RNF99通过TAK1/NF-κB信号通路参与泛素化与脓毒症性休克的潜在联系  

Involvement of RNF99 in potential link between ubiquitination and septic shock via TAK1/NF⁃κB signal⁃ing pathway

作　　者：张弛 胡赛[1] 王静[1] 夏凤强[1] 程晓英 甘泽英 ZHANG Chi;HU Sai;WANG Jing;XIA Fengqiang;CHENG Xiaoying;GAN Zeying(Department of Critical Care Medicine,Changsha Fourth Hospital(Changsha Hospital Affiliated to Hunan Normal University),Changsha 410006,China)

机构地区：[1]长沙市第四医院(湖南师范大学附属长沙医院)重症医学科,长沙410006

出　　处：《实用医学杂志》2024年第5期615-620,626,共7页The Journal of Practical Medicine

基　　金：湖南省卫健委科研计划项目(编号:202202331255)。

摘　　要：目的 探讨环指蛋白99(RNF99)介导的转化生长因子激酶1(TAK1)/核因子-κB(NF-κB)信号通路参与泛素化与脓毒症性急性呼吸窘迫综合征(ARDS)的潜在联系。方法 进行质粒和siRNA转染以过表达或敲低小鼠肺泡上皮细胞(MLE12)中RNF99,分析磷酸p65和p65蛋白表达。免疫沉淀分析RNF99与TRAF6和TAK1的蛋白相互作用关系。将40只小鼠随机分成WT+PBS、WT+LPS、RNF99特异性表达(TG)+PBS和TG+LPS组,每组10只。通过腹膜内注射30 mg/kg LPS诱导脓毒症。结果 与Vector组相比,RNF99组MLE12细胞中TRAF6和TAK1的蛋白表达水平显著降低(P<0.05)。泛素化TRAF6蛋白在RNF99敲低的MLE12细胞中增加。与LPS+Vector组相比,在LPS+RNF99组MLE12细胞中p65的磷酸化水平明显降低(P <0.05)。与si-NC组相比,si-RNF99组MLE12细胞中RNF99、IκBα的蛋白表达水平显著降低(P <0.05)。与LPS+si-NC组相比,在LPS+si-RNF99组MLE12细胞中p65的磷酸化水平明显增加(P <0.05)。TG+LPS组小鼠肺组织中CD68巨噬细胞染色百分比较WT+LPS组显著降低(P <0.05)。TG+LPS组小鼠肺组织中p65的磷酸化水平显著低于WT+LPS组小鼠(P <0.05)。结论 RNF99通过与NF-κB信号通路的关键调节因子(TRAF6/TAK1)相互作用来调节NF-κB信号通路,并改善小鼠腹腔注射LPS后肺损伤。Objective To explore the potential relationship between ubiquitination of transforming growth factor kinase 1(TAK1)/nuclear factor-κB(NF-κB)signaling pathway mediated by ring finger protein 99(RNF99)and septic acute respiratory distress syndrome(ARDS).Methods Plasmid and siRNA transfection were conducted to overexpress or knock down RNF99 in MLE12,and expressions of p65 phosphate and p65 protein were analyzed.The protein interaction between RNF99 and TRAF6 or TAK1 was analyzed by immunoprecipitation assay.Forty mice were randomly divided into WT plus PBS,WT plus LPS,RNF99 specific expression(TG)plus PBS,and TG plus LPS groups,with 10 mice in each group.Sepsis was induced by intraperitoneal injection of 30 mg/kg LPS.Results As compared with vector group,protein expression levels of TRAF6 and TAK1 in MLE12 cells decreased significantly in RNF99 group(P<0.05).Ubiquitinated TRAF6 protein increased in MLE12 cells with RNF99 knockdown.As compared with LPS plus vector group,phosphorylation level of p65 in MLE12 cells was signifi-cantly lower in LPS plus RNF99 group(P<0.05).As compared with si-NC group,protein expression levels of RNF99 and IκBαin si-RNF99 group decreased significantly(P<0.05).As compared with LPS plus si-NC group,phosphorylation level of p65 in LPS plus si-RNF99 group increased significantly(P<0.05).The staining percentage of CD68 macrophages in lung tissues was significantly lower in TG plus LPS group than in WT plus LPS group(P<0.05).Phosphorylation level of p65 in lung tissues was significantly lower in TG plus LPS group than in WT plus LPS group(P<0.05).Conclusion RNF99 regulates NF-κB signaling pathway by interacting with the key regulator of NF-κB signaling pathway(TRAF6/TAK1),and improves lung injury after intraperitoneal injection of LPS in mice.

关 键 词：环指蛋白99 转化生长因子激酶1 核因子-ΚB 脂多糖 肺泡上皮细胞 

分 类 号：R631.2[医药卫生—外科学]