He Junbing, Zhao Tian, Liu Lizhen, Liao Shuanglin, Yang Shuai, Lu Furong, Hong Yuan, Wei Ning, Cheng Hongxiao, Zhang Wenying, Shao Yiming
The Intensive Care Unit, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, Guangdong, China; The Intensive Care Unit, The Medical Research Center, Jieyang Affiliated Hospital, Sun Yat-sen University, Jieyang, Guangdong, China.
The Intensive Care Unit, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, Guangdong, China.
Int Immunopharmacol. 2022 Jan;102:108385. doi: 10.1016/j.intimp.2021.108385. Epub 2021 Nov 30.
A disintegrin and metalloproteinase 17 (ADAM17) is a proteolytic cleaving protein with a crucial function in the inflammatory responses, especially sepsis. But the clear role of ADAM17 in sepsis and the underlying mechanism remained unknown. In this study, we aim to determine the clinical association of ADAM17 -172A > G (rs12692386) promoter polymorphism with sepsis and to further explore the effect and mechanism of the early growth response 1 (EGR1)/ADAM17 pathway in inflammatory process during sepsis.
A total of 477 sepsis patients and 750 controls were enrolled in this study to determine the association of ADAM17 -172A > G polymorphism with sepsis. The transcription factor binding to the promoter region of ADAM17 gene was predicted by bioinformatics analysis and verified by Chromatin Immunoprecipitation (ChIP) and luciferase assays. Quantitative real-time PCR and Western blot were performed to detect EGR1 and ADAM17 expression. Cytokine production was detected by enzyme-linked immunosorbent assay. The effect of EGR1/ADAM17 pathway on sepsis-induced inflammatory responses was evaluated in EGR1-silenced cells and endotoxemia mouse model.
The frequencies of non-survivors among the sepsis patients with the -172AG/GG genotypes and G allele were distinctly higher than those among patients with the AA genotype (53.9% vs. 39.7%, OR = 1.779, 95% CI = 1.119-2.829, P = 0.0142) and A allele (30.9% vs. 22.2%, OR = 1.570, 95% CI = 1.095-2.251, P = 0.0136). The Kaplan-Meier survival analysis indicated that the 28-day survival in septic patients with -172AG/GG genotypes of this functional ADAM17 promoter polymorphism was much worse than in the AA genotype carriers (log-rank = 5.358, P = 0.021). The results of in vitro lipopolysaccharide-stimulated and luciferase assays indicated that the -172 A-to-G variation could functionally upregulate promoter activity and transcription of ADAM17 gene via enhancing the binding affinity of its promoter region with the EGR1. The ChIP assay identified the direct interaction. Further studies demonstrated that inhibition of EGR1 significantly decreased ADAM17 expression and the pro-inflammatory cytokine secretion in vitro, and improved the survival and inflammatory response of sepsis mouse model.
These results provided evidence that the ADAM17 -172A > G polymorphism functionally promoted ADAM17 expression and enhanced sepsis-induced inflammatory responses via the EGR1/ADAM17 pathway, which ultimately conferred susceptibility to sepsis mortality and poor prognosis.
解整合素金属蛋白酶17（ADAM17）是一种蛋白水解切割蛋白，在炎症反应尤其是脓毒症中起关键作用。但ADAM17在脓毒症中的明确作用及潜在机制仍不清楚。在本研究中，我们旨在确定ADAM17 -172A>G（rs12692386）启动子多态性与脓毒症的临床关联，并进一步探讨早期生长反应1（EGR1）/ADAM17通路在脓毒症炎症过程中的作用及机制。
本研究共纳入477例脓毒症患者和750例对照，以确定ADAM17 -172A>G多态性与脓毒症的关联。通过生物信息学分析预测与ADAM17基因启动子区域结合的转录因子，并通过染色质免疫沉淀（ChIP）和荧光素酶测定进行验证。采用定量实时PCR和蛋白质印迹法检测EGR1和ADAM17的表达。通过酶联免疫吸附测定检测细胞因子的产生。在EGR1沉默的细胞和内毒素血症小鼠模型中评估EGR1/ADAM17通路对脓毒症诱导的炎症反应的影响。
-172AG/GG基因型和G等位基因的脓毒症患者中非幸存者的频率明显高于AA基因型患者（53.9%对39.7%，OR = 1.779，95%CI = 1.119 - 2.829，P = 0.0142）和A等位基因患者（30.9%对22.2%，OR = 1.570，95%CI = 1.095 - 2.251，P = 0.0136）。Kaplan-Meier生存分析表明，具有这种功能性ADAM17启动子多态性的-172AG/GG基因型脓毒症患者的28天生存率远低于AA基因型携带者（对数秩= 5.358，P = 0.021）。体外脂多糖刺激和荧光素酶测定结果表明，-172 A到G的变异可通过增强其启动子区域与EGR1的结合亲和力在功能上上调ADAM17基因的启动子活性和转录。ChIP测定确定了直接相互作用。进一步的研究表明，抑制EGR1可显著降低体外ADAM17的表达和促炎细胞因子的分泌，并改善脓毒症小鼠模型的生存率和炎症反应。
这些结果提供了证据，表明ADAM17 -172A>G多态性通过EGR1/ADAM17通路在功能上促进ADAM17表达并增强脓毒症诱导的炎症反应，最终导致脓毒症死亡易感性和不良预后。
