Eicosapentaenoic Acid Modulates Transient Receptor Potential V1 Expression in Specific Brain Areas in a Mouse Fibromyalgia Pain Model

Abstract

Pain is an unpleasant sensory and emotional experience accompanied by tissue injury. Often, an individual's experience can be influenced by different physiological, psychological, and social factors. Fibromyalgia, one of the most difficult-to-treat types of pain, is characterized by general muscle pain accompanied by obesity, fatigue, sleep, and memory and psychological concerns. Fibromyalgia increases nociceptive sensations via central sensitization in the brain and spinal cord level. We used intermittent cold stress to create a mouse fibromyalgia pain model via a von Frey test (day 0: 3.69 ± 0.14 g; day 5: 2.13 ± 0.12 g). Mechanical pain could be reversed by eicosapentaenoic acid (EPA) administration (day 0: 3.72 ± 0.14 g; day 5: 3.69 ± 0.13 g). A similar trend could also be observed for thermal hyperalgesia. The levels of elements in the transient receptor potential V1 (TRPV1) signaling pathway were increased in the ascending pain pathway, including the thalamus, medial prefrontal cortex, somatosensory cortex, anterior cingulate cortex, and cerebellum. EPA intake significantly attenuated this overexpression. A novel chemogenetics method was used to inhibit SSC and ACC activities, which presented an analgesic effect through the TRPV1 downstream pathway. The present results provide insights into the role of the TRPV1 signaling pathway for fibromyalgia and its potential as a clinical target.

Keywords: TRPV1; eicosapentaenoic acid; fibromyalgia; mPFC; thalamus.

Figure 1

(A) Mechanical force measured with the von Frey filament test. (B) Thermal latency time via Hargreaves test. * p < 0.05 means significant difference in comparison to the normal group. ^# p < 0.05 means significant differences with the FM group. n = 9.

Figure 2

The expression levels of TRPV1 and related molecules in the mouse thalamus. Western blotting results of (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pJNK, pp38, pCREB, pNF-κB, and CB1 protein levels (refer to Supplementary Figure S1 for the Western blot bands). Red arrow means immune-positive signals. * p < 0.05 means significant difference in comparison to the normal group. ^# p < 0.05 means significant differences with the FM group. n = 9. (C) Immunofluorescence labeling of TRPV1, pERK, and double staining in the mouse THA (green, red, or yellow, respectively). Bar, 100 μm. n = 3 in all groups.

Figure 3

The expression levels of TRPV1 and related molecules in the mouse SSC. Western blotting results of (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pJNK, pp38, pCREB, pNF-κB, and CB1 protein levels (refer to Supplementary Figure S2 for the Western blot bands). Red arrow means immune-positive signals. * p < 0.05 means significant difference in comparison to the normal group. ^# p < 0.05 means significant differences with the FM group. n = 9. (C) Immunofluorescence labeling of TRPV1, pERK, and double staining in the mouse SSC (green, red, or yellow, respectively). Bar, 100 μm. n = 3 in all groups.

Figure 4

The expression levels of TRPV1 and related molecules in the mouse ACC. Western blotting results of (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pJNK, pp38, pCREB, pNF-κB, and CB1 protein levels (refer to Supplementary Figure S3 for the Western blot bands). Red arrow means immune-positive signals. * p < 0.05 means significant difference in comparison to the normal group. ^# p < 0.05 means significant differences with the FM group. n = 9. (C) Immunofluorescence labeling of TRPV1, pERK, and double staining in the mouse ACC (green, red, or yellow, respectively). Bar, 100 μm. n = 3 in all groups.

Figure 5

The expression levels of TRPV1 and related molecules in the mouse mPFC. Western blotting results of (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pJNK, pp38, pCREB, pNF-κB, and CB1 protein levels (refer to Supplementary Figure S4 for the Western blot bands). Red arrow means immune-positive signals. * p < 0.05 means significant difference in comparison to the normal group. ^# p < 0.05 means significant differences with the FM group. n = 9. (C) Immunofluorescence labeling of TRPV1, pERK, and double staining in the mouse mPFC (green, red, or yellow, respectively). Bar, 100 μm. n = 3 in all groups.

Figure 6

The expression levels of TRPV1 and related molecules in mouse cerebellum 5. Western blotting results of (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pJNK, pp38, pCREB, pNF-κB, and CB1 protein levels (refer to Supplementary Figure S5 for the Western blot bands). Red arrow means immune-positive signals. * p < 0.05 means significant difference in comparison to the normal group. ^# p < 0.05 means significant differences with the FM group. n = 9. (C) Immunofluorescence labeling of TRPV1, pERK, and double staining in mouse cerebellum 5 (green, red, or yellow, respectively). Bar, 100 μm. n = 3 in all groups.

Figure 7

The expression levels of TRPV1 and related molecules in mouse cerebellum 6. Western blotting results of (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pJNK, pp38, pCREB, pNF-κB, and CB1 protein levels (refer to Supplementary Figure S6 for the Western blot bands). Red arrow means immune-positive signals. * p < 0.05 means significant difference in comparison to the normal group. ^# p < 0.05 means significant differences with the FM group. n = 9. (C) Immunofluorescence labeling of TRPV1, pERK, and double staining in mouse cerebellum 6 (green, red, or yellow, respectively). Bar, 100 μm. n = 3 in all groups.

Figure 8

The expression levels of TRPV1 and related molecules in mouse cerebellum 7. Western blotting results of (A) TRPV1, pPKA, pPI3K, pPKC, pAkt, and pmTOR. (B) pERK, pJNK, pp38, pCREB, pNF-κB, and CB1 protein levels (refer to Supplementary Figure S7 for the Western blot bands). Red arrow means immune-positive signals. * p < 0.05 means significant difference in comparison to the normal group. ^# p < 0.05 means significant differences with the FM group. n = 9. (C) Immunofluorescence labeling of TRPV1, pERK, and double staining in mouse cerebellum 7 (green, red, or yellow, respectively). Bar, 100 μm. n = 3 in all groups.

Figure 9

Pain behaviors of FM and FM mice treated with chemogenetics method (hM4Di). (A) Mechanical hyperalgesia (von Frey test). (B) Thermal hyperalgesia (Hargreaves test). * p < 0.05 means significant difference in comparison to basal condition. Protein percentages of CB1, pPKA, pPI3K, pPKC, pAkt, pmTOR, pERK, and pNFkB were measured in mice (C) SSCs and (D) ACCs. * p < 0.05 means significant difference in comparison to the FM group.