Objective: Sulfur mustard (SM) is a vesicant alkylating agent. There are still biochemical mechanisms underlying SM damage that are unknown. This study was to determine the differences of apoptosis and pulmonary fibrosis underlying SM-induced acute pulmonary injury via intraperitoneal injection and intratracheal instillation in rats. Methods: An acute pulmonary injury model was established in a preliminary experiment using intraperitoneal injection and intratracheal instillation of SM in rats, in order to determine the equal toxicity dose of SM (1LD50). Then, SM-induced changes in apoptosis and pulmonary fibrosis were observed by immunohistochemical staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Masson staining and electron microscopic observationin intraperitoneal and tracheal SM groups that were injected intraperitoneally and instilled intratracheally with diluted SM [0.1 mL per rat (0.98 LD50 = 2 mg/kg)] and SM [0.1 mL per rat (0.96 LD50 = 8 mg/kg)] respectively, and an untreated control group. Results: In the alveolar septum, the positive expression ratio of apoptotic cells, Bax, caspase-3, and caspase-9 by TUNEL and Immunohistochemistry (streptavidin-perosidase method) in the intraperitoneal SM group was increased compared with the tracheal SM group (P < 0.05), but a significantly lower positive expression ratio of Bcl-2 was detected (P < 0.05). Electron microscopic observations confirmed that the type I and II alveolar epithelial cells in lungs exhibited apoptotic morphologic features, such as cracked, lost, and disordered microvilli of membranes, fuzzy mitochondrial cristae, and detached, dissociated ribosomes from the surface of the rough endoplasmic reticulum. A significantly higher positive expression ratio of MMP-2, MMP-9, TIMP-1, TIMP-2, collagen type I, collagen type III, TGF-beta 1, and Smad7 by immunohistochemical staining in the alveolar septum were detected in the intraperitoneal SM group compared with the tracheal SM group (P < 0.05). Conclusions: These data demonstrated increased apoptosis and pulmonary fibrosis via intraperitoneal injection under equal toxicity doses SM (1LD(50)) in rats.

目的 经腹腔和气管建立大鼠芥子气(SM)肺损伤的动物模型,比较两种大鼠急性肺损伤模型炎性反应的差异.方法 选取Sprague Dawley大鼠136只,分为5组,正常对照组8只,其他4个组(腹腔SM组、腹腔丙二醇对照组、气管SM组、气管丙二醇对照组)每组32只.腹腔SM组腹腔内注入稀释的SM 0.1 mL(0.96 LD50＝8 mg/kg),气管SM组气管内注入稀释的SM 0.1 mL(0.98 LD50 =2 mg/kg),正常对照组不做任何处理.ELISA法检测支气管肺泡灌洗液和血液标本,HE染色和免疫组织化学判断炎性反应情况.结果 腹腔SM组各时间段支气管肺泡灌洗液蛋白含量和细胞计数与气管SM组相比显著升高(P＜0.05);腹腔SM组各时间段血清TNF-α、IL-1β、IL-6与气管SM组相比显著升高(P＜0.05);腹腔SM组各时间段肺泡间隔T、B淋巴细胞和巨噬细胞阳性表达率与气管SM组相比显著增加(P＜0.05).结论 大鼠在SM LD50相似的情况下,腹腔SM组支气管肺泡灌洗液、肺泡间隔及血清炎性反应指标明显高于气管SM组.

目的 经腹腔和气管建立大鼠芥子气(SM)急性肺损伤动物模型,比较两种大鼠急性肺损伤模型细胞凋亡的差异.方法 选取Sprague Dawley大鼠136只,随机分为5组,正常对照组8只,其他4个组为腹腔SM组、腹腔丙二醇对照组、气管SM组、气管丙二醇对照组,每组32只.腹腔SM组腹腔内注入稀释的SM0.1 m1(0.96 LD50=8 mg/kg),气管SM组气管内注入稀释的SM 0.1 m1(0.98 LD50=2mg/kg),正常对照组未做任何处理.采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)染色和免疫组织化学法及电镜观察,判断细胞凋亡情况.结果 ①腹腔SM组各时间段肺泡间隔TUNEL染色阳性细胞表达率较气管SM组增多(P＜0.05).②腹腔SM组各时间段肺泡间隔凋亡蛋白Bax阳性表达率较气管SM组升高(P＜0.05);腹腔SM组各时间段肺泡间隔凋亡蛋白Bcl-2阳性表达率较气管SM组降低(P＜0.05).③腹腔SM组各时间段肺泡间隔凋亡蛋白酶Caspase-3、Caspase-9阳性表达率较气管SM组增多(P＜0.05).④电镜显示,染毒72h,腹腔SM组和气管SM组Ⅰ型和Ⅱ型肺泡上皮凋亡细胞形态特征为上皮细胞膜附着的微绒毛断裂缺失,排列紊乱;线粒体嵴模糊,粗面内质网表面附着的核糖体脱离,并游离于细胞质中.结论 SM经腹腔和气管染毒致大鼠急性肺损伤,通过内源性通道引发细胞凋亡调节异常,SM经腹腔染毒大鼠各项细胞凋亡指标比经气管明显升高,推测可能与SM腹膜腔的快速吸收有关.

Objective To establish the sulfur mustard (SM ) induced tracheal injury model in rat and to investigate its mecha-nism .Methods Male rats (SD) were anesthetized and intra-tracheally intubated .The SM group was intra-tracheally injected by 2 mg/kg of diluted SM ,while the propylene glycol control group only by 0 .1mL of propylene glycol and the normal control group had no any treatment .The tissue and blood samples were taken for conducting the HE and immunohistochemical staining and measuring serum enzymes and andinflammatory factors .Results In the SM group ,a large number of lymphocytes infiltration in submucosa were observed;the positive expression of caspase-3 and caspase-9 were observed in epithelium and submucosa ;serum levels of TNF-α,IL-1β,IL-6 reached the peak in 24 h;serum levels of LDH ,GP ,BARS reached the peak in 6h ,so did GGT in 24 h .In the propyl-ene glycol control group and the normal control group ,lymphocytes ,macrophages and neutrophils were rare in submucosa .Conclu-sion The mechanism of SM (2 mg/kg) induced acute tracheal injury involves the inflammatory reaction ,apoptosis and oxidative stress ,moreover the lesion degree has the correlation with time .