Fig 1.
Schematic illustration of the experimental design.
This study was composed of four major components. First, Cav1 expression in human central nervous system tissues was analyzed using bulk transcriptomic data from the GTEx database. Second, the inflammatory pain model was established via subcutaneous injection of complete Freund’s adjuvant (CFA) or saline into the right hind paw of adult mice, followed by behavioral testing from Day –3 (baseline) to Day 7. On Day 4, spinal cord tissues were collected for high-throughput RNA sequencing and subsequent differential gene expression (DEG) analysis, Gene Ontology (GO) enrichment, and single-sample gene set enrichment analysis (ssGSEA). On Day 7, molecular validation (qPCR, Western blot, immunofluorescence, HE staining) was performed ex vivo. Finally, in vitro validation was conducted using BV2 microglial cells through Cav1 silencing or overexpression, followed by Western blot and qPCR analysis.
Table 1.
List of qPCR primer sequences.
Fig 2.
Upregulation of Cav1 in the spinal cord dorsal horn during CFA-induced inflammatory pain.
(A) Analysis of public databases (GTEx) showing high expression of Cav1 in spinal cord tissue. (B) RT-qPCR analysis of Cav1 mRNA expression in the spinal cord dorsal horn at different time points after CFA injection. Data are presented as mean ± SEM. n = 6 mice per group. One-way ANOVA followed by Tukey’s post hoc test was performed. (C) Western blot analysis of Cav1 protein levels in the spinal cord dorsal horn at different time points after CFA injection. Relative protein levels were quantified. Data are presented as mean ± SEM. n = 5 mice per group. One-way ANOVA followed by Tukey’s post hoc test was performed. (D) Western blot analysis of Cav1 protein expression in wild-type (WT) and Cav1⁻/⁻ mice. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3.
Cav1 knockout attenuated CFA-induced pain behaviors and inflammatory responses.
(A) Paw withdrawal threshold (PWT) in wild-type (WT) and Cav1-/- mice injected with CFA or saline. (B) Thermal withdrawal latency in WT and Cav1-/- mice injected with CFA or saline. (C) Right hind paw edema measured by caliper in WT and Cav1-/- mice following CFA or saline injection. Data are presented as mean ± SEM. n = 15 mice per group. Statistical analysis for (A–C) was performed using two-way repeated measures ANOVA followed by Bonferroni post hoc test. “#” denotes comparisons between WT+Saline and WT + CFA. #P < 0.05; ##P < 0.01; ###P < 0.001. “*” denotes comparisons between Cav1-/- + CFA and WT + CFA. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Relative right/left paw area in wild-type (WT) and Cav1-/- mice after CFA or saline injection. Data are presented as mean ± SEM. n = 10 mice per group. (E) Representative H&E staining of right foot tissue from WT and Cav1-/- mice after CFA or saline injection (15 × magnification, scale bar = 100 μm). Bar graph shows inflammation scores for each group. Data are presented as mean ± SEM. n = 4 mice per group. One-way ANOVA followed by Tukey’s post hoc test was performed for(D-E). *P < 0.05; ***P < 0.001.
Fig 4.
Cav1 deficiency inhibited microglial activation in CFA-induced inflammatory pain.
(A) Heatmap analysis of differentially expressed genes associated with microglia signaling in spinal cord tissues of Cav1-/- and WT mice 4 days after CFA injection. Key genes included Sqstm1, Cox6a1, Sncb, Cav1, Enho, and Ap3m1. (B) Single-sample genomic enrichment analysis (ssGSEA) of differentially expressed genes, showing enrichment in pathways related to microglial differentiation, activation, and proliferation. (C-D) RT-qPCR analysis of microglial activation markers Aif1 and Csf1r in spinal cord tissues of WT and Cav1-/- mice. Data are presented as mean ± SEM. n = 6 mice per group. (E) Representative immunofluorescence staining of IBA-1 in spinal cord tissues from WT and Cav1 ⁻ / ⁻ mice injected with CFA or saline. Scale bar = 100 μm. IBA-1 activity is shown as relative density/area. Data are presented as mean ± SEM; n = 3 mice per group. (F-H) RT-qPCR analysis of inflammatory cytokines TNF-α, IL-1β, and IL-6 mRNA levels in the spinal cord tissues of WT and Cav1-/- mice following CFA or saline injection. Data are presented as mean ± SEM. n = 6 mice per group. One-way ANOVA followed by Tukey’s post hoc test was performed for(C-E). *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 5.
Cav1 modulated the cGAS-STING pathway and autophagy in inflammatory pain.
(A) GO enrichment analysis of upregulated genes in Cav1-/- mice after CFA treatment, showing enrichment in ciliary protein localization and protein self-ubiquitination (BP), cytoplasmic components of cell leading edge and membrane bundle (CC), and GTPase binding and nuclear transport carrier activity (MF) and GO enrichment analysis of downregulated genes, showing enrichment in type I interferon signaling and response (BP), cell membrane ribosome and mitochondrial H + -ATP synthase complex (CC), and cationic channel and growth factor activity (MF). (B) Western blot images of cGAS and STING protein expression in the spinal cord tissues of wild-type (WT) and Cav1-/- mice following CFA or saline injection. Data are presented as mean ± SEM. n = 4 mice per group. One-way ANOVA followed by Tukey’s post hoc test was performed. (C) Volcano plot analysis of differentially expressed genes between Cav1-/- + CFA and WT + CFA groups, highlighting Sqstm1 and Map1lc3a as closely associated with autophagy. (D) Western blot images of autophagy markers LC3 and p62 in the spinal cord tissues of wild-type (WT) and Cav1-/- mice following CFA or saline injection. Data are presented as mean ± SEM. n = 4 mice per group. One-way ANOVA followed by Tukey’s post hoc test was performed. (E) TEM of mitochondria (red arrowheads), lysosomes (green arrowheads), and autophagosomes (yellow arrowheads). scale bar = 5 μm. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 6.
Overexpression of Cav1 enhanced inflammation through cGAS-STING activation and autophagy inhibition.
(A) RT-qPCR analysis of Cav1 mRNA levels in BV2 microglial cells stimulated with LPS for 0, 1, 3, 6, 9, 12, and 24 hours. Data are presented as mean ± SEM. n = 5 independent samples per time point. (B) Western blot analysis of Cav1 protein levels in BV2 microglial cells stimulated with LPS for 0, 1, 3, 6, 9, 12, and 24 hours. Data are presented as mean ± SEM. n = 4 independent samples per time point. (C) RT-qPCR confirmation of Cav1 overexpression in Cav1-overexpressing (OE-Cav1) BV2 microglial cells. Data are presented as mean ± SEM. n = 3 independent samples per group. (D-F) RT-qPCR analysis of proinflammatory cytokine mRNA levels (TNF-α, IL-1β, and IL-6) in OE-Cav1 and control BV2 microglial cells. Data are presented as mean ± SEM. n = 3 independent samples per group. (G) Western blot analysis of cGAS and STING protein expression in OE-Cav1 and control BV2 microglial cells. Quantification of protein levels is shown. Data are presented as mean ± SEM. n = 4 independent samples per group. (H) Western blot analysis of LC3 and p62 protein expression in OE-Cav1 and control BV2 microglial cells. Quantification of the LC3-II/LC3-I ratio and p62 protein levels is shown. Data are presented as mean ± SEM. n = 4 independent samples per group. One-way ANOVA followed by Tukey’s post hoc test was performed. **P < 0.01; ***P < 0.001.
Fig 7.
si-Cav1 alleviated inflammation by inhibiting the cGAS-STING pathway and activating autophagy.
(A) RT-qPCR analysis of Cav1 mRNA levels in BV2 microglial cells transfected with different Cav1-targeting siRNAs. Data are presented as mean ± SEM. n = 3 independent samples per group. (B-D) RT-qPCR analysis of pro-inflammatory cytokine mRNA levels (TNF-α, IL-1β, and IL-6) in BV2 microglial cells transfected with si-501-Cav1 or si-297-Cav1. Data are presented as mean ± SEM. n = 3 independent samples per group. (E) Western blot analysis of cGAS and STING protein expression in BV2 microglial cells transfected with si-501-Cav1 or si-297-Cav1. Quantification of protein levels is shown. Data are presented as mean ± SEM. n = 4 independent samples per group. (F) Western blot analysis of autophagy markers LC3 and p62 in BV2 microglial cells transfected with si-501-Cav1 or si-297-Cav1. Quantification of the LC3-II/LC3-I ratio and p62 protein levels is shown. Data are presented as mean ± SEM. n = 4 independent samples per group. One-way ANOVA followed by Tukey’s post hoc test was performed. *P < 0.05; **P < 0.01; ***P < 0.001.