ANA-12 Targets and Inhibits BDNF/TrkB Signaling to Alleviate Pain Behaviors in Rheumatoid Arthritis Mice

Abstract

Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory disease. Sensitization of central pain pathways by pro-inflammatory mediators has been implicated in RA pain. Locus coeruleus (LC) functions in pain pathways. Brain-derived neurotrophic factor (BDNF) participates in the modulation of nociception and pain. A mouse model of RA immunized with collagen-induced arthritis (CIA) was used for investigating the mechanisms of pain relief by administration of the tropomyosin receptor kinase B (TrkB) receptor antagonist ANA-12. We measured the pain behaviors and locomotor activity and found increased pain sensitivity and locomotor deficit in RA mice; ANA-12 treatment reduced pain behaviors and promoted locomotor function recovery. The glial activation and increased activities of BDNF/TrkB and MAPK signal pathways were found in LC of RA mice. The components of NLRP3 inflammasome were all increased and consequently enhanced the production of pro-inflammatory cytokine interleukin (IL)-1β. Upon ANA-12 treatment, glial cell activation was reduced, BDNF/TrkB and MAPK pathways were suppressed, and the expression levels of the above-mentioned proteins were reduced. Finally, U251 cells were conducted to further confirm the regulatory mechanisms of ANA-12 on inflammation. The results showed the colocalization of BDNF/NLRP3/IL-1β and GFAP. ANA-12 treatment decreased the protein levels of BDNF, TrkB, MAPK, NLRP3, and caspase-1 in IL-1β-induced cells. Besides, ANA-12 treatment decreased NLRC4 and AIM2 inflammasomes both in RA mice and IL-1β-induced cells. These results suggested that ANA-12 alleviates hyperalgesia in RA mice by inhibiting BDNF/TrkB signaling in LC, thereby reducing glial cell activation and inflammatory cytokine release.

Keywords: ANA-12; Arthritis pain; Brain-derived neurotrophic factor; Inflammatory factor; Locus coeruleus.

Fig. 1

Pain-related behaviors in mice from each group. (A) Establishment of the CIA model, behavioral testing and histological analysis (n = 9). (B) Comparison of paw swelling between the Control and CIA groups. (C, E, G, I) Schematic diagram of spontaneous flinches, PWT test, PWL test and rotarod test. (D, F, H, J) Statistical analysis of the numbers of spontaneous flinches, PWT values in response to von Frey filaments, PWL values in response to thermal stimuli and the latency to fall of rotarod motor test. (K-M) Linear analysis of motor coordination with spontaneous flinches (K), PWT (L) and PWL (M). *P < 0.05 compared to control mice; ^#P < 0.05 compared to CIA mice. Data were expressed as the mean ± SEM. CIA, collagen-induced arthritis; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; ANA, ANA-12

Fig. 2

Activation status of glial cells in each group. (A, C) Representative immunofluorescence images of GFAP and Iba-1 in LC. Scale bar = 50 μm. (B, D) Quantitative analysis of the average fluorescence intensity in the left and right LC regions of GFAP and Iba-1. Data are presented as mean ± SD, n = 3, *P < 0.05 compared to control mice; ^#P < 0.05 compared to CIA mice. LC, locus coeruleus; CIA, collagen-induced arthritis; ANA, ANA-12

Fig. 3

Changes in BDNF/TrkB signaling pathway in each group. (A, C) Representative immunofluorescence images of BDNF and pTrkB in the in LC. Scale bar = 50 μm. (B, D) Quantitative analysis of the average fluorescence intensity in the left and right LC regions of BDNF and pTrkB. Data are presented as mean ± SD, n = 3, *P < 0.05 compared to control mice; ^#P < 0.05 compared to CIA mice. LC, locus coeruleus; CIA, collagen-induced arthritis; ANA, ANA-12

Fig. 4

Alterations of downstream signaling pathways of BDNF/TrkB in each group. (A, C, E, G) Representative immunofluorescence images of pMAPK, NLRP3, Caspase-1, and IL-1β in LC. Scale bar = 50 μm. (B, D, F, H) Quantitative analysis of the average fluorescence intensity in the left and right LC regions of pMAPK, NLRP3, caspase-1, and IL-1β. Data are presented as mean ± SD, n = 3, *P < 0.05 compared to control mice; ^#P < 0.05 compared to CIA mice. LC, locus coeruleus; CIA, collagen-induced arthritis; ANA, ANA-12

Fig. 5

Changes of NLRC4 and AIM2 inflammasomes in each group. (A, C) Representative immunofluorescence images of NLRC4 and AIM2 in the LC. Scale bar = 50 μm. (B, D) Quantitative analysis of the average fluorescence intensity in the left and right LC regions of NLRC4 and AIM2. Data are presented as mean ± SD, n = 3, *P < 0.05 compared to control mice; ^#P < 0.05 compared to CIA mice. CIA, collagen-induced arthritis; ANA, ANA-12

Fig. 6

The effect of ANA-12 treatment on inflammatory signaling in U251 cells upon IL-1β. (A, B, C) Colocalization of BDNF/NLRP3/IL-1β (green) and GFAP (red) in U251 cells. Scale bar = 50 μm. (D) Quantitative analysis of the immunofluorescence intensity of BDNF, NLRP3, IL-1β, and GFAP in cells. (E) Immunoblot analysis of GFAP and Iba-1 expression in U251 cells. (F) Quantitative analysis of GFAP and Iba-1 protein level. (G) Immunoblot analysis of BDNF, TrkB, and pTrkB expression in U251 cells. (H) Quantitative analysis of BDNF, pTrkB/TrkB protein level. (I) Immunoblot analysis of MAPK, pMAPK, NLRP3, and caspase-1 expression in U251 cells. (J) Quantitative analysis of pMAPK/MAPK, NLRP3, and caspase-1 protein level. (K) Immunoblot analysis of NLRC4 and AIM2 expression in U251 cells. (L) Quantitative analysis of NLRC4 and AIM2 protein level. Data are presented as mean ± SD, n = 3, *P < 0.05 compared to control groups; ^#P < 0.05 compared to IL-1β groups. CIA, collagen-induced arthritis; ANA, ANA-12

Fig. 7

The mechanism diagram of ANA-12 in reducing glial cell activation and the release of pro-inflammatory cytokines by inhibiting the BDNF/TrkB signaling pathway in LC, thereby alleviating hyperalgesia in RA mice