Abstract：
Current studies have demonstrated that disintegrin and metalloproteinase 17 (ADAM17) plays a critical role in the pathogenesis of sepsis. MicroRNA (miR)-145 is known to control immune responses as an anti-inflammatory modulatory molecule. However, a fundamental understanding of how miR-145 regulates ADAM17 and, more broadly, sepsis-induced inflammatory response remains unknown.
We used western blotting and quantitative real-time PCR (qRT-PCR) to measure expression levels of ADAM17 and miR-145. Enzyme-linked immunosorbent assays (ELISA) were performed to measure cytokine production. To determine if ADAM17 is a target gene of miR-145, bioinformatics analyses and luciferase reporter assays were conducted. The impacts of ADAM17 and miR-145 on sepsis-induced inflammatory responses were accessed in vitro using human umbilical endothelial cells (HUVECs) treated with lipopolysaccharide (LPS). Sepsis-induced inflammatory response was measured in vivo using a polymicrobial septic mouse model induced by cecal ligation and puncture (CLP) with pre-injection of a miR-145 agomir.
In HUVECs treated with LPS, miR-145 expression was downregulated and miR-145 negatively regulated ADAM17 expression through direct binding to the ADAM17 transcript 3\'-UTR. MiR-145 overexpression markedly reduced LPS-induced inflammatory cytokine production by targeting ADAM17 in HUVECs. In comparison to CLP-induced septic mice treated with a control agomir, treatment with a miR-145 agomir significantly reduced the expression of ADAM17, numerous downstream cytokines such as IL-6, TNF-α, IL-1β and MCP-1, and the endothelial injury factors ICAM-1, VCAM-1. The miR-145 agomir also alleviated acute lung and kidney injury and improved the survival rate of septic mice.
This study showed that miR-145, by specifically targeting ADAM17, negatively regulates sepsis-induced inflammatory responses and vascular endothelial injury, and ultimately improved organ injury and survival during sepsis. The underlying mechanism for the regulation of ADAM17 expression by miR-145 and sepsis-induced inflammatory reactions may offer sepsis patients a novel therapeutic option.
We used western blotting and quantitative real-time PCR (qRT-PCR) to measure expression levels of ADAM17 and miR-145. Enzyme-linked immunosorbent assays (ELISA) were performed to measure cytokine production. To determine if ADAM17 is a target gene of miR-145, bioinformatics analyses and luciferase reporter assays were conducted. The impacts of ADAM17 and miR-145 on sepsis-induced inflammatory responses were accessed in vitro using human umbilical endothelial cells (HUVECs) treated with lipopolysaccharide (LPS). Sepsis-induced inflammatory response was measured in vivo using a polymicrobial septic mouse model induced by cecal ligation and puncture (CLP) with pre-injection of a miR-145 agomir.
In HUVECs treated with LPS, miR-145 expression was downregulated and miR-145 negatively regulated ADAM17 expression through direct binding to the ADAM17 transcript 3\'-UTR. MiR-145 overexpression markedly reduced LPS-induced inflammatory cytokine production by targeting ADAM17 in HUVECs. In comparison to CLP-induced septic mice treated with a control agomir, treatment with a miR-145 agomir significantly reduced the expression of ADAM17, numerous downstream cytokines such as IL-6, TNF-α, IL-1β and MCP-1, and the endothelial injury factors ICAM-1, VCAM-1. The miR-145 agomir also alleviated acute lung and kidney injury and improved the survival rate of septic mice.
This study showed that miR-145, by specifically targeting ADAM17, negatively regulates sepsis-induced inflammatory responses and vascular endothelial injury, and ultimately improved organ injury and survival during sepsis. The underlying mechanism for the regulation of ADAM17 expression by miR-145 and sepsis-induced inflammatory reactions may offer sepsis patients a novel therapeutic option.
摘要：
背景：目前的研究表明，去整合素和金属蛋白酶17（ADAM17）在脓毒症的发病机制中起着至关重要的作用。已知微小RNA(miR)-145作为抗炎调节分子控制免疫应答。然而,对miR-145如何调节ADAM17和，更广泛地说，脓毒症诱导的炎症反应仍然未知.
方法：我们使用蛋白质印迹和定量实时PCR（qRT-PCR）来测量ADAM17和miR-145的表达水平。进行酶联免疫吸附测定(ELISA)以测量细胞因子产生。为了确定ADAM17是否是miR-145的靶基因,进行了生物信息学分析和荧光素酶报告基因测定。使用脂多糖（LPS）处理的人脐内皮细胞（HUVEC）在体外研究了ADAM17和miR-145对脓毒症诱导的炎症反应的影响。使用预注射miR-145agomir的盲肠结扎和穿孔(CLP)诱导的多微生物败血症小鼠模型在体内测量败血症诱导的炎症反应。
结果：在用LPS处理的HUVEC中，miR-145表达下调，miR-145通过直接结合ADAM17转录物3'-UTR负调控ADAM17表达。MiR-145过表达通过靶向HUVEC中的ADAM17显著减少LPS诱导的炎性细胞因子产生。与用对照agomir处理的CLP诱导的败血症小鼠相比，用miR-145agomir治疗显著降低ADAM17的表达，许多下游细胞因子，如IL-6，TNF-α，IL-1β和MCP-1，以及内皮损伤因子ICAM-1，VCAM-1。miR-145agomir还减轻了急性肺和肾损伤，并提高了脓毒症小鼠的存活率。
结论：这项研究表明，miR-145通过特异性靶向ADAM17负调节脓毒症诱导的炎症反应和血管内皮损伤，并最终改善脓毒症期间的器官损伤和存活率。miR-145调节ADAM17表达和脓毒症诱导的炎症反应的潜在机制可能为脓毒症患者提供新的治疗选择。
方法：我们使用蛋白质印迹和定量实时PCR（qRT-PCR）来测量ADAM17和miR-145的表达水平。进行酶联免疫吸附测定(ELISA)以测量细胞因子产生。为了确定ADAM17是否是miR-145的靶基因,进行了生物信息学分析和荧光素酶报告基因测定。使用脂多糖（LPS）处理的人脐内皮细胞（HUVEC）在体外研究了ADAM17和miR-145对脓毒症诱导的炎症反应的影响。使用预注射miR-145agomir的盲肠结扎和穿孔(CLP)诱导的多微生物败血症小鼠模型在体内测量败血症诱导的炎症反应。
结果：在用LPS处理的HUVEC中，miR-145表达下调，miR-145通过直接结合ADAM17转录物3'-UTR负调控ADAM17表达。MiR-145过表达通过靶向HUVEC中的ADAM17显著减少LPS诱导的炎性细胞因子产生。与用对照agomir处理的CLP诱导的败血症小鼠相比，用miR-145agomir治疗显著降低ADAM17的表达，许多下游细胞因子，如IL-6，TNF-α，IL-1β和MCP-1，以及内皮损伤因子ICAM-1，VCAM-1。miR-145agomir还减轻了急性肺和肾损伤，并提高了脓毒症小鼠的存活率。
结论：这项研究表明，miR-145通过特异性靶向ADAM17负调节脓毒症诱导的炎症反应和血管内皮损伤，并最终改善脓毒症期间的器官损伤和存活率。miR-145调节ADAM17表达和脓毒症诱导的炎症反应的潜在机制可能为脓毒症患者提供新的治疗选择。
