3-吲哚乙酸通过调控应激颗粒介导的NLRP3炎性小体激活减轻新生隐球菌诱导的脑微血管内皮细胞焦亡

doi:10.12122/j.issn.1673-4254.2025.12.15

摘要/Abstract

摘要：

目的探究3-吲哚乙酸（IAA）通过调控应激颗粒（SGs）形成和炎症小体NLRP3激活来缓解新生隐球菌（Cn）诱导的脑微血管内皮细胞焦亡的作用机制。方法采用10⁷/mL浓度的Cn处理脑微血管内皮细胞构建体外细胞模型，并使用IAA进行干预。通过Western blotting、细胞免疫荧光、ELISA和CCK-8活性检测评估G3BP1、DDX3X、NLRP3及焦亡相关蛋白、细胞因子、细胞活性的变化。在免疫抑制的C57BL/6小鼠中，尾静脉注射Cn，连续7 d给予IAA灌胃治疗，构建体内动物实验模型。小鼠随机分为对照组、模型组及IAA处理组（3只/组）。通过依文思蓝实验检测血脑屏障通透性，取小鼠大脑皮层组织检测相关蛋白表达情况。结果 Cn感染可诱导脑微血管内皮细胞中G3BP1表达下调（P<0.05）、DDX3X和NLRP3表达上调（P<0.05）。IAA干预恢复了G3BP1、DDX3X和NLRP3的异常表达（P<0.05），有效抑制焦亡相关蛋白NT-GSDMD/GSDMD（P<0.05）和P20/Caspase1（P<0.01）的活化，大幅减少炎性细胞因子IL-18（P<0.001）、IL-1β（P<0.01）的释放。IAA干预可逆转Cn引起的脑微血管内皮细胞DDX3X向NLRP3的转移（P<0.05），促进DDX3X与G3BP1的结合（P<0.05）。体内实验结果显示：IAA治疗可缓解血脑屏障的损伤，减少依文思蓝的渗出量（2.64±0.32 vs 1.06±0.12，P<0.001），和大脑皮层中ZO-1的表达量（P<0.05）。同时有效抑制VEGFR2、G3BP1、DDX3X、NLRP3和焦亡相关蛋白NT-GSDMD/GSDMD、P20/Caspase1的异常表达（P<0.05）。结论 IAA通过调控SGs的形成以及炎症小体NLRP3的活化，有效缓解Cn引起的脑微血管内皮细胞焦亡。

关键词: 新生隐球菌, 焦亡, 3-吲哚乙酸, 应激颗粒, NLRP3炎症小体

Abstract:

Objective To investigate whether indole-3-acetic acid (IAA) alleviates Cryptococcus neoformans (Cn)‑induced pyroptosis in cerebral microvascular endothelial cells by modulating stress granules (SGs) formation and the NLRP3 inflammasome. Methods In vitro cultured cerebral microvascular endothelial cells were pretreated with different concentrations of IAA before Cn infection (10⁷/mL), and the changes in cellular expresisons of G3BP1, DDX3X, NLRP3 and pyroptosis-related proteins, cytokines, and cell viability were deceted using Western blotting, immunofluorescence staining, ELISA, and CCK-8 assay. In the animal experiment, C57BL/6 mice with cyclophosphamide-induced immunosuppression were pretreated with saline or IAA gavage for 7 days before intravenous Cn injection. The changes in blood-brain barrier (BBB) integrity of the mice was assessed with Evans blue assay, and the brain cortical tissues were analyzed for changes in protein expressions. Results Cn infection significantly downregulated G3BP1 expression and upregulated the expressions of DDX3X and NLRP3 in cultured cerebral microvascular endothelial cells. IAA intervention not only restored normal expressions of G3BP1, DDX3X, and NLRP3, but also effectively suppressed the activation of pyroptosis-related proteins, including NT-GSDMD/GSDMD and P20/caspase-1, and reduced the release of IL-18 and IL-1β. IAA treatment also inhibited the translocation of DDX3X to NLRP3 induced by Cn infection and promoted the binding between DDX3X and G3BP1. In Cn-infected C57BL/6 mice, IAA treatment significantly alleviated BBB injury, decreased the expression of ZO-1 in the cerebral cortex, and effectively ameliorated abnormal expressions of VEGFR2, G3BP1, DDX3X, NLRP3, NT-GSDMD/GSDMD and P20/caspase-1. Conclusion IAA effectively alleviates Cn infection-induced pyroptosis of cerebral microvascular endothelial cells by modulating the formation of SGs and activating the NLRP3 inflammasome.

Key words: Cryptococcus neoformans, pyroptosis, indole-3-acetic acid, stress granules, NLRP3 inflammasome

陈晶宇, 邹金虎, 周炳亮, 高雪锋, 黄鹏伟, 曹虹. 3-吲哚乙酸通过调控应激颗粒介导的NLRP3炎性小体激活减轻新生隐球菌诱导的脑微血管内皮细胞焦亡[J]. 南方医科大学学报, 2025, 45(12): 2679-2689.

Jingyu CHEN, Jinhu ZOU, Bingliang ZHOU, Xuefeng GAO, Pengwei HUANG, Hong CAO. Indole-3-acetic acid alleviates Cryptococcus neoformans-induced pyroptosis in cerebral microvascular endothelial cells by regulating stress granule-mediated NLRP3 inflammasome activation[J]. Journal of Southern Medical University, 2025, 45(12): 2679-2689.

图/表 6

图1 细胞免疫荧光分析Cn感染后脑微血管内皮细胞中G3BP1、DDX3X和NLRP3蛋白表达水平变化

Fig.1 Immunofluorescence sataining for G3BP1 (A,B), DDX3X (C,D) and NLRP3 (E,F) in cerebral microvascular endothelial cells with Cn infection (Original magnification: ×100). *P<0.05, **P<0.01.

图2 Western blotting检测Cn处理脑微血管内皮细胞后细胞G3BP1, NLRP3, DDX3X表达情况

Fig.2 Western blotting for detecting G3BP1 (A), DDX3X(B) and NLRP3(C) protein expressions in cerebral microvascular endothelial cells following Cn infection. D: Protein bands in Western blotting. *P<0.05, ***P<0.001.

图3 IAA可缓解Cn引起的脑微血管内皮细胞焦亡

Fig.3 IAA alleviates Cn-induced pyroptosis of cerebral microvascular endothelial cells. A: Survival rates of Cn-infected cerebral microvascular endothelial cells treated with gradient concentrations of IAA. ***P<0.001 vs 0 mmol/L. B: The growth curve of Cn after IAA treatment. C-G: Expressions of G3BP1, DDX3X, NLRP3, NT-GSDMD /GSDMD, and P20/caspase-1 in Cn-infected cerebral microvascular endothelial cells with IAA treatment. H: Protein bands by Western blotting. I-J: IL-18 and IL-1β levels in the cell culture supernatant determined by ELISA. *P<0.05, **P<0.01, ***P<0.001.

图4 细胞免疫荧光检测IAA可减轻Cn引起的G3BP1、DDX3X和NLRP3的变化

Fig.4 IAA alleviates Cn-induced changes in G3BP1, DDX3X and NLRP3 expressions in cerebral microvascular endothelial cells (Immunofluorescence staining, ×100). A, D: Effect of IAA on G3BP1 expression in Cn-infected cerebral microvascular endothelial cells. B,E: Effect of IAA on DDX3X expression in Cn-infected cerebral microvascular endothelial cells. C,F: Effect of IAA on NLRP3 expression in Cn-infected cerebral microvascular endothelial cells. *P<0.05, **P<0.01.

图5 IAA调控DDX3X对Cn诱导的脑微血管内皮细胞中SGs生成和NLRP3炎性小体活化

Fig.5 IAA regulates translocation of DDX3X in the formation of SGs and activation of NLRP3 inflammasome in Cn-infected cerebral microvascular endothelial cells (Immunofluorescence staining, ×200). A,B: Co-localization of G3BP1 and DDX3X in cells treated with Cn and IAA. C, D: Co-localization of NLRP3 and DDX3X in cells treated with Cn and IAA. *P<0.05, **P<0.01.

图6 体内实验验证IAA调控SGs的形成对Cn诱导的BBB焦亡的作用

Fig.6 Regulatory effect of IAA on formation of SGs and Cn-induced pyroptosis in the blood-brain barrier (BBB) in mice. A: Evans blue staining showing changes in permeability of the BBB after Cn infection and IAA treatment. B: Expression level of ZO-1 in the BBB of mice with Cn infection and IAA treatment detected by Western blotting. C-F: Expression levels of VEGFR2, G3BP1, DDX3X, and NLRP3 in the cerebral cortex of the mice detected by Western blotting. G: Expression levels of NT-GSDMD and GSDMD. H: Expression levels of P20 and caspase-1. I: Portein bands by Western blotting. *P<0.05, **P<0.01, ***P<0.001.

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