巨噬细胞HO-1调控炎症小体在小鼠下肢缺血损伤修复中的作用及机制

Macrophages, as the important inflammatory cells, play a critical role in tissue injury and repair after hindlimb ischemia. Heme oxygenase-1 (HO-1) in macrophages has pro-inflammatory effect, however, its role in hindlimb ischemia remains unreported. Our preliminary research showed that HO-1 protein was highly expressed in ischemic hindlimb tissue of mouse model, and mainly located in the infiltrated macrophages. We found that myeloid-specific HO-1 deficiency significantly improved blood perfusion in the ischemic hindlimbs of mouse model. We further found that myeloid cell deficiency of HO-1 significantly inhibited the activation of Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Meanwhile, in vitro study demonstrated that HO-1 inhibition prevented tumor necrosis factor-α-induced activation of NLRP3 inflammasome in macrophages. Therefore, we speculate that HO-1 in macrophages may participate in the injury and repair of hindlimb ischemia by regulating NLRP3 inflammasome activation. We will apply the myeloid-specific HO-1 knockout mice, co-immunoprecipitation, cell co-culture and other techniques, to elucidate for the first time the role of HO-1 in macrophages in hindlimb ischemia and its mechanism by regulating NLRP3 inflammasome activation, providing a novel therapeutic target for the treatment of hindlimb ischemia.

巨噬细胞是重要的炎症细胞，在下肢缺血后组织损伤及修复中起着重要作用。巨噬细胞血红素加氧酶-1(HO-1)具有促炎作用，但其在下肢缺血中的作用尚未报道。我们的预实验发现小鼠下肢缺血组织HO-1蛋白表达显著升高，并主要定位于巨噬细胞；髓系特异性HO-1敲除显著改善了小鼠缺血下肢的血流灌注，并抑制核苷酸结合寡聚化结构域样受体3(NLRP3)炎症小体的活化。同时，体外实验发现HO-1抑制剂可阻止肿瘤坏死因子-α诱导的巨噬细胞NLRP3炎症小体活化。因此我们推测：巨噬细胞HO-1可能通过调控NLRP3炎症小体活化参与下肢缺血损伤和修复。本项目拟应用髓系特异性HO-1敲除小鼠，结合免疫共沉淀、细胞共培养等技术，首次阐明巨噬细胞HO-1在下肢缺血中的作用及其调控NLRP3炎症小体活化的机制，为下肢缺血性疾病寻找新的干预靶点奠定研究基础。

巨噬细胞介导的炎症反应在调控下肢缺血后组织修复及血管新生中起着重要作用。近期研究发现巨噬细胞来源的血红素加氧酶-1（HO-1）可以促进炎症反应和心肌组织损伤，然而巨噬细胞HO-1在缺血后修复中的作用尚无报道。本研究旨在探讨巨噬细胞HO-1在下肢缺血后调控炎症反应、血管新生的作用和机制。生物信息学分析GEO数据库来源的转录组测序结果发现，与健康对照组相比，HO-1在严重下肢缺血患者的腓肠肌活检样本中表达显著升高。构建小鼠下肢缺血模型，western blot和免疫荧光染色证实缺血组织HO-1表达显著增高，并定位在浸润的巨噬细胞。构建髓系特异性HO-1敲除小鼠，HO-1敲除小鼠下肢缺血后修复显著改善，表现为组织灌注增强、缺血损伤减轻、运动功能保留，免疫荧光显示缺血腓肠肌内毛细血管密度增加。进一步研究发现髓系HO-1敲除对缺血的保护作用伴随着NLRP3炎症小体的减少。机制探讨发现阻断骨髓来源的巨噬细胞HO-1可以降低TNF-α诱导的NLRP3蛋白水平，而不影响其mRNA水平。而且巨噬细胞HO-1调控的NLRP3蛋白降解依赖于自噬溶酶体途径。此外，成管实验显示阻断巨噬来源的HO-1可以减轻NLRP3炎症小体激活对内皮细胞管腔形成的破坏作用。该研究首次阐明了巨噬细胞来源的HO-1在下肢缺血后修复中的作用及机制。阻断HO-1可以促进NLRP3蛋白的降解，抑制缺血后NLRP3炎症小体信号，促进血管新生和组织修复。该研究发现为寻找下肢缺血性疾病的治疗靶点提供了依据。