以宿主炎症调控分子A7R为靶点的美金刚抗新生儿败血症和脑膜炎的实验研究

Despite the availability of antibiotics over the last several decades, an unmet need still exists for new effective therapeutic approaches to treat neonatal infections. Most of these deaths are caused by bacterial sepsis and meningitis (BSM), which is characteristic of high mortality and adverse prognosis. The most common gram negative bacterial pathogens causing BSM is E. coli. We have demonstrated that nAchR (α7R) played a detrimental role in the genesis of bacteremia and pathogen penetration across the blood-brain barrier (BBB), but the pathogenic mechanism of BSM is still unknown. Using the α7R knockout mice, we found that α7R is critical for the pathogenesis of BSM. The findings support the notion that α7R could serve as a unique drug target for therapeutic interventions against BSM. Using in vitro/in vivo models of BBB and RNA-seq, our drug repositioning studies have shown that MEM could very efficiently block BSM caused by E. coli E44 and enhance phagocytosis and neutrophil extracellular trap (NET)-mediated bacterial activity in a manner dependent on SA9 protein and A20 protein. Therefore, we hypothesize that MEM is a novel α7R target drug for treatment of BSM through enhancing the activity of NETS which will be examined by cell culture systems and KO animal models.

抗生素应用已取得巨大成果，但细菌感染引起的败血症和脑膜炎(BSM)仍是新生儿死亡主要原因，且大肠埃希菌(E.coli)是BSM最常见病菌。研究发现，宿主α7乙酰胆碱受体(α7R)在E.coli穿越血脑屏障(BBB)引发BSM过程起关键作用，但机制不明。通过体内外BBB模型、α7R敲除动物实验和RNA测序，我们发现美金刚(MEM)可阻断由脑膜炎E.coli E44感染引起的α7R活化。通过化学阻断或基因敲除(KO)屏蔽α7R功能均可显著降低BSM造成的脑损伤，其机制可能是上调宿主蛋白SA9和A20提高中性粒细胞胞外杀菌网络(NET)活性。本项目将从细胞、离体(脐血)及整体(α7R/SA9敲除小鼠模型)层面，采用实时动态成像、单细胞分析法(SCP)、RABT和双转录组/蛋白组等技术，研究MEM以α7R为靶点的NET抗菌机理，结合MEM药物学实验及药物间联合应用，寻找有效防治BSM策略。

细菌感染引起的败血症和脑膜炎(BSM)是新生儿死亡主要原因，其中大肠埃希菌(E.coli E44)是引起BSM的最常见病菌。我们前期研究发现，宿主α7乙酰胆碱受体(A7R)在E.coli穿越血脑屏障(BBB)引发BSM过程起关键作用，且美金刚(MEM)可阻断由脑膜炎E.coli E44感染引起的A7R活化。研究MEM以A7R为靶点的抗菌机理，可为MEM治疗BSM的新用途提供理论基础。本研究运用脑微血管内皮细胞(HBMEC)和A7R敲除小鼠等模型，通过RNA测序，阻断或基因敲除(KO)A7R等技术，深入研究MEM作为一种新型的A7R靶向药物通过增强宿主天然免疫及抵抗感染的机制。我们主要发现：(1)MEM靶向抑制A7R，促进外周血中性粒细胞胞外诱捕网(NETs)的形成和释放，从而增强宿主天然免疫的病原体捕获能力，改善新生儿E.coli E44菌血症诱发的相关损伤；(2) MEM通过抑制A7R而促进HBMEC负向炎症调控蛋白CISH的表达，继而抑制炎症通路JAK2/STAT5通路活化发挥抗炎效应；(3)E.coli E44感染将通过内源性A7R配体SLURP1激活A7R并促进新生儿败血症病程进展，而阻断SLURP1-A7R的相互作用可能是一种E.coli E44脑膜炎的新治疗策略；(4)MEM与维生素D3(VD3)协同使用可明显降低E.coli E44对靶细胞的侵袭作用，VD3和MEM协同处理组相比单独VD3治疗组降低了相对侵袭率。以上结论表明A7R在E44感染BSM过程中的重要调节作用，MEM可通过对A7R的抑制增强宿主免疫的抗炎和抗菌能力。总之，本项研究深入探究了MEM改善E44感染引起BSM当中的作用机制，发现A7R在BSM当中的重要调控作用，并指出MEM对A7R的阻断效应增强宿主免疫的机制，为MEM旧药新用治疗BSM提供理论基础。