ATP/P2X7轴介导的K^(+)外排促进NDV感染的ECA109细胞中NLRP3炎性小体激活  

The ATP/P2X7 axis-mediated K^(+)efflux promotes NLRP3 inflammasome activation in NDV-infected ECA109 cells

作　　者：曹旭 武彩霞 兰金苹 王静 贾朝霞 刘浩 刘开扬 Cao Xu;Wu Caixia;Lan Jinping;Wang Jing;Jia Zhaoxia;Liu Hao;Liu Kaiyang(Life Science Research Center of Hebei North University,Zhangjiakou 075000)

机构地区：[1]河北北方学院生命科学研究中心,张家口075000

出　　处：《安徽医科大学学报》2023年第1期42-47,共6页Acta Universitatis Medicinalis Anhui

基　　金：河北省自然科学基金(编号:H2018405044);河北北方学院省属高校基本科研业务费青年基金(编号:301010300105219);张家口市重点研发计划项目(编号:1921013D)。

摘　　要：目的探究新城疫病毒(NDV)感染食管癌ECA109细胞后,NLRP3炎性小体是否激活、其激活是否与K^(+)外排有关及ATP/P2X7轴对NLRP3炎性小体激活的影响。方法Western blot检测NLRP3和IL-1β表达情况;ELISA检测上清液中IL-1β含量;荧光免疫技术检测ASC斑点的形成情况;荧光探针技术检测细胞内K^(+)浓度变化;使用ATP酶、ATP及P2X7受体抑制剂进行干预,研究其在NLRP3炎性小体激活中的作用。结果与control组比较,NDV F3感染细胞后,细胞内NLRP3、IL-1β和ASC蛋白表达上调;胞内钾离子浓度随感染时间延长而降低(P<0.05)。P2X7受体抑制剂干预后,胞内K^(+)外流受阻,随抑制剂浓度的增加,K^(+)外流在10 mol/L处受最大抑制(P<0.05)。ATP酶及ATP干预结果显示,ATP酶抑制K^(+)外流,ATP促进K^(+)外流。Western blot结果显示,与control组比较,抑制P2X7受体,NLRP3和IL-1β蛋白表达下调;ATP酶干预细胞后,NLRP3和IL-1β表达降低;ATP干预细胞后,NLRP3、IL-1β蛋白表达上调(P<0.05)。结论NDV F3感染ECA109细胞能激活NLRP3炎性小体,其机制可能与ATP/P2X7轴有关。Objective To explore whether the NLRP3 inflammasome is activated after Newcastle disease virus(NDV)exposure to esophageal cancer ECA109 cells,whether its activation is related to K^(+)efflux,and the effect of ATP/P2X7 axis on the activation of NLRP3 inflammasome.Methods The expression of NLRP3 and IL-1βwas detected by Western blot;the content of IL-1βin the supernatant was detected by ELISA;the formation of ASC spots was detected by fluorescence immunoassay;the change of intracellular K^(+)concentration was detected by fluorescent probe technology;Interventions with ATPase,ATP and P2X7 receptor inhibitors were used to investigate their role in NLRP3 inflammasome activation.Results Compared with the control group,the expression of NLRP3,IL-1βand ASC protein in cells was up-regulated after NDV F3 infection;the intracellular potassium concentration decreased with the prolongation of infection time(P<0.05).After the intervention of P2X7 receptor inhibitor,the efflux of intracellular K^(+)was blocked.With the increase of inhibitor concentration,the efflux of K^(+)was maximally inhibited at 10μmol/L(P<0.05).The results of ATPase and ATP intervention showed that ATPase inhibited K^(+)efflux,while ATP promoted K^(+)efflux.Western blot results showed that compared with the control group,P2X7 receptor was inhibited,and the expressions of NLRP3 and IL-1βwere down-regulated;after ATPase intervened cells,the expressions of NLRP3 and IL-1βdecreased;After ATP intervention in cells,the protein expressions of NLRP3 and IL-1βwere up-regulated(P<0.05).Conclusion NDV F3 infection of ECA109 cells can activate the NLRP3 inflammasome,the mechanism may be related to the ATP/P2X7 axis.

关 键 词：新城疫病毒 ATP P2X7 K^(+) NLRP3炎性小体 ECA109细胞 

分 类 号：R737.33[医药卫生—肿瘤]