【摘要】 为探讨干扰素调节因子(interferon regulatory factor 1, IRF-1)对小胶质细胞BV2焦亡和炎症小体的激活，以及BV2细胞吞噬β淀粉样蛋白(amyloid β-protein, Aβ)功能的影响，利用脑单细胞RNA测序(single cell RNA sequencing, scRNA-seq)数据集筛选出阿尔茨海默病(Alzheimer’s disease, AD)患者脑组织与正常脑组织中差异表达基因IRF-1,并分析其在各个细胞类型中的表达情况，对其进行功能富集分析。用Aβ1-42刺激BV2细胞模拟AD的炎性环境，分为对照组、模型组(Aβ1-42处理)、 Aβ1-42+sh-NC组、 Aβ1-42+sh-IRF-1组和Aβ1-42+sh-IRF-1+ML385(Nrf2抑制剂)组。采用CCK-8法检测细胞活力，乳酸脱氢酶(lactic dehydrogenase, LDH)释放法、 Annexin V-FITC/PI双染色法评估细胞焦亡水平。Western blotting法检测NOD样受体家族含pyrin结构域蛋白3(NOD-like receptor family pyrin domain-containing protein 3, NLRP3)、凋亡相关斑点样蛋白(adaptin apoptosis-associated spot-like protein, ASC)、 Caspase-1、消皮素D N端结构域(Gasdermin D N-terminal domain, GSDMD-N)、核因子E2相关因子2(transcription factor nuclear factor E2 related factor 2, Nrf2)和血红素加氧酶-1(heme oxygenase-1, HO-1)表达水平。ELISA法测定IL-1β和IL-18水平。DCFH-DA荧光染色法检测BV2细胞中活性氧(reactive oxygen, ROS)水平。流式细胞术检测BV2细胞对Aβ的吞噬能力。结果显示，与对照组相比，模型组BV2细胞活力显著降低，炎症小体相关分子NLRP3、 ASC、 Caspase-1、 GSDMD-N表达水平显著增加，IL-1β、 IL-18水平显著升高，LDH释放量及细胞焦亡比例显著升高，细胞内ROS显著增多，Nrf2和HO-1蛋白表达显著降低(P<0.01)。与模型组相比，Aβ1-42+sh-IRF-1组细胞活力显著增高，吞噬Aβ能力显著升高，IL-1β、 IL-18水平降低，炎症小体及焦亡相关蛋白表达下调，LDH的释放量和细胞焦亡比例降低，ROS生成减少，Nrf2和HO-1蛋白表达升高(P<0.01)。与Aβ1-42+sh-IRF-1组相比，Aβ1-42+sh-IRF-1+ML385组BV2细胞焦亡水平被逆转。该研究表明，干预IRF-1可降低Aβ1-42诱导的BV2细胞NLRP3炎症小体活化和焦亡，其机制可能为激活Nrf2/HO-1信号通路。更多还原

【Abstract】 This study aims to investigate the effects of interferon regulatory factor 1(IRF-1) on pyroptosis and inflammasome activation in BV2 microglial cells,as well as its impact on BV2 cell phagocytosis of amyloid β-protein(Aβ).A single-cell brain dataset was used to screen for the differentially expressed gene IRF-1 in brain tissues of Alzheimer’s disease(AD) patients and normal controls,analyze its expression across various cell types,and perform functional enrichment analysis.BV2 cells were stimulated with Aβ1-42 to mimic the inflammatory environment of AD and were divided into the control group,the model group(treated with Aβ1-42),the Aβ1-42+sh-NC group,the Aβ1-42+sh-IRF-1 group,and the Aβ1-42+sh-IRF-1+ML385(Nrf2 inhibitor) group.Cell viability was assessed using the CCK-8 assay.Pyroptosis levels were evaluated using lactate dehydrogenase(LDH) release assay and Annexin V-FITC/PI double staining.Western blotting was performed to detect the expression levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing a CARD(ASC),Caspase-1,Gasdermin D N-terminal domain(GSDMD-N),nuclear factor erythroid 2-related factor 2(Nrf2),and heme oxygenase-1(HO-1).ELISA was used to measure,the levels of IL-1β and IL-18.Reactive oxygen species(ROS) levels in BV2 cells were detected using DCFH-DA fluorescence staining.The phagocytic capacity of BV2cells for Aβ was assessed using flow cytometry.Compared with the control group,the model group showed decreased BV2 cell viability,increased expressions of inflammasome-related molecules,including NLRP3,ASC,Caspase-1,and GSDMD-N,elevated levels of IL-1β,IL-18,LDH release,and pyroptosis rate,increased intracellular ROS production,and decreased protein expressions of Nrf2 and HO-1(P<0.01).Compared with the model group,the Aβ1-42+sh-IRF-1 group exhibited significantly increased cell viability and Aβ phagocytic capacity,decreased levels of IL-1β and IL-18,downregulated expression of inflammasome and pyroptosis-related proteins,reduced LDH release,pyroptosis rate,and ROS production,as well as increased protein expressions of Nrf2 and HO-1(P <0.01).Compared to those of the Aβ1-42+sh-IRF-1 group,BV2 cell pyroptosis levels were reversed in the Aβ1-42+sh-IRF-1+ML385 group.The results suggest that intervening IRF-1 reduces NLRP3 inflammasome activation and pyroptosis in Aβ1-42-induced BV2 cells,and the mechanism may involve activation of the Nrf2/HO-1 signaling pathway.更多还原