G Protein-Coupled Receptor 30 Attenuates Neuronal Pyroptosis Induced by Subarachnoid Hemorrhage

Abstract

Background: Pyroptosis is implicated as a pathogenic mechanism in early brain injury (EBI) after subarachnoid hemorrhage (SAH). This study aimed to investigate the regulatory role of G protein-coupled receptor 30 (GPR30) in neuronal pyroptosis during SAH. Methods: SAH was induced in rats via intravascular perforation and hemin-treated neurons modeled SAH in vitro. GPR30 agonist G1 and antagonist G15 were administered to assess functional impacts. Neurological deficits (Garcia score), SAH severity, and cerebral edema (brain water content) were evaluated. Pyroptotic markers (cleaved caspase-1, gasdermin D (GSDMD)-N, interleukin (IL)-1β, and IL-18) were quantified. Inflammasome activation (NLRC4 and IFI16) and Toll-like receptor 4/nuclear factor kappa-B (TLR4/NF-κB) signaling were analyzed via immunofluorescence (IF) and immunoblotting. The TLR4 antagonist LPS-RS (lipopolysaccharide from Rhodobacter sphaeroides) was applied to validate pathway involvement. Results: GPR30 expression increased post-SAH. G15 exacerbated hemorrhage severity, neurological deficits, and cerebral edema, whereas G1 modestly attenuated SAH. G15 upregulated pyroptotic markers, enhanced neuronal pyroptosis, and activated NLRC4/IFI16 inflammasomes. Concurrently, G15 stimulated TLR4/MyD88 expression and NF-κB phosphorylation. Conversely, G1 suppressed pyroptosis, inactivated inflammasomes, and inhibited TLR4/NF-κB signaling. LPS-RS cotreatment reversed G15-induced pyroptotic and inflammatory cascades. Conclusion: GPR30 mitigates NLRC4- and IFI16-driven neuronal pyroptosis in SAH by modulating TLR4/NF-κB signaling. Pharmacological targeting of GPR30 represents a promising therapeutic strategy to ameliorate SAH-associated brain injury.

Keywords: IFI16; NLRC4; TLR4/NF-κB; inflammasome; pyroptosis; subarachnoid hemorrhage.

Figure 1

GPR30 agonist attenuates EBI after SAH. (A) Representative images of cerebral hemorrhage in rat brains. (B) Quantification of SAH severity grades across experimental groups. (C) Neurological function assessed by Garcia scoring system. (D) Brain water content difference (%) reflecting cerebral edema. (E) Western blot analysis of GPR30 protein expression levels. ⁣^∗p  < 0.05.

Figure 2

GPR30 regulates neuronal pyroptosis during SAH. (A) Protein expression levels of cleaved caspase-1, GSDMD-N, IL-1β, and IL-18 in brain tissues assessed by western blot. Immunofluorescence (IF) analysis of caspase-1+/NeuN+ (B) and GSDMD+/NeuN+ (C) colocalization in cortical brain tissue. White arrows denote cells exhibiting colocalization of the indicated markers. Scale bar = 50 μm (200x) or 25 μm (400x). (D) Transmission electron microscopy (TEM) images of pyoptotic neurons. Blue arrows indicate membrane rupture; red arrows denote swollen mitochondria with disrupted cristae. (E) Dose-dependent effects of G1 (0–100 μM) and G15 (0–10 μM) on GPR30 expression in neuronal cells using western blot. (F) Neuronal viability under G1 and G15 treatment measured by CCK-8 assay. (G) Quantitative analysis of GPR30 protein levels in neuronal cells. (H) Caspase-1-positive neurons quantified from IF images. Scale bar = 50 μm (up) and 25 μm (down). (I) Flow cytometric quantification of pyroptotic neurons. ⁣^∗p  < 0.05.

Figure 3

GPR30 modulates inflammasome activation after SAH. (A) Western blot analysis of inflammasome sensors AIM2, NLRP3, NLRC4, and IFI16. (B) IF staining of NLRC4 and IFI16 expression in neurons. Scale bar = 50 μm (up) and 25 μm (down). (C) Western blot analysis of cleaved caspase-1, GSDMD-N, IL-1β, and IL-18. ⁣^∗p  < 0.05.

Figure 4

GPR30 suppression enhances TLR4/NF-κB signaling activation. (A) Dose-dependent effects of G1 (0–100 μM) and G15 (0–10 μM) on TLR4 expression in neuronal cells using western blot. (B) Western blot analysis of TLR4, MyD88, p-NF-κB, and NF-κB. ⁣^∗p  < 0.05.

Figure 5

TLR4/NF-κB pathway inhibition suppresses IFI16 and NLRC4 inflammasome activity. (A) Western blot analysis of IFI16 and NLRC4. (B) IF analysis of GSDMD expression in neurons. Scale bar = 50 μm (up) and 25 μm (down). ⁣^∗p  < 0.05.

Figure 6

GPR30 silencing activates IFI16/NLRC4 inflammasomes through TLR4/NF-κB signaling. (A) Western blot analysis of IFI16 and NLRC4. (B) GSDMD expression visualized by IF staining. Scale bar = 50 μm (up) and 25 μm (down). (C) Pyroptosis rates quantified using flow cytometry. (D) TEM images of pyroptotic neurons. Red arrows indicate cytoplasmic vacuolization. ⁣^∗p  < 0.05.