芥子气经腹腔和气管致大鼠急性肺损伤细胞凋亡的变化

Changes of apoptosis in acute pulmonary injury of rats induced by intraperitoneal and tracheal injection of sulfur mustard

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 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),正常对照组未做任何处理。采用末端脱氧核苷酸转移酶介导的d UTP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72 h,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 rat models of sulfur mustard （SM）-induced acute lung injury via intraperitoneal and tracheal injection, and compare the difference in apoptosis of the two models. Methods A total of 136 male Sprague Dawley rats were selected and randomly divided into control group with 8 cases and other four groups （i.e. intraperitoneal SM group, intraperitoneal glycol propylene group, tracheal SM group and tracheal glycol propylene group with 32 cases in each group）. The intraperitoneal SM group was intraperi- toneally injected with 0.1 ml diluted SM （0.96 LD50= 8 mg/kg）, the tracheal SM group had intratracheal injec- tion of 0.1 ml diluted SM （0.98 LD50 = 2 mg/kg）, meanwhile the status quo was kept with the control group. SM-induced apoptosis was observed by TUNEL staining and immunohistochemical staining as well as electron microscopy. Results In the alveolar septum, the expression rate of positive cells by TUNEL staining in the intraperitoneal SM group was increased compared with that in the tracheal SM group at the same period of time （P〈 0.05）. In the alveolar septum, a significantly higher positive expression rate of Bax protein was detected by immunohistochemical staining in the intraperitoneal SM group at different periods of time compared with that in the tracheal SM group at the corresponding period （P〈 0.05）; while a significantly lower positive expression rate of Bcl-2 protein was detected by immunohistochemical staining in the intraperitoneal SM group at different periods of time compared with that in the tracheal SM group at the corresponding period （P 〈 0.05）. In the alveolar septum, the expression rates of caspase-3 and caspase-9 by immunohistochemical stain- ing in the intraperitoneal SM group at different periods of time were increased compared those with the tra- cheal SM group at the corresponding period （P〈 0.05）. Electron microscopic observation confirmed that both type I and type II alveolar epithelial cells in the lungs exhibited apoptotic morphologic features, such as break, loss and disarrangement of the microvilli on cell membrane, blurred mitochondrial cristae, and detach- ment and dissociation of the ribosomes from the surface of the rough endoplasmic reticula. Conclusions Our results showed that dysregulation of apoptosis via intrinsic pathways in the intraperitoneal SM group and the tracheal SM group leads to up-regulation of apoptosis. In SM-induced acute lung injury in rats via intraperitoneal route, the index of apoptosis is significantly higher than that via tracheal route, which may be related to fast absorption of SM in the peritoneal cavity.