Inhibition of Pyroptosis by Hydroxychloroquine as a Neuroprotective Strategy in Ischemic Stroke

Abstract

Hydroxychloroquine (HCQ), a well-known antimalarial and anti-inflammatory drug, has demonstrated potential neuroprotective effects in ischemic stroke by inhibiting pyroptosis, a programmed cell death associated with inflammation. This study investigates the impact of HCQ on ischemic stroke pathology using both in vivo and in vitro models. In vivo, C57BL/6 mice subjected to middle cerebral artery occlusion (MCAO) were treated with HCQ. Neurological deficits, infarct volume, and the expression of pyroptosis markers were evaluated. The results demonstrated that HCQ significantly improved motor function and reduced infarct volume in the MCAO mouse model. In vitro, BV2 microglial cells exposed to lipopolysaccharide (LPS) and oxygen-glucose deprivation (OGD) were treated with HCQ. Western blot and immunofluorescence analyses revealed that HCQ effectively suppressed the expression of pyroptosis markers GSDMD and NLRP3 in both in vivo and in vitro models. These findings suggest that HCQ mitigates ischemic stroke damage by inhibiting pyroptosis, highlighting its potential as a therapeutic agent for ischemic stroke. This study provides novel insights into the molecular mechanisms by which HCQ exerts its neuroprotective effects, offering a promising new avenue for developing safe, cost-effective, and widely applicable stroke treatments. The potential of HCQ to modulate neuroinflammatory pathways presents a significant advancement in ischemic stroke therapy, emphasizing the importance of targeting pyroptosis in stroke management and the broader implications for treating neuroinflammatory conditions.Significance Statement Ischemic stroke remains a leading cause of disability and death globally, with limited effective treatments. This study reveals that HCQ significantly mitigates ischemic stroke damage by inhibiting pyroptosis, a form of programmed cell death. Using in vivo and in vitro models, HCQ was shown to improve motor function and reduce infarct volume, highlighting its potential as a neuroprotective agent. These findings offer a promising new therapeutic approach for ischemic stroke, emphasizing the importance of targeting pyroptosis in stroke treatment.

Figure 1.

MCAO-induced neuronal cell pyroptosis in mouse brain tissue. A, Protein immunoblot analysis of inflammatory bodies NLRP3, pyroptosis-associated proteins GSDMD-FL, N-GSDMD, Caspase-1 p20, and inflammatory factor IL-1β in mouse brain tissue at 1–5 d post-MCAO treatment. B–D, Western blot quantitative analysis of NLRP3, pyroptosis protein GSDMD-FL, and N-GSDMD. E, F, Quantitative analysis of Pro-IL-1β and mIL-1β by Western blot. G, Quantitative analysis of Caspase-1 by Western blot at Days 1–5 post-MCAO. n = 8. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the Sham group.

Figure 2.

GO and pathway enrichment analysis of proteins associated with HCQ and ischemic stroke. A, A Venn diagram depicting the relationship between predicted target proteins of HCQ and proteins associated with ischemic stroke. B, Results of KEGG analysis for the 71 common genes. C, Results of GO analysis for the 71 common genes.

Figure 3.

Effect of HCQ on motor and balance function impairment in MCAO mice within 21 d poststroke. A, Impact of HCQ on the mNSS neurofunctional scoring of MCAO mice at 1, 3, 5, 7, 14, and 21 d poststroke. B, Influence of HCQ on the rotarod retention time of MCAO mice at 1, 3, 5, 7, 14, and 21 d poststroke. C, D, Effects of HCQ on the motor balance function of mice poststroke in terms of grip strength (C) and foot fault test (D) at 1, 3, 5, 7, 14, and 21 d. *p < 0.05, **p < 0.01, compared with the injury group. n = 8, data presented as mean ± SD.

Figure 4.

TTC staining showing the impact of HCQ on infarct volume in stroke mice. A, Comparison between MCAO + HCQ and MCAO groups reveals a significant decrease in the white infarct area, which also appears darker. B, ***p < 0.001 compared with the MCAO group. n = 8, data represented as mean ± SD.

Figure 5.

Inhibition Effect of HCQ on cell pyroptosis in mice on the third day after MCAO. A, Representative images of the expression levels of NLRP3, GSDMD-FL, N-GSDMD, Caspase-1, and IL-1β in the brain tissues of mice on the third day after MCAO, detected by Western blot. β-Actin was used as the internal control. B–F, Western blot quantitative analysis graphs of NLRP3, GSDMD-FL, N-GSDMD, Caspase-1, and IL-1β on the third day after MCAO. ^###p < 0.001, ^####p < 0.0001, compared with the Sham group; **p < 0.01, ***p < 0.001, compared with the MCAO injury group; NS indicates no statistical difference between the MCAO + HCQ and MCAO groups. n = 4, data presented as mean ± SD.

Figure 6.

Immunofluorescence staining of cell pyroptosis in the brain ischemic area 3 d after MCAO treatment. A, Colocalization imaging of pyroptosis-related protein GSDMD (in red) with Iba-1 (microglial cell marker), GFAP (astrocyte marker), and NeuN (neuronal marker). Scale bar, 100 µm. B–D, Quantitative analysis of different cell types: The number of microglial cells (Iba-1) significantly increased after cerebral ischemia (B), while the number of neurons (NeuN) decreased (C), and the number of astrocytes (GFAP) increased (D). E, Quantitative analysis of GSDMD expression.

Figure 7.

Impact of HCQ on necroptosis-related proteins in BV2 cells. A, Protein immunoblot images representing the effects of different concentrations of HCQ (5, 10, 20 µM) treatment on the expression levels of NLRP3, GSDMD-FL, N-GSDMD, and the inflammatory factor IL-1β in BV2 cells detected by Western blot technique. B–E, Quantitative analysis of protein immunoblots for NLRP3, GSDMD-FL, N-GSDMD, and IL-1β. ^#p < 0.05, ^###p < 0.001, compared with the Control group; *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significant difference, compared with the LPS + OGD group. n = 4, data are presented as mean ± SD.

Figure 8.

Effect of HCQ (20 µM) on the inhibition of NLRP3 inflammasome assembly in BV2 cells post LPS + OGD treatment. A, Representative immunofluorescence images displaying the NLRP3 expression in BV2 cells in different groups. Scale bar, 100 µm, with a 4× magnification of the inset region showing a scale bar of 25 µm. B, Proportion of NLRP3 protein-positive spots. Statistical analysis showed a significant difference ***p < 0.001 compared with the Control group; ^##p < 0.01 compared with the LPS + OGD group. n = 4, data are presented as mean ± SEM.

Figure 9.

Potential neuroprotective effects of HCQ in ischemic stroke: inhibiting cell apoptosis and improving neural function.