Indole-3-acetic acid alleviates Cryptococcus neoformans-induced pyroptosis in cerebral microvascular endothelial cells by regulating stress granule-mediated NLRP3 inflammasome activation

doi:10.12122/j.issn.1673-4254.2025.12.15

Abstract

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

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.

Figures/Tables 6

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.

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.

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.

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.

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.

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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