未折叠蛋白反应在严重烧伤大鼠淋巴液诱导肺内皮细胞凋亡中的作用

Lymph of rats with severe scalds induces apoptosis in vascular endothelial cell via the unfolded protein response

目的探讨未折叠蛋白反应介导的凋亡通路在严重烧伤大鼠淋巴液对大鼠肺微血管内皮细胞损伤中的作用。方法选成年Wistar雄性大鼠20只,分为烫伤组(行30%体表面积Ⅲ度烫伤)和假伤组,建立大鼠胸导管淋巴瘘模型,收集伤后24 h淋巴液,用于体外实验。将细胞实验分为烫伤淋巴液刺激组(A组)、烫伤淋巴液+4苯基丁酸(4-PBA)处理组(B组)和假伤淋巴液处理组(C组)。刺激6 h后,Annexin V/PI标记法流式细胞术检测细胞凋亡。western blot检测caspase12、葡萄糖调节蛋白78(GRP78)和C/EBP同源蛋白(CHOP)等蛋白表达的情况。结果 A组细胞凋亡水平显著高于C组(P<0.01),B组细胞凋亡水平显著低于A组(P<0.01)。A组GRP78、CHOP和caspase12蛋白表达水平显著高于C组,经PBA处理后3种蛋白水平显著降低(P<0.01)。结论烧伤大鼠淋巴液可以引起血管内皮细胞凋亡,未折叠蛋白反应可能是其主要通路之一。

Objective To investigate the effects of the unfolded protein response （UPR） in endoplasmic reticulum stress on the apoptosis of rat pulmonary microvascular endothelial cells （rPMECs） induced by lymph from rats with severe scalds. Methods Twenty Wistar rats were divided into bum group （n = 10） and sham burn group （n = 10）. Lymph was harvested within 24 hours after the establishment of scalds model. Cultured rPMECs were divided into scald lymph stimulated group （ A, stimulated by 5 % scalded rat lymph）, scald lymph and 4-PBA group （ B, stimulated by 5 % scalded rat lymph and 5mM 4-PBA） and control group（ C ,5% sham scalded rats lymph）. Cells apoptosis was determined by flow cytometery with annexin V/PI. Expressions of protein of GRP78, CHOP and Caspase 12 were determined by western blotting. Results Significant apoptotic cells were determined in group A, which was higher than that of group C（P 〈 0.01 ）. There was significant decrease of apoptosis of cell treated with 4-PBA and burn lymph. Protein level of GRP78, CHOP and Caspasel2 increased significantly when treated with burn lymph, but decreased when treated by 4- PBA（P 〈 0.01 ）. Conclusion Lymph from rats with severe scalds can induce significant apoptosis in rPMECs. UPR may be the main pathway of apoptosis, which can be improved by 4-PBA.