Ganoderma lucidum spore powder alleviates rheumatoid arthritis-associated pain hypersensitivity through inhibiting accumulation, N1 polarization, and ROS production of neutrophils in mice

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic condition characterized by joint pain that significantly impairs patients' work and daily lives. The limited understanding of the pathological mechanisms underlying RA-related pain poses challenges for effective clinical pain management. Ganoderma lucidum spore powder (GLSP) has demonstrated therapeutic benefits in various diseases, with no reported toxicity or adverse effects.

Methods: This study investigates the role of neutrophils in the pathological mechanisms of RA-related pain using collagen-induced arthritis (CIA) mice and an ex vivo neutrophil model. A combination of techniques, including animal models, flow cytometry, behavioral testing, cell adoptive transfer, and network pharmacology analysis, was employed to evaluate the effects and targets of GLSP on pain symptoms and neutrophil activity in CIA mice.

Results: Flow cytometric analysis revealed the accumulation and activation of neutrophils in the paws of CIA mice. Furthermore, the levels of pro-inflammatory CD95⁺ neutrophil subpopulations (N1 state) and ROS⁺ cells in the affected paws were positively correlated with the severity of mechanical allodynia and heat hyperalgesia observed in these mice. Our findings indicate that oral administration of GLSP significantly alleviates joint destruction, paw swelling, and pain hypersensitivity in CIA mice. Notably, GLSP reversed CIA-induced neutrophil accumulation, N1 polarization, and reactive oxygen species (ROS) production. Both network pharmacology target prediction and in vivo/in vitro experimental validation indicated that GLSP inhibits N1 polarization and ROS production in neutrophils by modulating the TNF-α signaling pathway, thus exerting RA-specific analgesic effects.

Discussion: In summary, this study offers new insights into the pathological mechanisms of RA-related pain and demonstrates that neutrophil accumulation, N1 polarization, and ROS production contribute to RA-related pain. GLSP alleviates RA-related pain by inhibiting the pro-inflammatory phenotype of neutrophils, highlighting its potential for clinical translation in the treatment of RA.

Keywords: Ganoderma lucidum spore powder; neutrophils; pain; reactive oxygen species; rheumatoid arthritis.

Figure 1

Ganoderma lucidum spore powder demonstrates the potential to alleviate joint damage and pain associated with RA. (A) Schematic representation of the experimental protocol. (B) Representative images illustrating the paws of mice from distinct experimental groups. (C, D) Hematoxylin and eosin (HE) staining data indicate that GLSP mitigates joint damage in CIA mice, with damage highlighted by arrows. The scale bar measures 100 µm. (E, F) GLSP treatment reduces paw swelling and arthritis scores induced by CIA. (G–I) GLSP treatment alleviates mechanical allodynia and thermal hyperalgesia in CIA mice, but has no effect on cold allodynia. (J–L) GLSP treatment reverses CIA-induced changes in temperature preference in mice. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA assay followed by Tukey’s post hoc test (D), two-way ANOVA assay followed by Tukey’s post hoc test (E–I, L). NS, no significance.

Figure 2

GLSP treatment alleviates the hyperexcitability of DRGs neurons in CIA mice. (A) Representative images of immunofluorescence (IF) staining for lumbar DRGs neurons from various experimental groups of mice are presented. (B) IF data reveals that GLSP attenuates the CIA-induced upregulation of TRPV1 expression in DRGs neurons. (C) IF data indicate that GLSP decreases the secretion of CGRP elevated by CIA in DRGs neurons. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA assay followed by Tukey’s post hoc test (B, C).

Figure 3

GLSP reduced neutrophil accumulation in the paws of CIA mice. (A) Representative flow cytometry images of affected paw samples from CIA and naïve mice. (B, C) GLSP decreases the proportion and number of CD45⁺ leukocytes in the affected paw tissues of CIA mice. (D) Representative flow cytometry images depict various immune cell populations within paw samples from CIA mice and naive mice. (E, F) GLSP reduced the proportion and count of neutrophils, while not affecting other immune cell populations in the affected paws of CIA mice. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA assay followed by Tukey’s post hoc test (B, C), two-way ANOVA assay followed by Tukey’s post hoc test (E, F). NS, no significance.

Figure 4

GLSP treatment reversed the N1-polarized state of neutrophils in the affected paws of CIA mice. (A–C) GLSP treatment reduced both the proportion and absolute count of N1-polarized (CD95⁺) neutrophils in the paws of mice with CIA. (D–F) GLSP treatment upregulated both the proportion and number of N2-state (CD206⁺) neutrophils within the paws of CIA mice. (G, H) In vehicle-treated CIA mice, N1 polarization of joint neutrophils exhibits a negative correlation with mechanical and thermal pain thresholds. (I, J) In GLSP-treated CIA mice, N1 polarization of joint neutrophils demonstrates no correlation with mechanical or thermal pain thresholds. (K, L) In vehicle-treated CIA mice, N2 polarization of joint neutrophils shows no significant correlation with mechanical or thermal pain thresholds. (M, N) N2 polarization of joint neutrophils in GLSP-treated CIA mice correlates positively with both mechanical and thermal pain thresholds. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA assay followed by Tukey’s post hoc test (B, C, E, F), Pearson correlation analysis (G–N). NS, no significance.

Figure 5

Identify the intersection targets shared by RA and GLSP for biological function analysis. (A) Total ion chromatogram of GLSP in positive ion mode. (B) 134 intersection targets of RA and GLSP. (C) The PPI network of intersection targets between disease targets of RA and the target proteins of active components in GLSP. (D) Key targets of GLSP for the treatment of RA were identified through CentiScaPe analysis utilizing Degree, Closeness, and Betweenness algorithms. (E–F) GO biological functional enrichment analysis and KEGG pathway analysis of key targets of GLSP in the treatment of RA.

Figure 6

Docking patterns of core targets and active compounds of GLSP. (A–O). Molecular docking analysis predicts the binding of compounds extracted from GLSP to key targets of RA. The red numerals in the upper right corner correspond to GLSP compounds 1–6 listed in Table 1 . The crystal structure of PTGS2 (PDB ID: 3HS5), MMP9 (PDB ID: 1L6J), XIAP (PDB ID: 8W59) and MMP3 (PDB ID: 1G49) was obtained from the Protein Data Bank.

Figure 7

GLSP attenuates TNF-α-induced nociceptive effects in neutrophils by reversing their N1 polarization. (A) Schematic representation of the experimental protocol designed to investigate the nociceptive effects of neutrophil N1 and N2 polarization states. (B, C) TNF-α challenge increased the proportion of CD95⁺ neutrophils in dHL-60 cells, an effect reversed by Co-treatment with GLSP extract, as shown by flow cytometry. (D, E) Co-treatment with GLSP extract reversed the TNF-α-challenged reduction in the proportion of CD206⁺ neutrophils in dHL-60 cells. (F, G) Intraplantar injection of TNF-α-challenged neutrophils induced mechanical allodynia and thermal hyperalgesia. Co-treatment with GLSP extract attenuated this effect. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA assay followed by Tukey’s post hoc test (C, E), two-way ANOVA assay followed by Tukey’s post hoc test (F, G). NS, no significance.

Figure 8

GLSP attenuates TNF-α-induced nociceptive effects in neutrophils by inhibiting the production of ROS. (A) The gating strategy employed to ROS levels in TNF-α-treatment neutrophils, along with representative plots of ROS levels across different groups, is presented. (B) TNF-α or H₂O₂treatment increases the proportion of ROS⁺ neutrophils in dHL-60 cells in vitro, while co-treatment with GLSP or XJB-5–131 extract reverses this increase. (C, D) The intraplantar injection of TNF-α-treatment neutrophils induces mechanical allodynia and thermal hyperalgesia. Co-treatment with either GLSP extract or XJB-5–131 mitigates this effect. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA assay followed by Tukey’s post hoc test (B), two-way ANOVA assay followed by Tukey’s post hoc test (C, D).

Figure 9

GLSP exerts therapeutic effects by reducing ROS levels in the paws cell of mice with CIA. (A) The gating strategy employed to ROS levels in paw immune cells, along with representative plots of ROS levels across different groups, is presented. (B, C) In vivo studies demonstrate that GLSP treatment reduces both the proportion and number of ROS-positive cells within the affected paw tissues of CIA mice. (D–I) The ratio of ROS-positive cells in the vehicle group, but not in the GLSP or naive control group, negatively correlates with mechanical and thermal pain thresholds. (J, K) The ROS scavenger XJB-5–131 alleviates mechanical allodynia and thermal hyperalgesia in CIA mice treated with the vehicle, but does not exert a similar effect in mice treated with GLSP. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA assay followed by Tukey’s post hoc test (B, C), Pearson correlation analysis (D–I), two-way ANOVA assay followed by Tukey’s post hoc test (J, K).