摘要
目的探讨Wnt-1诱导信号通路蛋白3/调节蛋白6(WISP-3/CNN6)与半胱氨酸蛋白酶-8(Caspase-8)共结合在高氧诱导肺上皮细胞凋亡中的作用与可能机制.方法利用小干扰RNA和质粒转染实现对人肺上皮Beas-2B细胞中WISP-3/CCN6基因的沉默或过表达,分别将低表达或过表达WISP-3/CCN6的Beas-2B细胞置高氧(95%O2和5%CO2)或空气中处理0、24、48 h,通过蛋白免疫印迹法和流式细胞仪检测各组细胞中Caspase途径凋亡相关蛋白Caspase-8、Caspase-3及活性片段cleaved Caspase-8、cleaved Caspase-3和cleaved多聚ADP核糖聚合酶(PARP)的表达量及Caspase-8活性,通过免疫共聚焦和免疫共沉淀检测WISP-3/CCN6与Caspase-8的蛋白相互作用.结果高氧刺激48 h,WISP-3/CCN6沉默的Beas-2B细胞中cleaved Caspase-8、cleaved Caspase-3和cleaved PARP蛋白表达量均比0 h时增加(P均<0.017),且Caspase-8的活性增高(P<0.05);过表达WISP-3/CCN6的Beas-2B细胞中cleaved Caspase-8蛋白表达量比0 h时减少(P<0.017),cleaved Caspase-3和cleaved PARP的蛋白表达量在24 h时比0 h时升高、在48 h时比24 h时下降(P均<0.017).高氧刺激下,Beas-2B细胞中WISP-3/CCN6蛋白和Caspase-8蛋白结合减少.结论WISP-3/CCN6通过Caspase途径参与高氧诱导肺上皮细胞凋亡的调控,其作用可能通过与Caspase-8蛋白结合方式实现.
Objective To investigate the role and the molecular mechanism of WISP-3/CCN6 binding with Caspase-8 in the hyperoxia-induced apoptosis of the lung epithelial cells.Methods The silencing and overexpression of WISP-3/CCN6 in the human lung bronchial epithelial cells(Beas-2B)were performed by plasmid transfection and siRNA.The Beas-2B cells with silencing or overexpressing WISP-3/CCN6 were maintained in hyperoxia(95%O2 and 5%CO2)or air for 0,24 and 48 h,respectively.The expression levels of key proteins of Caspase signaling pathway(Caspase-8,Caspase-3,cleaved Caspase-8,cleaved Caspase-3 and PARP)were detected with Western blot and the activity of Caspase-8 was determined with flow cytometry.The protein interaction between WISP-3/CCN6 and Caspase-8 was analyzed by immunoconfocal and immunocoprecipitation.Results After 48-h hyperoxia treatment,the expression levels of cleaved Caspase-8,cleaved Caspase-3 and cleaved PARP in the Beas-2B cells with silencing WISP-3/CCN6 were significantly up-regulated(all P<0.017)and the activity of Caspase-8 was remarkably increased compared with those at 0 h(P<0.05).The expression of cleaved Caspase-8 in the Beas-2B cells with overexpressing WISP-3/CCN6 was significantly down-regulated than that at 0 h(P<0.017).The expression levels of cleaved Caspase-3 and cleaved PARP at 24 h were significantly up-regulated than those at 0 h, whereas the expression levels at48 h were significantly down-regulated compared with those at 24 h( all P < 0.017). The protein interactionbetween WISP-3/CCN6 and Caspase-8 was signigficantly decreased in the Beas-2B cells after hyperoxia.Conclusion WISP-3/CCN6 participates in the regulation of hyperoxia-induced apoptosis of lung epithelialcells via the Caspase signaling pathway, probably by binding with Caspase-8 protein.
作者
黄晓梅
王康玮
唐万娜
赵祝香
赵子文
魏树全
Huang Xiaomei;Wang Kangwei;Tang Wanna;Zhao Zhuxiang;Zhao Ziwen;Wei Shuquan(Department of Pulmonary and Critical Care Medicine,Guangzhou First People’s Hospital,the Second Affiliated Hospital of South China University of Technology,Guangzhou 510180,China)
出处
《新医学》
2020年第4期267-273,共7页
Journal of New Medicine
基金
广东省医学科学技术研究基金(A2015385)。
