Janus激酶/信号转导和转录激活因子3对重症急性胰腺炎大鼠肺泡Ⅱ型上皮细胞损伤的体外作用研究

摘要

Objective To study the role of Janus kinase/signal transducer and activator of transcription 3(JAK/STAT3)in injury of alveolar typeⅡepithelial cells(ATⅡ)in severe acute pancreatitis(SAP).Methods The rat model of SAP was reproduced intraductal injection of sodium taurocholate.Serum was collected for examinations.ATⅡcells in primary culture were randomized to be treated with serum from rats with SAP(SAP group),or SAP serum+AG490(JAK inhibitor,SAP+AG490 group),while the normal cell control was cultured with Dulbecco improvemed Eagle medium(DMEM).ATⅡcells were collected after the treatment to determine the activation of STAT3 by electrophoretic mobility shift assay(EMSA),STAT3 mRNA expression by reverse transcription-polymerase chain reaction(RT-PCR),STAT3 protein expression by Western blotting,surfactant protein C(SP-C)level in ATⅡand apoptosis of ATⅡwith flow cytometry.Results Compared with control group,activation of STAT3 was enhanced in SAP group,and expression of STAT3 mRNA and protein was also enhanced,level of SP-C protein declined(2 hours SP-C fluorescence index 0.69±0.02 vs.1.02±0.03,P＜0.01),and apoptosis of ATⅡincreased[(11.55±1.10)%vs.(5.30±0.36)%,P＜0.053.Compared with SAP group,activation of STAT3 was attenuated in SAP+AG490 group,expression of STAT3 mRNA and protein was lowered,and level of SP-C protein declined(2 hours SP-C fluorescence index 0.48±0.10 vs.0.69±0.02,P＜0.01),and the apoptosis of AT Ⅱ increased[(13.92±0.82)%vs.(11.55±1.10)%,P＜0.05].Conclusion The results suggest that JAK/STAT3 pathway might be involved in the pathogenesis of injury to ATⅡin SAP.%目的 探讨Janus激酶/信号转导和转录激活因子3(JAK/STAT3)信号转导通路在重症急性胰腺炎(SAP)肺泡Ⅱ型上皮细胞损伤中的作用.方法 用牛磺胆酸钠建立SAP大鼠模型,取血清备用.将经原代培养的肺泡Ⅱ型上皮细胞随机分组,对照组加入不含SAP大鼠血清的Dulbecco改良Eagle培养基(DMEM)培养液;SAP组加入含SAP大鼠血清的DMEM培养液;SAP+AG490组用JAK激酶抑制剂AG490预处理细胞,加入含SAP大鼠血清的DMEM培养液.用电泳迁移率改变分析法(EMSA)检测STAT3活化状态;逆转录一聚合酶链反应(RT-PCR)检测mRNA表达;蛋白质免疫印迹法(Western blotting)检测蛋白水平;流式细胞技术检测肺泡表面活性物质相关蛋白C(SP-C)表达和肺泡Ⅱ型上皮细胞凋亡.结果 与对照组比较,SAP组STAT3活性增强,STAT3 mRNA和蛋白表达均增强,SP-C蛋白表达下降(2 h SP-C荧光指数0.69±0.02比1.02±0.03,P＜0.01),肺泡Ⅱ型上皮细胞凋亡增加[(11.55±1.10)%比(5.30±0.36)%,P＜0.053;与SAP组比较,SAP+AG490组STAT3活性减弱,STAT3 mRNA和蛋白表达减弱,SP-C蛋白表达下降(2 h SP-C荧光指数0.48±0.10比0.69±0.02,P＜0.01),肺泡Ⅱ型上皮细胞凋亡增加[(13.92±0.82)%比(11.55±1.10)%,P＜0.053.结论 提示JAK/STAT3信号转导通路参与了SAP肺泡Ⅱ型上皮细胞损伤的病理生理过程.