硫化氫吸入干預(yù)大鼠棉花煙霧吸入性肺損傷中的氧化應(yīng)激

姜 毅，韓志海，王曉陽(yáng)，方庭正，黃 燕，段蘊(yùn)鈾

硫化氫吸入干預(yù)大鼠棉花煙霧吸入性肺損傷中的氧化應(yīng)激

姜 毅1,2，韓志海3，王曉陽(yáng)1，方庭正1，黃 燕1，段蘊(yùn)鈾1

目的探討吸入硫化氫(hydrogen sulfide, H2S)干預(yù)大鼠棉花煙霧吸入性肺損傷的氧化應(yīng)激反應(yīng)機(jī)制。方法24只雄性SD大鼠隨機(jī)分成對(duì)照組、H2S組、煙霧組、煙霧+H2S組，每組6只。復(fù)制大鼠棉花煙霧吸入性損傷模型，在煙霧吸入或模擬煙霧吸入后，H2S組、煙霧+H2S組大鼠予以持續(xù)吸入H2S 80 ppm+30%氧氣6 h，對(duì)照組、煙霧組予以吸入30%氧氣6 h，ELISA法檢測(cè)肺組織勻漿中MDA、NO、iNOS、NF-κB p65濃度，免疫組化檢測(cè)肺組織NF-κB p65并進(jìn)行半定量分析，熒光定量PCR法行肺組織iNOS mRNA定量。結(jié)果煙霧組大鼠肺組織勻漿中MDA、NO、iNOS、NF-κB p65濃度和肺組織中NF-κB p65的累積光密度、iNOS mRNA的相對(duì)表達(dá)量均明顯高于對(duì)照組，而煙霧+H2S組的上述指標(biāo)較煙霧組均明顯降低，如肺組織勻漿中NF-κB p65濃度(8123.51±2095.33) pg/mlvs(13803.19±2196.37) pg/ml，P<0.001；肺組織中iNOS mRNA的相對(duì)表達(dá)量(1.04±0.24)vs(2.20±0.21)，差異有統(tǒng)計(jì)學(xué)意義(P<0.001)；H2S組iNOS濃度、iNOS mRNA的相對(duì)表達(dá)量、NF-κB p65的累積光密度高于對(duì)照組，但MDA、NO、iNOS、NF-κB p65濃度與對(duì)照組比較無(wú)明顯差別。結(jié)論吸入H2S的干預(yù)機(jī)制可能是吸入H2S可抑制NF-κB p65的激活，使iNOS mRNA的轉(zhuǎn)錄合成減少，從而減少iNOS、NO生成，減輕氧化應(yīng)激反應(yīng)和減輕大鼠肺損傷。

急性肺損傷；煙霧吸入性損傷；氧化應(yīng)激；硫化氫

煙霧吸入性損傷主要發(fā)生在呼吸道和肺實(shí)質(zhì)，嚴(yán)重者產(chǎn)生中毒性肺炎或肺水腫，可迅速進(jìn)展為急性肺損傷/急性呼吸窘迫綜合征(acute lung injury/acute respiratory distress syndrome，ALI/ARDS)，使救治難度加大，病死率增加[1]。在煙霧吸入性損傷的發(fā)病機(jī)制中，氧化應(yīng)激占有很大比重。高溫?zé)熿F中的很多成分是強(qiáng)氧化劑，并且炎性反應(yīng)失控時(shí)大量的炎性反應(yīng)細(xì)胞聚集在肺內(nèi)，產(chǎn)生過(guò)量的活性氧(reactive oxygen species，ROS)，也可誘導(dǎo)氧化應(yīng)激損傷。

近十年來(lái)的研究發(fā)現(xiàn)，H2S具有抗氧化應(yīng)激[2]、調(diào)節(jié)炎性反應(yīng)[3]、舒張血管[4-7]、抗纖維化、參與調(diào)節(jié)內(nèi)分泌及生殖系統(tǒng)功能等作用[8]。吸入H2S 80 ppm 6 h，在內(nèi)毒素誘導(dǎo)的ALI小鼠模型中可抑制全身炎性反應(yīng)，提高小鼠生存率[9, 10]；在過(guò)度通氣誘導(dǎo)的ALI小鼠模型中可抑制肺內(nèi)炎性反應(yīng)和肺泡上皮細(xì)胞凋亡而保護(hù)肺臟[11]。本課題前期實(shí)驗(yàn)研究證實(shí)，大鼠棉花煙霧吸入性肺損傷時(shí)氧化應(yīng)激反應(yīng)增強(qiáng)，本實(shí)驗(yàn)經(jīng)預(yù)實(shí)驗(yàn)后觀察吸入80 ppm H2S 6 h氣體對(duì)大鼠煙霧吸入性肺損傷時(shí)氧化應(yīng)激反應(yīng)的影響。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物及分組 清潔級(jí)健康成年雄性SD大鼠共24只，體重150～250 g，由軍事醫(yī)學(xué)科學(xué)院實(shí)驗(yàn)動(dòng)物中心[SCXK-(軍)-2012-0004]提供，海軍總醫(yī)院實(shí)驗(yàn)動(dòng)物中心[SCXK-(軍)-2012-0012]飼養(yǎng)。遵守實(shí)驗(yàn)動(dòng)物條例處置動(dòng)物，按照隨機(jī)化原則將動(dòng)物分組，分為對(duì)照組、H2S組、煙霧組、煙霧+H2S組，每組6只。

1.2 大鼠煙霧吸入性損傷模型的復(fù)制及H2S的吸入 參照文獻(xiàn)[12-14]，本課題組已成功制作大鼠煙霧吸入性損傷模型，操作如下：大鼠2只分別置入吸煙瓶中，錫爐預(yù)熱至300 ℃恒溫，稱取2 g棉花放入錫爐內(nèi)，立即蓋上集煙筒，啟動(dòng)風(fēng)扇，開(kāi)始計(jì)時(shí)2 min，見(jiàn)大鼠足底皮膚逐漸出現(xiàn)櫻紅色至紫紅色，并有躁動(dòng)、呼吸急促、張口呼吸，至出現(xiàn)呼吸深慢、張口喘鳴時(shí)或滿2 min時(shí)限時(shí)，予以停止吸煙，立即敞開(kāi)吸煙瓶口，吸入空氣7 min，重復(fù)上述步驟3～5次，至大鼠吸入空氣7 min仍舊昏迷不醒時(shí)結(jié)束。4組大鼠均先后置于煙霧吸入裝置中給予上述類似處置，其中煙霧組、煙霧+H2S組給予煙霧輸入，對(duì)照組、H2S組無(wú)煙霧輸入；在煙霧吸入或模擬煙霧吸入后，4組大鼠均給予30%氧氣吸入6 h，但H2S組、煙霧+H2S組大鼠還同時(shí)予以吸入H2S 80 ppm 6 h。實(shí)驗(yàn)中大鼠予以自由進(jìn)食及飲水。

1.3 ELISA檢測(cè) 實(shí)驗(yàn)結(jié)束后大鼠腹腔注射戊巴比妥鈉致死。采用雙抗體夾心ABC-ELISA法檢測(cè)右肺下葉勻漿中一氧化氮(nitric oxide，NO)、誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase，iNOS)、核轉(zhuǎn)錄因子-κB p65(nuclear factor-kappaB，NF-κB p65)濃度。比色法檢測(cè)右肺下葉肺組織勻漿中丙二醛(malondialdehyde，MDA)濃度。ELISA試劑盒廠家為嘉美生物(Jiamay Biotech Co.Ltd)，按試劑盒說(shuō)明操作。

1.4 右肺中葉肺組織NF-κB p65免疫組化檢測(cè)及半定量分析 右肺中葉4%多聚甲醛溶液浸泡72 h后常規(guī)石蠟包埋、切片，厚度3 μm，60～65 ℃烤片4 h，脫蠟、水化、PBS緩沖液洗滌，高壓修復(fù)組織抗原，3%H2O2滅活過(guò)氧化物酶，正常山羊血清封閉，滴加第一抗體Anti-NF-κB p65 antibody (abcam，ab16502) 50 μl，稀釋度1∶200，4 ℃孵育過(guò)夜，滴加復(fù)合二抗HRP-Polymer anti Mouse/Rabbit IgG(Maixin.Bio，KIT-5020)，室溫靜置20 min；DAB顯色，蘇木精復(fù)染，陰性對(duì)照以血清代替一抗。鏡檢陽(yáng)性染色為黃色或棕黃色染色。應(yīng)用Image Pro Plus 6.0圖像分析系統(tǒng)(美國(guó)Media Cybernetics公司)進(jìn)行半定量分析，每張玻片顯微鏡下隨機(jī)選擇5個(gè)高倍鏡視野(×1000)，以相同參數(shù)攝取圖像，IPP6.0軟件分析得出每個(gè)視野陽(yáng)性染色的平均光密度(mean density)、積分光密度(integrated optical density，IOD)的總和即累積光密度(sum IOD)，計(jì)算出每個(gè)標(biāo)本上述指標(biāo)的均值，從而對(duì)染色濃度進(jìn)行半定量分析。

1.5 熒光定量PCR方法檢測(cè)大鼠右肺上葉iNOS mRNA轉(zhuǎn)錄水平 目的基因iNOS的PCR檢測(cè)引物序列由嘉美生物設(shè)計(jì)合成，iNOS上下游引物分別是5′-ACACCGATTCCACTCAACTA-3′和5′-ACCACCTGTTAGTTCAAGCC-3′，擴(kuò)增產(chǎn)物長(zhǎng)度為159bp，內(nèi)參為β-actin(CWbio.Co.Ltd，Cat#CW0918)。用超純RNA提取試劑盒(CWbio.Co.Ltd，Cat#CW0581)提取組織樣本中總RNA。取5 μl RNA用1%瓊脂糖凝膠進(jìn)行電泳。用HiFi-MMLVcDNA第1鏈合成試劑盒(CWbio.Co.Ltd，Cat#CW0744)進(jìn)行反轉(zhuǎn)錄，用UltraSYBR Mixture(with Rox)(CWbio.Co.Ltd，Cat#CW0956)進(jìn)行擴(kuò)增，擴(kuò)增程序?yàn)椋?5 ℃ 10 min，(95 ℃15 s+60 ℃ 60 s) 40個(gè)循環(huán)。用LightCycler-480II型熒光定量PCR儀測(cè)量，采用2-△△CT法進(jìn)行數(shù)據(jù)的相對(duì)定量分析。

2 結(jié) 果

2.1 大鼠肺組織勻漿ELISA檢測(cè)結(jié)果 由表1可見(jiàn)，煙霧組大鼠肺組織勻漿中MDA、NO、iNOS、NF-κB p65濃度較對(duì)照組均明顯升高(P<0.001)，煙霧+H2S組上述指標(biāo)較煙霧組降低(P<0.001)。H2S組上述指標(biāo)僅iNOS濃度高于對(duì)照組，其他指標(biāo)與對(duì)照組比較無(wú)明顯差別，H2S組上述指標(biāo)均低于煙霧組。

表1 各組大鼠肺組織中MDA、NO、iNOS、NF-κB p65濃度檢測(cè)結(jié)果 ；n=6)

注：與煙霧組比較，①P<0.05；與對(duì)照組比較， ②P<0.05

2.2 大鼠體重和肺組織iNOSmRNA的相對(duì)表達(dá)量 由表2可見(jiàn)，各組大鼠體重沒(méi)有統(tǒng)計(jì)學(xué)差異(P>0.05)，大鼠平均體重為(186.68±28.79) g，可以認(rèn)為大鼠體重對(duì)各組間實(shí)驗(yàn)結(jié)果沒(méi)有影響。煙霧組、煙霧+H2S組和H2S組大鼠肺組織iNOSmRNA的相對(duì)表達(dá)量較對(duì)照組明顯升高(P<0.001)，煙霧+H2S組和H2S組較煙霧組降低(P<0.001)。

2.3 大鼠肺組織iNOS的免疫組化分析結(jié)果 大鼠肺組織NF-κB p65的免疫組化染色結(jié)果見(jiàn)圖1。由表2可見(jiàn)，大鼠肺組織NF-κB p65的累積光密度(sum IOD)煙霧組較對(duì)照組高(P<0.001)；煙霧+H2S組和H2S組較煙霧組低(P<0.01)，但比對(duì)照組高(P<0.001)。大鼠肺組織NF-κB p65的平均光密度(mean density) H2S組、煙霧組、煙霧+H2S組無(wú)統(tǒng)計(jì)學(xué)差異(P>0.05)，但均低于對(duì)照組(P<0.01)。

表2 各組大鼠體重、肺組織iNOS mRNA相對(duì)表達(dá)量、NF-κB p65累積吸光度和平均光密度的比較 ；n=6)

注：與煙霧組比較，①P<0.05；與對(duì)照組比較， ②P<0.05

圖1 肺組織NF-κB p65免疫組化染色鏡下觀察(×1000)

3 討 論

煙霧成分的復(fù)雜性決定了煙霧吸入性肺損傷的發(fā)病機(jī)制的復(fù)雜性，尤以氧化應(yīng)激更為重要[15]。煙霧吸入可激活肺內(nèi)巨噬細(xì)胞、中性粒細(xì)胞、內(nèi)皮細(xì)胞及血管平滑肌細(xì)胞釋放出大量細(xì)胞因子，如腫瘤壞死因子-α(TNF-α)、白介素-1β(IL-1β)、IL-6、IL-8等，從而激活NF-κB，其在胞漿中分解后的活性片段NF-κB p65進(jìn)入細(xì)胞核，促進(jìn)下游iNOS mRNA的轉(zhuǎn)錄，導(dǎo)致iNOS的合成增加，iNOS可分解精氨酸產(chǎn)生大量NO，可催化合成過(guò)氧化亞硝酸鹽，引起生物膜的脂質(zhì)過(guò)氧化反應(yīng)[16]。同時(shí)，煙霧中的NO、N2O、SO2、氧化性顆粒物均是強(qiáng)氧化劑，粒細(xì)胞受刺激30 s內(nèi)即可釋放出大量的氧自由基，缺氧后吸氧治療也增加氧自由基的產(chǎn)生。氧自由基同樣可使生物膜發(fā)生脂質(zhì)過(guò)氧化反應(yīng)，破壞膜結(jié)構(gòu)，激活炎性介質(zhì)的合成，影響能量代謝，并使蛋白質(zhì)變性而發(fā)生功能障礙。上述損傷性因素使肺泡-毛細(xì)血管通透性增高，血液中有形成分滲出到肺泡腔，形成肺水腫。此外, 煙霧吸入性肺損傷產(chǎn)生的活性氧可導(dǎo)致NO合成過(guò)量[17]，NO可使血管外漏、缺氧性肺血管收縮功能喪失[18]，同時(shí)使具有細(xì)胞毒性的活性氮(reactive nitrogen species，RNS)生成增加[19]，進(jìn)一步加重肺損傷。既往本課題組的實(shí)驗(yàn)研究已證實(shí)，大鼠棉花煙霧吸入后6 h即可出現(xiàn)典型的肺損傷表現(xiàn)，本實(shí)驗(yàn)發(fā)現(xiàn)，煙霧吸入后6 h大鼠肺組織中NF-κB p65濃度、NF-κB p65的累積光密度、iNOSmRNA的相對(duì)表達(dá)量、iNOS和NO濃度均增高，完全符合上述煙霧吸入后氧化應(yīng)激反應(yīng)的表現(xiàn)，即煙霧吸入后激活NF-κB，NF-κB p65濃度增加并進(jìn)入細(xì)胞核，促進(jìn)iNOS mRNA的轉(zhuǎn)錄增加，使iNOS表達(dá)增加和NO合成增多。同時(shí)，MDA作為自由基與生物膜多價(jià)不飽和脂肪酸發(fā)生反應(yīng)而生成的過(guò)氧化物，其含量的同步升高也說(shuō)明氧化應(yīng)激反應(yīng)加劇，脂質(zhì)過(guò)氧化反應(yīng)增強(qiáng)，組織損傷加重。

火災(zāi)后煙霧吸入性肺損傷的早期救治尤為關(guān)鍵，從其發(fā)病機(jī)制可以看出，打斷氧化應(yīng)激反應(yīng)通路，抑制NF-κB的激活從而減少其下游產(chǎn)物的生成，從理論上看有可能減輕肺損傷。有研究表明，連續(xù)靜脈點(diǎn)滴低劑量精氨酸血管加壓素，抑制iNOS產(chǎn)生過(guò)量的NO，可明顯減輕燒傷和煙霧吸入導(dǎo)致的肺損傷[20]。

近些年的研究中，H2S一改既往“具有臭雞蛋氣味的神經(jīng)毒氣”形象，被稱為繼NO、CO之后的第3種氣體信號(hào)分子[21, 22]。動(dòng)物實(shí)驗(yàn)證實(shí)，靜脈點(diǎn)滴硫氫化鈉(NaHS)或吸入H2S氣體，在多種原因所致的肺損傷動(dòng)物模型研究中具有抗氧化應(yīng)激、抗炎性反應(yīng)、抗凋亡、減輕肺損傷的作用[2, 9-11, 23]，但吸入H2S氣體對(duì)煙霧吸入性肺損傷的作用研究尚無(wú)報(bào)道。在脂多糖激活的離體巨噬細(xì)胞中，H2S可抑制NF-κB信號(hào)通路的激活，減少NO的產(chǎn)生，而起到抗氧化應(yīng)激的作用[24]。因此，筆者設(shè)計(jì)實(shí)驗(yàn)研究吸入H2S氣體對(duì)煙霧吸入性肺損傷的干預(yù)效果。

實(shí)驗(yàn)發(fā)現(xiàn)，在煙霧吸入后立即予以吸入H2S 80 ppm 6 h，可明顯減輕大鼠的肺損傷，在大鼠肺組織中NF-κB p65濃度、NF-κB p65的累積光密度、iNOSmRNA的相對(duì)表達(dá)量、iNOS和NO濃度均明顯降低，MDA下降，提示吸入H2S減輕大鼠棉花煙霧吸入性肺損傷的機(jī)制可能是吸入H2S抑制了NF-κB p65的激活，使iNOSmRNA的轉(zhuǎn)錄合成減少，減少了iNOS、NO的生成，從而減輕氧化應(yīng)激反應(yīng)，減輕了大鼠的肺損傷。大鼠肺組織NF-κB p65的平均光密度(mean density) 代表了切片上免疫組化陽(yáng)性染色的深淺，但不能代表陽(yáng)性染色的總量變化，因而無(wú)指示意義。

細(xì)胞類型、氧自由基的類型、NF-κB通路上眾多氧化應(yīng)激敏感位點(diǎn)的狀態(tài)、上游的信號(hào)通路等因素的變化均可影響到NF-κB的激活，從而干預(yù)氧化應(yīng)激反應(yīng)的進(jìn)程[25]，因此，NF-κB是細(xì)胞對(duì)氧化應(yīng)激狀態(tài)極為敏感的核轉(zhuǎn)錄因子。H2S調(diào)節(jié)NF-κB蛋白活性的上游通路包括ERK通路[26,27]、p38-MAPK通路[28]、血紅蛋白加氧酶-1和熱休克蛋白70[24]等。近年來(lái)對(duì)H2S的作用機(jī)制的研究發(fā)現(xiàn)，H2S信號(hào)轉(zhuǎn)導(dǎo)的蛋白質(zhì)巰基化機(jī)制，即H2S通過(guò)與靶蛋白的半胱氨酸活性殘基結(jié)合，發(fā)生蛋白質(zhì)巰基化反應(yīng)，改變蛋白質(zhì)的活性，可能是H2S作用機(jī)制的中心環(huán)節(jié)[29,30]。在本實(shí)驗(yàn)的煙霧+H2S組，煙霧為外部刺激，吸入后作用在肺內(nèi)巨噬細(xì)胞、中性粒細(xì)胞、內(nèi)皮細(xì)胞及血管平滑肌細(xì)胞，釋放出大量細(xì)胞因子，如TNF-α、IL-1β、IL-6、IL-8等。同時(shí)，煙霧中的氧化劑也導(dǎo)致細(xì)胞內(nèi)氧自由基明顯增加，上述因素可激活NF-κB通路，使iNOS的表達(dá)及NO濃度均顯著升高，并使細(xì)胞處于強(qiáng)氧化狀態(tài)。由于機(jī)體抗氧化調(diào)節(jié)不足以對(duì)抗氧化應(yīng)激[31]，機(jī)體損傷加劇。在此基礎(chǔ)上，外源性H2S的吸入表現(xiàn)出抑制NF-κB通路的作用，使iNOS表達(dá)及NO合成減少，而減輕了大鼠的肺損傷。說(shuō)明大鼠棉花煙霧吸入后立即吸入H2S 80 ppm 6 h可減輕大鼠的肺損傷，H2S的應(yīng)用具有研究意義。

本實(shí)驗(yàn)中H2S組MDA、NO、iNOS、NF-κB p65濃度與對(duì)照組比較無(wú)統(tǒng)計(jì)學(xué)差異，大鼠未見(jiàn)明顯的肺損傷，說(shuō)明單純吸入80 ppm H2S 6 h對(duì)健康大鼠是相對(duì)安全的。本實(shí)驗(yàn)充實(shí)了H2S吸入研究的應(yīng)用范疇。H2S屬較強(qiáng)的還原劑，氣道吸入后可直接與氧化劑發(fā)生中和反應(yīng)，它至少能與超氧陰離子、過(guò)氧化氫、超氧化氮及次氯酸4種氧自由基起反應(yīng)[32, 33]，從而保護(hù)膜結(jié)構(gòu)免受自由基損傷，這也是吸入H2S氣體治療煙霧吸入性肺損傷的便利條件，在以后的研究中可繼續(xù)探討。在本次實(shí)驗(yàn)研究中，只設(shè)計(jì)了6 h的治療時(shí)間段的比較，缺乏長(zhǎng)期動(dòng)態(tài)效果的觀察。另外，H2S可激活A(yù)TP敏感鉀通道而舒張血管[6]，調(diào)節(jié)Fas/FasL死亡受體通路而減輕凋亡[34]，抑制神經(jīng)源性炎性反應(yīng)，3種氣體信號(hào)分子相互作用等[35]，這些機(jī)制也可能在煙霧性肺損傷的治療中發(fā)揮作用，可進(jìn)一步探討。

本研究揭示了吸入H2S 80 ppm 6 h減輕大鼠棉花煙霧吸入性肺損傷的機(jī)制可能是吸入H2S抑制了NF-κB p65的激活，使iNOSmRNA的轉(zhuǎn)錄合成減少，減少了NO的生成，減輕氧化應(yīng)激反應(yīng)，從而減輕了大鼠的肺損傷。

[1] Ballard-Croft C, Sumpter L R, Broaddus R,etal. Ovine smoke/burn ARDS model: a new ventilator-controlled smoke delivery system [J]. J Surg Res, 2010, 164(1): 155-162.

[2] Esechie A, Kiss L, Olah G,etal. Protective effect of hydrogen sulfide in a murine model of acute lung injury induced by combined burn and smoke inhalation [J]. Clin Sci (Lond), 2008, 115(3): 91-97.

[3] Li P C, Chen W C, Chang L C,etal. Substance P(SP) acts via the neurokinin receptor 1 to elicit bronchoconstriction, oxidative stress, and upregulated ICAM-1 expression after oil smoke exposure [J]. Am J Physiol Lung Cell Mol Physiol, 2008, 294(5): 912-920.

[4] Papapetropoulos A, Pyriochou A, Altaany Z,etal. Hydrogen sulfide is an endogenous stimulator of angiogenesis [J]. Proc Natl Acad Sci USA, 2009, 106(51): 21972-21977.

[5] Szabo C, Papapetropoulos A. Hydrogen sulphide and angiogenesis: mechanisms and applications [J]. Br J Pharmacol, 2011, 164(3): 853-865.

[6] Yang G, Wu L, Jiang B,etal. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase [J]. Science, 2008, 322(5901): 587-590.

[7] Muzaffar S, Jeremy J Y, Sparatore A,etal. H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G [J]. Br J Pharmacol, 2008, 155(7): 984-994.

[8] Zhu X Y, Gu H, Ni X. Hydrogen sulfide in the endocrine and reproductive systems [J]. Expert Rev Clin Pharmacol, 2011, 4(1): 75-82.

[9] Tokuda K, Kida K, Marutani E,etal. Inhaled hydrogen sulfide prevents endotoxin-induced systemic inflammation and improves survival by altering sulfide metabolism in mice [J]. Antioxid Redox Signal, 2012, 17(1): 11-21.

[10] Faller S, Zimmermann K K, Strosing K M,etal. Inhaled hydrogen sulfide protects against lipopolysaccharide-induced acute lung injury in mice [J]. Med Gas Res, 2012, 2(1): 26-31.

[11] Faller S, Ryter S W, Choi A M,etal. Inhaled hydrogen sulfide protects against ventilator-induced lung injury [J]. Anesthesiology, 2010, 113(1): 104-115.

[12] Lee H M, Greeley G H, Herndon D N,etal. A rat model of smoke inhalation injury: influence of combustion smoke on gene expression in the brain [J]. Toxicol Appl Pharmacol, 2005, 208(3): 255-265.

[13] Huang P S, Tang G J, Chen C H,etal. Whole-body moderate hypothermia confers protection from wood smoke-induced acute lung injury in rats: the therapeutic window [J]. Crit Care Med, 2006, 34(4): 1160-1167.

[14] Zou Y Y, Lu J, Poon D J,etal. Combustion smoke exposure induces up-regulated expression of vascular endothelial growth factor, aquaporin 4, nitric oxide synthases and vascular permeability in the retina of adult rats [J]. Neuroscience, 2009, 160(3): 698-709.

[15] Rehberg S, Maybauer M O, Enkhbaatar P,etal. Pathophysiology, management and treatment of smoke inhalation injury [J]. Expert Rev Respir Med, 2009, 3(3): 283-297.

[16] Enkhbaatar P, Traber D L. Pathophysiology of acute lung injury in combined burn and smoke inhalation injury [J]. Clin Sci (Lond), 2004, 107(2): 137-143.

[17] Cox R A, Jacob S, Oliveras G,etal. Pulmonary expression of nitric oxide synthase isoforms in sheep with smoke inhalation and burn injury [J]. Exp Lung Res, 2009, 35(2): 104-118.

[18] Westphal M, Enkhbaatar P, Schmalstieg F C,etal. Neuronal nitric oxide synthase inhibition attenuates cardiopulmonary dysfunctions after combined burn and smoke inhalation injury in sheep [J]. Crit Care Med, 2008, 36(4): 1196-1204.

[19] Rehberg S, Maybauer M O, Maybauer D M,etal. The role of nitric oxide and reactive nitrogen species in experimental ARDS [J]. Front Biosci (Schol Ed), 2010, 2: 18-29.

[20] Westphal M, Rehberg S, Maybauer M O,etal. Cardiopulmonary effects of low-dose arginine vasopressin in ovine acute lung injury [J]. Crit Care Med, 2011, 39(2): 357-363.

[21] Calvert J W. The summer of hydrogen sulfide: highlights from two international conferences [J]. Med Gas Res, 2013, 3(1): 5-9.

[22] Gadalla M M, Snyder S H. Hydrogen sulfide as a gasotransmitter [J]. J Neurochem, 2010, 113(1): 14-26.

[23] Liu W L, Liu Z W, Li T S,etal. Hydrogen sulfide donor regulates alveolar epithelial cell apoptosis in rats with acute lung injury [J]. Chin Med J (Engl), 2013, 126(3): 494-499.

[24] Oh G S, Pae H O, Lee B S,etal. Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide [J]. Free Radic Biol Med, 2006, 41(1): 106-119.

[25] Janssen-Heininger Y M, Poynter M E, Baeuerle P A. Recent advances towards understanding redox mechanisms in the activation of nuclear factor kappaB [J]. Free Radic Biol Med, 2000, 28(9): 1317-1327.

[26] Zhi L, Ang D, Zhang H,etal. Hydrogen sulfide induces the synthesis of proinflammatory cytokines in human monocyte cell line U937 via the ERK-NF-kappaB pathway [J]. J Leukoc Biol, 2007, 81(5): 1322-1332.

[27] Jeong S O, Pae H O, Oh G S,etal. Hydrogen sulfide potentiates interleukin-1beta-induced nitric oxide production via enhancement of extracellular signal-regulated kinase activation in rat vascular smooth muscle cells [J]. Biochem Biophys Res Commun, 2006, 345(3): 938-944.

[28] Hu L F, Wong P T, Moore P K,etal. Hydrogen sulfide attenuates lipopolysaccharide-induced inflammation by inhibition of p38 mitogen-activated protein kinase in microglia [J]. J Neurochem, 2007, 100(4): 1121-1128.

[29] Paul B D, Snyder S H. H2S signalling through protein sulfhydration and beyond [J]. Nat Rev Mol Cell Biol, 2012, 13(8): 499-507.

[30] Sen N, Paul B D, Gadalla M M,etal. Hydrogen sulfide-linked sulfhydration of NF-kappaB mediates its antiapoptotic actions [J]. Mol Cell, 2012, 45(1): 13-24.

[31] LaLonde C, Nayak U, Hennigan J,etal. Plasma catalase and glutathione levels are decreased in response to inhalation injury [J]. J Burn Care Rehabil, 1997, 18(6): 515-519.

[32] Whiteman M, Armstrong J S, Chu S H,etal. The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite 'scavenger'? [J]. J Neurochem, 2004, 90(3): 765-768.

[33] Whiteman M, Cheung N S, Zhu Y Z,etal. Hydrogen sulphide: a novel inhibitor of hypochlorous acid-mediated oxidative damage in the brain? [J]. Biochem Biophys Res Commun, 2005, 326(4): 794-798.

[34] Mustafa A K, Gadalla M M, Suyder S H. Signaling by gasotransmitters[J]. Sci Signal, 2009,2(68):re2.

[35] Liu W, Wang D, Liu K,etal. Nrfz as a converging node for cellular signaling pathways of gasotransmitters[J]. Med Hypotheses, 2012,79(3):308-310.

(2014-01-23收稿 2014-03-02修回)

(責(zé)任編輯 岳建華)

Inhaledhydrogensulfideinhibitsoxidativestressofcottonsmokeinhalation-inducedacutelunginjuryinrats

JIANG Yi1,2, HAN Zhihai3, WANG Xiaoyang1, FANG Tingzheng1, HUANG Yan1，and DUAN Yunyou1. 1. Department of Respiratory Medicine, Clinical Medical College of Navy, Second Military Medical University, Beijing 100048, China; 2. Political Department Clinic of Shenyang Military Area Command, Shenyang 110032, China; 3. Pulmonary and Critical Care Medicine of PLA Navy General Hospital, Beijing 100048, China

ObjectiveTo investigate the mechanisms of inhaled hydrogen sulfide inhibiting oxidative stress of cotton smoke inhalation-induced acute lung injury in rats.MethodsTwenty-four male SD rats were randomly allocated into control group, H2S group, smoke group and smoke+H2S group. The rat model of cotton smoke inhalation injury was established. After smoke inhalation or simulated smoke inhalation, rats inhaled H2S 80 ppm, 30% oxygen for 6 hours (H2S group and smoke+H2S group), or rats inhaled 30% oxygen for 6 hours (control group and smoke group). Then rats were mercy killed. In each group of rats we observed the concentration of NO，iNOS，NF-κB p65，MDA in homogenized lung tissue by ELISA，used the method of fluorescence quantitative PCR to detect the expression of iNOS mRNA in homogenized lung tissue, and immunohistochemically detected the relative expression of NF-κB p65 with Image Pro Plus 6.0 software.ResultsCompared with the control group, concentrations of MDA, NO, iNOS, NF-κB p65, relative expression of iNOS mRNA and sum IOD of NF-κB p65 in the smoke group rats’ homogenized lung tissue were significantly elevated, and those in the smoke+H2S group were relatively lower, for example, concentrations of NF-κB p65 were (8123.51±2095.33) pg/ml vs (13803.19±2196.37) pg/ml,P<0.001; relative expression of iNOS mRNA was (1.04±0.24)vs(2.20±0.21),P<0.001. Concentrations of iNOS, relative expression of iNOS mRNA and sum IOD of NF-κB p65 in the H2S group were higher than those in the control group, meanwhile concentrations of MDA, NO, NF-κB p65 in the H2S group were similar to those in the control group.ConclusionsThe mechanisms of inhaled 80 ppm hydrogen sulfide for 6 hours protecting against cottn smoke inhalation-induced ALI in rats potentially is inhaled hydrogen sulfide inhibiting the activation of NF-κB p65, so the expression of iNOS mRNA, iNOS and NO grow downwards and as a result, it relieves oxidative stress and reduces pathological damage to lung tissue.

acute lung injury；smoke inhalation injury；oxidative stress；hydrogen sulfide

全軍醫(yī)學(xué)科研“十二五”計(jì)劃課題(CWS11J180)

姜 毅，博士，主治醫(yī)師，E-mail：jiangyi1974@sohu.com

1. 100048北京，第二軍醫(yī)大學(xué)海軍臨床醫(yī)學(xué)院呼吸內(nèi)科；2. 110032沈陽(yáng)， 沈陽(yáng)軍區(qū)政治部門(mén)診部；3. 100048北京，海軍總醫(yī)院呼吸內(nèi)科

通迅作者：段蘊(yùn)鈾，E-mail：duanyunyou@126.com

R363.274