.720±0.028*10.920±0.023*11.020±0.054*11.120±0.039*1P組60.520±0.023*1*20.650±0.037*1*20.730±0.032*1*20.810±0.020*1*2

表3 3組大鼠血清TNF-α與IL-6含量Tab.3 Serum content of TNF-α and IL-6 in three groups of rats ±s

表3 3組大鼠血清TNF-α與IL-6含量Tab.3 Serum content of TNF-α and IL-6 in three groups of rats ±s

與C組比較,*1P＜0.01;與L組比較,*2P＜0.01,*3P＜0.05Compared with group C,*1P＜0.01;compared with group L,*2P＜0.01,*3P＜0.05

組別與時(shí)間例數(shù)TNF-α/(ng·mL-1) IL-6/(pg·mL-1) C組6 1 h1.12±0.24108.22±23.84 3 h1.12±0.30107.27±30.48 6 h1.14±0.28106.44±25.30 12 h1.13±0.27107.36±24.92 L組6 1 h6.62±1.12*1252.68±32.22*13 h4.27±0.48*1316.61±36.08*16 h2.43±0.43*1184.52±29.93*112 h1.26±0.28137.48±25.35 P組6 1 h4.48±0.85*1*2196.59±28.37*1*33 h2.50±0.68*1*2230.88±33.41*1*36 h1.75±0.30*1*3157.20±22.98*112 h1.19±0.32124.58±21.88

3 討論

腹腔注射LPS是建立大鼠ALI模型的經(jīng)典方法,本實(shí)驗(yàn)中大鼠注射LPS 1 h后,呼吸加快,四肢、口唇紫紺,個(gè)別大鼠發(fā)出喘鳴音,口鼻內(nèi)涌出粉紅色泡沫,拒食,豎毛,輕度腹瀉,W/D增加。光鏡下肺泡結(jié)構(gòu)基本消失,肺間質(zhì)及肺泡毛細(xì)血管明顯擴(kuò)張、片狀出血,支氣管壁、肺泡間隔及毛細(xì)血管周圍有大量炎癥細(xì)胞浸潤(rùn),說(shuō)明ALI模型建立成功[4]。LPS主要與巨噬細(xì)胞Toll受體結(jié)合,啟動(dòng)胞內(nèi)信號(hào)傳遞鏈,促進(jìn)κIBα亞型的降解并活化NF-κB的DNA結(jié)合能力,啟動(dòng)基因轉(zhuǎn)錄,表達(dá)和釋放多種細(xì)胞因子,發(fā)揮其毒性作用[5-7]。

本實(shí)驗(yàn)中L組在注射內(nèi)毒素后,肺組織ICAM-1mRNA,血清中TNF-α、IL-6含量均有增高(P＜0.05或P＜0.01),提示ICAM-1、TNF-α和IL-6作為炎癥因子在LPS誘導(dǎo)的ALI發(fā)病過(guò)程中起重要作用。ICAM-1通過(guò)介導(dǎo)細(xì)胞與細(xì)胞間,細(xì)胞與細(xì)胞外基質(zhì)間的粘附作用,使大量的多形核白細(xì)胞(polymorphonuclear, PMN)黏附于肺血管內(nèi)皮細(xì)胞、在肺內(nèi)扣押并釋放生物活性遞質(zhì)[8]。TNF-α能誘導(dǎo)肺內(nèi)上皮細(xì)胞活化、白細(xì)胞遷移和毛細(xì)血管滲漏,積聚的水腫液進(jìn)一步阻礙肺泡內(nèi)細(xì)胞的灌流和氧氣的交換,引起肺損傷。TNF-α的含量改變可反映其他炎癥因子的改變情況[7,9]。IL-6是一種遲發(fā)性細(xì)胞因子,可以誘導(dǎo)活化的B細(xì)胞增殖和分化,激活T細(xì)胞,在誘導(dǎo)和維持炎癥反應(yīng)中起重要作用。在急性炎性反應(yīng)中,IL-6的水平與病情嚴(yán)重程度密切相關(guān)[10]。TNF-α在模型建立后1 h達(dá)到峰值,隨后降低,提示TNF-α可能是導(dǎo)致ALI和ARDS的始動(dòng)因素。IL-6在3 h達(dá)到高峰,可能與炎癥的持續(xù)和進(jìn)一步發(fā)展有關(guān)。

PDTC是一種抗氧化藥,近年來(lái)的研究證明它對(duì)NF-κB的活性有很強(qiáng)的抑制作用,被認(rèn)為是NF-κB的特異性抑制藥,它的金屬鰲合特性和二硫基基團(tuán)清道夫作用能夠選擇性的抑制NF-κB活化[2-3,11]:①直接抑制NF-κBP65亞單位;②抑制NF-κB抑制藥κIB的降解;③減少NF-κB的核移位。ICAM-1、TNF-α和IL-6的基因啟動(dòng)子上都含有NF-κB結(jié)合位點(diǎn),NF-κB的激活,促進(jìn)其表達(dá);它們又可反過(guò)來(lái)進(jìn)一步活化NF-κB,使炎癥不斷放大。在大鼠注射LPS以后活化了NF-κB,促使肺血管內(nèi)皮細(xì)胞上三類基因的表達(dá)明顯上調(diào),促進(jìn)白細(xì)胞粘附、扣押于肺泡區(qū)域,導(dǎo)致肺實(shí)質(zhì)細(xì)胞損傷及持續(xù)細(xì)胞因子釋放,使毛細(xì)血管滲漏,積聚的水腫液進(jìn)一步阻礙肺泡內(nèi)細(xì)胞的灌流和氧氣的交換,引起肺損傷加重肺組織損傷[12-13]。本實(shí)驗(yàn)應(yīng)用PDTC后的結(jié)果顯示,P組肺泡結(jié)構(gòu)破壞輕微,可看到代償增大的肺泡,無(wú)明顯的水腫、淤血,僅有少量炎癥細(xì)胞浸潤(rùn), W/D值增大,同時(shí)ICAM-1mRNA表達(dá)顯著減輕,血清TNF-α、IL-6含量減少。表明PDTC通過(guò)減少肺組織ICAM-1mRNA表達(dá),減輕PMN在肺組織內(nèi)的聚集和激活,減少炎癥因子TNF-α、IL-6的表達(dá)抑制肺損傷時(shí)的炎癥反應(yīng),產(chǎn)生肺保護(hù)作用[14]。但是在本實(shí)驗(yàn)中未對(duì)不同劑量的PDTC進(jìn)行比較,將來(lái)作為進(jìn)一步研究的目標(biāo)。

綜上所述,PDTC預(yù)先給藥對(duì)LPS誘導(dǎo)的大鼠ALI有一定的保護(hù)作用,與減少肺組織ICAM-1mRNA表達(dá),抑制TNF-α、IL-6的表達(dá)有關(guān)。

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DOI 10.3870/yydb.2014.03.009

Protective Effect of PDTC on Acute Lung Injury Induced by LPS in Rats

WANG Da-long1,LENG Yu-fang2
(1.Department of Anesthesiology,Shengli Oilfield Center Hospital,Dongying 257000,China;2.Department of Anesthesiology,the First Hospital of Lanzhou University,Lanzhou 730000, China)

Objective To explore the protective effect of pyrrolidine dithiocarbamate(PDTC)on acute lung injury induced by LPS.MethodsA total of 72 Wistar rats were randomly divided into the control(C),LPS model control(L),and PDTC+LPS(P)treatment groups.Rats in each group were further divided into 1,3,6,12 h subgroups(n=6 each)based on the time between intraperitoneal injection of endotoxin.To produce the endotoxin-induced lung injury model,group L and group P were intraperitoneally injected with LPS 8 mg·kg-1,group C injected with the same volume of normal saline.And group P was injected with PDTC 120 mg·kg-1from the tail vein and half an hour later,LPS was intraperitoneally injected in group P,and the same volume of normal saline was injected to groups C and L.The pathological changes in lung tissue,wet/dry weight(W/D)of lung,ICAM-1mRNA expression in lung and TNF-α and IL-6 in serum were detected.ResultsIn groups P and L,the structure of lungs was seriously injured,the wet/dry weight ratio(W/D)of the lungs was significantly increased,the expression of ICAM-1mRNA was enhanced,and the content of serum TNF-α,IL-6 was elevated.Compared with group L,the injury of lung structure was deceased,lung wet/dry weight ratio(W/D)was significantly declined(P＜0.05),the ICAM-1mRNA expression was alleviated(P＜0.05),and the serum level of TNF-α and IL-6 was lowered(P＜0.05 orP＜0.01)in group P.ConclusionPDTC could inhibit the expression of ICAM-1mRNA and reduce TNF-α and IL-6 secretion,which plays a protective role in LPS-induced acute lung injury of rats.

Pyrrolidine dithiocarbamate;Nuclear factor-kappa B;LPS;Injury,lung,acute

R453.9;R965

A

1004-0781(2014)03-0307-04

2013-03-20

2013-04-15

王大龍(1982-),男,山東泰安人,主治醫(yī)師,碩士,從事臨床麻醉學(xué)研究。電話:0546-8770301,E-mail：16719077@qq.com。

冷玉芳(1964-),女,甘肅蘭州人,主任醫(yī)師,博士,從事臨床麻醉學(xué)研究。電話:0931-8625200-6626,E-mail：lengyf@lzu.edu.cn。