蔡康興 汪 麗 王 婷 羅 莉 陳 龍 王 楠 史 源

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·論著·

一氧化碳釋放分子3通過抑制肺泡上皮細胞凋亡減輕脂多糖誘導的新生鼠急性肺損傷

蔡康興1汪 麗1王 婷2羅 莉2陳 龍1王 楠1史 源1

目的 探討一氧化碳釋放分子3(CORM3)對脂多糖(LPS)誘導的新生鼠急性肺損傷(ALI)肺泡上皮細胞凋亡的影響。方法 32只新生SD大鼠均分為對照組、LPS組、CORM3組和失活CORM3(iCORM3)組；LPS組、CORM3組和iCORM3組采用LPS氣管內滴注建立新生鼠ALI模型，分別腹腔注射生理鹽水、CORM3和iCORM3；對照組不建立ALI模型，腹腔注射生理鹽水。于建模后12 h取肺組織，蘇木素-伊紅染色觀察肺組織病理改變，濕干(W/D)比值測定，肺泡灌洗液(BALF)細胞計數及蛋白含量測定。體外培養(yǎng)A549細胞，LPS誘導細胞凋亡。MTT檢測細胞活性，Tunel染色觀察組織、細胞凋亡變化。結果 ①與對照組相比，LPS組肺組織形態(tài)結構明顯紊亂，肺泡壓縮，肺間質大量炎性細胞浸潤；CORM3組肺組織形態(tài)結構基本正常，間質炎性細胞浸潤少；iCORM3組見肺組織水腫及大量炎性細胞浸潤。②與對照組相比，LPS組肺組織W/D比值、BALF細胞數及蛋白含量明顯升高，肺泡上皮細胞凋亡率明顯增多[(37.3±4.5)%vs(3.0±1.0)%]；A549細胞活力下降，凋亡率明顯升高[(29.6±4.1)%vs(3.6±1.0)%]，差異均有統計學意義 (P<0.05)。CORM3組肺組織W/D比值、BALF細胞數和蛋白含量較LPS組顯著下降，肺泡上皮細胞凋亡率減少[(19.3±4.6)%]；A549細胞活力回升，凋亡率[(15.3±4.5)%]明顯降低，差異均有統計學意義 (P<0.05)。LPS組與iCORM3組間上述指標差異均無統計學意義。結論 CO通過抑制肺泡上皮細胞凋亡減輕新生鼠ALI。

一氧化碳釋放分子3； 脂多糖； 肺損傷； 凋亡； 肺泡上皮細胞

感染是引起新生兒急性肺損傷(ALI)的常見原因之一[1,2]，可表現為免疫系統激活，各種炎癥因子和趨化因子表達增加，引起中性粒細胞廣泛浸潤，導致肺血氣交換屏障破壞[3]。肺泡上皮細胞在穩(wěn)定肺泡結構，維持新生兒正常呼吸功能和應對外源刺激等方面均發(fā)揮重要作用[4]。在ALI過程中外來因素的直接損害和過度炎癥反應均可破壞肺泡上皮細胞[5.6]，而抑制ALI過程中肺泡上皮細胞凋亡能明顯緩解病程進展[7]，提示在ALI病程中，肺泡上皮細胞可能發(fā)揮了重要的作用。一氧化碳(CO)在體內微量存在，主要由血紅素氧合酶催化血紅素生成，低劑量CO具有抗炎、抗凋亡、抗增殖作用，也有舒張血管的作用[8]。在多種ALI的動物模型中，CO均有良好的肺保護效應[8,9]，在敗血癥新生兒也發(fā)現CO的水平顯著高于正常新生兒[10]。本研究建立新生鼠ALI模型，以CO釋放分子3(CORM3)作為干預藥物，探討CO的保護作用及其相關機制。

1 方法

1.1 試劑 LPS(0111:B4)購于Sigma公司,CORM3購于ApexBIO公司；Tunel試劑盒購于Roche公司；MTT購于Biosharp公司；Dapi購于碧云天生物公司；失活CORM3(iCORM3)為去除CO的羰基骨架結構，采用Stec等[11]方法制備，CORM3室溫下放置24 h，使用前用移液槍反復吸吹液體，排出釋放的CO氣體分子。

1.2 實驗動物 清潔級7日齡新生SD大鼠32只，雌雄不限，體重13～18 g，購自第三軍醫(yī)大學附屬大坪醫(yī)院實驗動物中心[動物許可證號：SGCK(渝)2012-0005]。

1.3 分組和處理 32只新生大鼠均分為4組: 對照組、LPS組、CORM3組和iCORM3組。新生鼠予10%水合氯醛麻醉，沿頸正中線剪開皮膚，鈍性分離肌肉及筋膜，暴露氣管。LPS組、CORM3組和iCORM3組予氣管內滴注LPS (10 mg·kg-1,生理鹽水溶解)20 μL制備新生鼠ALI模型[12]；分別腹腔注射100 μL生理鹽水、CORM3 (10 mg·kg-1L，生理鹽水溶解) 和iCORM3 (10 mg·kg-1L，生理鹽水溶解)；對照組氣管內滴注和腹腔注射100 μL生理鹽水。12 h后處死大鼠取肺組織備檢。實驗動物處置符合動物倫理學要求。

1.4 細胞培養(yǎng)及LPS誘導細胞凋亡 人肺腺癌細胞株A549細胞(第三軍醫(yī)大學野戰(zhàn)外科研究所惠贈)以含10%胎牛血清的DMEM培養(yǎng)液，37℃、5%CO2飽和濕度傳代培養(yǎng)，0.25%胰酶消化液傳代。所有實驗均采用對數生長期細胞。LPS和CORM3溶解于DMEM基礎培養(yǎng)基中， -20℃保存?zhèn)溆茫琇PS溶液加藥前震蕩渦旋，二者同時加藥。96孔板細胞接種濃度為每孔10×103，反應體系200 μL；6孔板細胞接種濃度為每孔4×105，反應體系2 mL。

1.5 肺組織濕干(W/D)比值測定 取右肺上葉生理鹽水漂洗，吸水紙吸去肺組織表面液體，電子天平稱重為肺組織濕重；70℃恒溫箱烘烤72 h[13]，待肺組織稱重不再發(fā)生變化，為肺組織干重。

1.6 肺組織病理觀察 取右肺中下葉用4%多聚甲醛固定，脫水，包埋，制備組織石蠟切片(4 μm)，脫蠟后蘇木素-伊紅染色，光鏡下觀察[14]。

1.7 肺泡灌洗液(BALF)蛋白含量及細胞計數測定 取左肺葉以24G留置針沿氣管兩環(huán)狀軟骨間插入，拔出針芯，1 mL注射器取200 μL冰凍生理鹽水緩慢灌洗肺組織3次，每次回收率達>80%。收集BALF，500g、4℃離心10 min，上清用于蛋白含量測定，細胞團塊重懸計數[12]。

1.8 細胞凋亡Tunel分析 按照羅氏公司Tunel試劑盒操作說明行染色[15]。將切好的石蠟切片常規(guī)脫蠟至水化(細胞標本采用細胞涂片于玻片上制備)，3%H2O2甲醇阻斷內源性過氧化物酶15 min，PBS漂洗5 min×3次，加入20 mg·mL-1蛋白酶K工作液常溫下反應20 min(細胞標本采用0.3% Triton-X-100作用10 min)，PBS漂洗5 min×3次，加入Tunel反應液(臨用前配置，避光包裝置于冰上)于37℃避光反應60 min，同時設置陽性對照和陰性對照，陽性對照在加入Tunel反應液前加入脫氧核糖核酸酶反應10 min，后續(xù)步驟同實驗組；陰性對照加入不含TdT酶，只含熒光素的液體反應，其余步驟同實驗組。PBS漂洗5 min×3次，加入Dapi染核，常溫下避光反應20 min；PBS漂洗5 min×3次，DAB顯色，當背景開始變深時停止反應,雙蒸水漂洗后甲基綠復染核，常規(guī)脫水、透明、中性樹膠封片。

細胞Tunel實驗不進行DAB顯色，其余步驟同組織Tunel染色。綠色通道(505～535 nm)可見凋亡細胞顯示綠色信號，藍色通道(475～490 nm)可見細胞核(Dapi染核)為藍色信號；以細胞胞核Dapi藍色和Tunel綠色熒光信號重疊為細胞凋亡陽性信號，任意選取3個視野，分別計數A549凋亡細胞和正常細胞。

1.9 MTT法檢測細胞活力 取對數生長期細胞，0.25%胰酶消化，將細胞懸液濃度調成5×104·mL-1接種至96孔板，每孔200 μL，細胞培養(yǎng)過夜后分別加入濃度0.000 8、0.004、0.02、0.1、0.5、2.5和12.5 mmol·L-1的CORM3, LPS 200 μg·L-1, 陰性和陽性對照分別加入等量培養(yǎng)液和LPS，每組設5個復孔，培養(yǎng)24 h后加入MTT溶液(5 mg·mL-1)20 μL，4 h后加入150 μL DMSO,搖床上振蕩溶解。酶標儀于490 nm波長測定吸光值[16]。

2 結果

2.1 新生鼠肺組織病理變化 對照組肺組織形態(tài)結構基本正常，肺間質炎性細胞浸潤現象不明顯(圖1A)；LPS組肺組織形態(tài)結構明顯紊亂、水腫，肺泡壓縮，肺間質大量炎性細胞浸潤(圖1B)；CORM3組肺組織水腫較LPS組明顯輕微，炎性細胞浸潤明顯少(圖1C)；iCORM3組肺組織水腫及炎性細胞浸潤同LPS組(圖1D)。

2.2 肺組織W/D比值和BALF蛋白含量、細胞計數比較 表1顯示，肺組織W/D比值、BALF細胞數目和蛋白含量4組間差異總體上有統計學意義(P均<0.001)，組間兩兩比較，上述3個指標LPS組均顯著高于對照組(P<0.05)；CORM3組與LPS組比較，W/D比值、BALF細胞計數和蛋白含量顯著下降，差異有統計學意義(P<0.05)，iCORM3組與LPS組3個指標差異均無統計學意義。

2.3 肺泡細胞凋亡變化 4組肺泡細胞凋亡見圖2。對照組、LPS組、CORM3組和iCORM3組肺泡細胞凋亡率分別為(3.0±1.0)%、(37.3±4.5)%、(19.3±4.6)%和(36.7±0.9)%，其中LPS組高于對照組，CORM3組低于LPS組，差異有統計學意義(P<0.05)；iCORM3組和LPS組差異無統計學意義。

GroupW/DratioBALF/×104BALFprotein/mg·mL-1Control5.33±0.2113±30.048±0.026LPS6.00±0.1863±90.103±0.028CORM35.59±0.2929±60.056±0.029iCORM35.93±0.3261±100.102±0.029F11.66385.68531.071P<0.001<0.001<0.001

Notes W/D ratio, BALF and BALF protein of LPS group siginificantly differed from control group, allPvalues were less than 0.001; whereas the observations of CORM3 group were siginificantly lower than LPS groups, the correspondingPvalues were 0.005, <0.001 and 0.001, respectively.

圖1 4組新生鼠肺組織病理變化(蘇木精-伊紅染色，20 ×)

Fig 1 The lung tissue pathology of four groups (HE staining, 20×)

Notes A: The control group. The lung tissue kept normal morphological structure, and inflammatory cells infiltrated in the pulmonary interstitium were not obvious. B: LPS group. The morphological structure of lung tissue disordered, alveoli were compressed, and a lot of inflammatory cells infiltrated in pulmonary interstitium. C: The CORM3 group. Compared with the LPS group, the edema of lung tissue was relieved significantly in CORM3 group, and inflammatory cells infiltrated in alveolar interstitium were less. D: The iCORM3 group. The condition of edema of lung tissue and infiltration of inflammatory cells had not obviously improved

圖2 4組Tunel檢測肺泡細胞凋亡(40×)

Fig 2 The index of AECs apoptosis by Tunel assay(40×)

Notes A: control group, B:LPS group, C: CORM3 group, D: iCORM3 group. Transfer Tunel stain to DAB signal, brown signal of Tunel positive cells could be seen, and normal cells kept pale green signal. The red arrows shown in figure were apoptotic AECs

2.4 LPS刺激對A549細胞活力影響及CORM3的保護作用 圖3顯示，陽性對照細胞活性顯著低于陰性對照，加入CORM3干預后細胞活力逐漸回升，當CORM3濃度>0.1 mmol·L-1時，MTT結果顯示OD值不再發(fā)生明顯變化。

2.5 4組A549細胞凋亡率比較 4組A549細胞凋亡情況如圖4所示，對照組細胞凋亡率為(3.6±1.0)%，LPS組細胞凋亡率[(29.6±4.1)%]較對照組增加，差異有統計學意義(P<0.05)；CORM3組細胞凋亡率為(15.3±4.5)%，與LPS組差異有統計學意義(P<0.05)；iCORM3組細胞凋亡率為(27.6±8.7)%，與LPS組差異無統計學意義。

圖3 MTT檢測A549細胞活力變化

Fig 3 Detection of A549 cell vitality by MTT

Notes The protective role of CORM3 which directed against the reduction of activity of A549 cells induced by LPS. 1)P<0.05,vsnegative control group; 2)P<0.05,vspositive control (LPS) group

圖4 4組Tunel檢測A549細胞凋亡(20×)

Fig 4 Detection of A549 cell apoptosis by Tunel assay (20×) in 4 groups

Notes To define the apoptotic A549 cells by Tunel (upper panel) and Dapi (middle panel) double dying. The apoptotic A549 cells appeared green signal after Tunel dying, and the nucleus of A549 cells were dyed blue. They were affirmed as apoptotic cells when Tunel dying and Dapi dying overlapped (lower panel)

3 討論

新生鼠的免疫系統未完全成熟，在應對外源性刺激時所表現出的反應與成年鼠存在較大差異[17]，不同給藥方式所造成的組織損傷也有明顯區(qū)別，在本研究前期的預實驗中已得到了證實。因此，構建較理想的新生鼠ALI模型尤為重要。Martin團隊研究發(fā)現，采用氣管直接給予LPS刺激新生鼠和成年鼠，新生鼠BALF中炎性細胞的浸潤遠低于成年鼠[18]。LPS常用于構建各種炎癥模型[19,20]，其刺激誘發(fā)ALI的方式有多種，包括腹腔給藥[21]、尾靜脈給藥[22]、霧化吸入給藥[11]和氣管直接給藥[12]。在預實驗發(fā)現，腹腔給藥方式建立ALI模型，其損傷程度較輕，個體差異較大；而采用氣管給藥方式直接作用于肺泡，造成局部的ALI更為穩(wěn)定，且對其他臟器功能的影響較小，從肺組織病理學來看，LPS刺激12 h后，肺正常組織結構明顯紊亂，并伴隨大量炎性細胞浸入肺間質和肺泡，肺間質水腫、增寬，肺泡壓縮，并伴隨有大量肺泡細胞的凋亡，與文獻報道的結果相一致[23]。

目前，CO對ALI的保護作用已得到廣泛證實，但CO在新生鼠ALI的研究尚少。本研究發(fā)現，CORM3組較LPS組其肺組織結構紊亂明顯好轉，中性粒細胞的浸潤明顯減少，BALF蛋白含量、細胞數目顯著下降；而iCORM3干預未起到治療作用，提示CORM3復合物中真正發(fā)揮作用的是其攜帶的CO分子，這與CO在成年鼠中的保護效應相一致[24]。既往認為CO的保護與其抗炎效應有關[24,25]，過度的炎癥反應被認為是造成ALI的主要損傷因素，低濃度CO具有良好的抗炎效果，其機制與多條信號通路均有密切的聯系。近年發(fā)現， CO還能抑制血管內皮細胞的凋亡，起到保護ALI的作用[26]。本研究肺組織Tunel染色結果證實CORM3抑制了ALI導致的肺泡細胞凋亡。A549具有肺泡上皮細胞的生物學特征，廣泛運用于ALI體外實驗[27]，本研究MTT結果顯示，LPS作用24 h后，細胞活力明顯下降，而予CORM3干預，細胞活力呈增強趨勢。細胞Tunel染色同樣證實，CORM3能明顯抑制LPS刺激誘導的A549細胞凋亡。

既往研究顯示，CO對ALI的保護主要是抑制肺泡巨噬細胞相關炎癥因子和趨化因子的釋放，減輕炎癥反應，同時保護肺血管內皮功能[8]。本研究證實了CO可通過抑制肺泡上皮細胞凋亡，從而發(fā)揮其保護肺損傷的作用，這為探討CO在ALI中的保護機制提供了一個新的研究方向。

本研究的不足之處：由于新生鼠取材受限，沒能取得血氣變化的結果以及監(jiān)測血液CO濃度的變化。

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(本文編輯：丁俊杰)

CORM3 attenuates acute lung injury induced by LPS via inhibiting AECs apoptosis in neonatal rats

CAIKang-xing1,WANGLi1,WANGTing2,LUOLI2,CHENLong1,WANGNan1,SHIYuan1

(1DepartmentofPediatrics,DapingHospital,ThirdMilitaryMedicalUniversity,Chongqing400042; 2Department1,InstituteofSurgeryResearch,DapingHospital,ThirdMilitaryMedicalUniversity,StateKeyLaboratoryofTrauma,BurnsandCombinedInjury,Chongqing400042,China)

SHI Yuan，E-mail:petshi530@vip.163.com

ObjectiveTo study the protective effects of CORM3 treatment on AECs apoptosis after LPS inducing acute lung injury in neonatal rats. MethodsThirty two SD neonate rats were divided equally into four groups, the control group, LPS group,CORM3 group and iCORM3 group. Neonate rat acute lung injury was induced by LPS intratracheal administration in LPS group, CORM3 group and iCORM3 group received treatment of itraperitoneal injection of saline, CORM3 and iCORM3 respectively. The control group received intraperitoneal injection of saline. Animals in each group were sacrificed after 12h modeling, the histopathologic changes were observed by H-E staining, and lung tissue was seperated and weighed, wet and dry ratio of lung tissue was calculated, lung tissue damage was detected by BALF cell counting and protein quantitative analysis. Cultivated A549 cell apoptosis was induced by LPS in vitro, cell activity was determined by MTT test, cell apoptosis was watched by Tunel dyeing.ResultsFirstly, compared with the control group, the morphological structure of lung tissue was disordered in model group, alveoli were compressed, and a lot of inflammatory cells in filtrated in pulmonary interstitium. CORM3 group kept basic normal morphological structure, and inflammatory cells infiltrated in alveolar interstitium were less, the iCORM3 group was consistent with the LPS group in lung morphological structure and inflammatory cells infiltration. Secondly, compared with the control group, the W/D ratio, BALF cell number and protein content increased significantly in LPS group, and the number of AECs apoptosis was increased obviously[(37.3±4.5)%vs(3.0±1.0)%]. The A549 cells activity was decreased, and percentage of apoptosis cells increased significantly[(29.6±4.1)%vs(3.6±1.0)%,P<0.05],with a statistically significant difference. In CORM3 group compared with the LPS group, the W/D ratio, BALF cell number and protein content decreased obviously, and the number of AECs apoptosis was decreased[(19.3±4.6)%]. The A549 cells activity rebounded, and the rate of cells apoptosis[(15.3±4.5)%] decreased significantly, the difference was statistically significant(P < 0.05).There were no significant changes between the iCORM3 group and the CORM3 group in these indexes.ConclusionCORM3 attenuates neonate rat acute lung injury induced by LPS via inhibiting AECs apoptosis.

Carbon monoxide-releasing molecule 3; Lipopolysaccharid; Lung injury; Apoptosis; Alveolar epithelial cell

重慶市科技計劃項目:cstc2013yykfA0193, 國家自然科學基金:81100458

1 第三軍醫(yī)大學附屬大坪醫(yī)院兒科 重慶，400042；2 第三軍醫(yī)大學附屬大坪醫(yī)院野戰(zhàn)外科研究所一室 重慶，400042

史源，E-mail:petshi530@vip.163.com

10.3969/j.issn.1673-5501.2015.06.012

2015-07-20

2015-11-23)