自噬在光气诱导急性肺损伤中的作用

Role of autophagy in phosgene-induced acute lung injury

目的建立光气鼻吸入染毒所致大鼠急性肺损伤(ALI)模型和光气染毒BEAS-2B细胞(人源支气管上皮细胞)损伤模型,探讨自噬在光气致ALI中的作用。方法在体内实验中,将40只SD大鼠随机分为4组:对照组,光气染毒后3 h组、6 h组和12 h组,对照组给予空气,光气组大鼠经鼻吸入1230 mg/m^(3)光气30 min。测定肺湿质量、肺体比、肺泡灌洗液(BALF)总蛋白含量;HE染色切片观察肺组织病理变化;Western blotting检测肺组织自噬相关蛋白LAMP2a、LC3-Ⅱ/Ⅰ、P62表达。在体外实验中,将BEAS-2B细胞分为对照组、光气组,对照组给予空气,光气组给予238.7 mg/m^(3)光气动态染毒细胞60 min,12 h后PCR检测IL-1β、IL-6、IL-8 mRNA的水平,使用自噬双标腺病毒(mRFP-GFP-LC3)检测支气管上皮细胞自噬流的变化;给予自噬激动剂雷帕霉素干预后,检测细胞内IL-1β、IL-6、IL-8 mRNA的水平。结果与对照组相比,光气染毒大鼠肺湿质量、肺体比和BALF中总蛋白明显增高(P<0.01);光镜下可见染毒组大鼠肺间质增厚,炎症细胞浸润,肺泡腔有渗出液;染毒组大鼠肺组织LAMP2a和LC3-Ⅱ/Ⅰ蛋白表达明显降低(P<0.01),P62蛋白表达明显增高(P<0.01);光气染毒细胞后12 h,细胞炎症因子升高(P<0.05),自噬流降低(P<0.05);给予雷帕霉素后,与光气组相比,支气管上皮细胞IL-1β、IL-6、IL-8 mRNA水平降低(P<0.05)。结论光气能造成大鼠ALI和支气管上皮细胞发生炎症反应,并抑制自噬;自噬激动剂雷帕霉素可缓解光气对细胞的炎症反应,提示光气染毒后自噬降低可能是光气所致ALI的重要机制。

Objective To establish a rat model of acute lung injury(ALI)caused by nasal inhalation of phosgene and a model of human bronchial epithelial cell line BEAS-2B injury induced by phosgene,and to explore the role of autophagy in ALI induced by phosgene.Methods In in vivo experiment,40 SD rats were randomly divided into 4 groups:control group,3 h group,6 h group and 12 h group after phosgene poisoning.Rats were exposed to air in the control group,and the phosgene groups were given 1230 mg/m^(3) phosgene nasally for 30 min.Then we determined lung wet weight,lung-to-body ratio,and total protein content in bronchoalveolar lavage fluid(BALF).Pathological changes of the lung were observed by HE staining.The expression of autophagy-related proteins was detected by Western blotting,such as LAMP2a,LC3-Ⅱ/Ⅰ,and P62 in the lung.Human-derived bronchial epithelial BEAS-2B cells were divided into control group and phosgene group in vitro.The control group was exposed to air,and the phosgene group was poisoned by 238.7 mg/m^(3) phosgene for 60 min.The mRNA levels of IL-1β,IL-6,and IL-8 were detected by PCR after 12 h.The change of autophagy flow in bronchial epithelial cells was detected using mRFP-GFP-LC3.The mRNA levels of IL-1β,IL-6 and IL-8 were detected after intervention with rapamycin,an autophagy agonist.Results Compared with the control group,the lung wet weight,lung-to-body ratio and total protein content in BALF were significantly higher in rats exposed to phosgene(P<0.01).The thickened lung interstitium,infiltrated inflammatory cells,and exudate in the alveolar cavity were found under the light microscope in rats exposed to phosgene.The expressions of LAMP2a and LC3-Ⅱ/Ⅰprotein went down(P<0.01),and the expression of P62 protein increased(P<0.01)in the lung tissue of rats exposed to phosgene.The inflammatory factors increased(P<0.05),and autophagy flow decreased(P<0.05)in 12 h group after phosgene poisoning.The expressions of IL-1β,IL-6 and IL-8 in bronchial epithelial cells reduced in the cells treated with rapamycin compared with the phosgene group(P<0.05).Conclusion Phosgene can cause ALI in rats and inflammation in human bronchial epithelial cells,and weaken autophagy.The autophagy agonist rapamycin can alleviate the inflammatory response in cells exposed to phosgene,which suggests that reduction of autophagy may be an important mechanism of ALI induced by phosgene.