Targeting ASIC3 for Relieving Mice Fibromyalgia Pain: Roles of Electroacupuncture, Opioid, and Adenosine

Abstract

Many scientists are seeking better therapies for treating fibromyalgia (FM) pain. We used a mouse model of FM to determine if ASIC3 and its relevant signaling pathway participated in FM pain. We demonstrated that FM-induced mechanical hyperalgesia was attenuated by electroacupuncture (EA). The decrease in fatigue-induced lower motor function in FM mice was also reversed by EA. These EA-based effects were abolished by the opioid receptor antagonist naloxone and the adenosine A1 receptor antagonist rolofylline. Administration of opioid receptor agonist endomorphin (EM) or adenosine A1 receptor agonist N⁶-cyclopentyladenosine (CPA) has similar results to EA. Similar results were also observed in ASIC3^-/- or ASIC3 antagonist (APETx2) injected mice. Using western blotting, we determined that pPKA, pPI3K, and pERK were increased during a dual acidic injection priming period. Nociceptive receptors, such as ASIC3, Nav1.7, and Nav1.8, were upregulated in the dorsal root ganglion (DRG) and spinal cord (SC) of FM mice. Furthermore, pPKA, pPI3K, and pERK were increased in the central thalamus. These aforementioned mechanisms were completely abolished in ASIC3 knockout mice. Electrophysiological results also indicated that acid potentiated Nav currents through ASIC3 and ERK pathway. Our results highlight the crucial role of ASIC3-mediated mechanisms in the treatment of FM-induced mechanical hyperalgesia.

Figure 1. Electroacupuncture (EA) reduced mechanical hyperalgesia induced by dual acid saline injection (fibromyalgia model, FM) as measured by von Frey filaments.

(A) Mechanical responses of saline-injected control mice. (B) Mechanical responses of FM group mice. (C) Mechanical responses of FM mice treated with EA (EA group). (D) Mechanical responses of FM mice treated with EA and naloxone (Nal group). (E) Mechanical responses of FM mice treated with EA and rolofyllin (Rol group). (F) Mechanical responses of FM mice treated with endomorphin (EM group). (G) Mechanical responses of FM mice treated with EA and N⁶-cyclopentyladenosine (CPA group). (H) Mechanical responses of FM in ASIC null mice (ASIC3 null group). (I) Mechanical responses of FM mice treated with APETx2 (APETx2 group). Mice were tested before injection (day 0), 4 hours after injection, day 1 (D1), day 5 (D5), day 6 (D6), and day 8 (D8). *p < 0.05 compared to baseline (n = 10 mice per group).

Figure 2. Expression levels of ASIC3-associated signaling pathway proteins in DRG and SC after first acid injection.

(A) ASIC3, (B) pPKA, (C) pPI3K, (D) pPKC, (E) pERK, (F) Nav1.7, and (G) Nav1.8 expression levels in tissues from the Con, FM, EA groups (from left to right). Con = Control; FM = Fibromyalgia group; EA = Electroacupuncture. *p < 0.05 compared with the Con group. ^#p < 0.05 compared with the FM group. The western blot bands at the top show the cropped target protein. The lower bands are cropped internal controls (β-actin or α-tubulin).

Figure 3. Expression levels of ASIC3-associated signaling pathway proteins in DRG and SC after second acid injection.

(A) ASIC3, (B) pPKA, (C) pPI3K, (D) pPKC, (E) pERK, (F) Nav1.7, and (G) Nav1.8 expression levels in tissues from the Con, FM, EA groups (from left to right). Con = Control; FM = Fibromyalgia group; EA = Electroacupuncture. *p < 0.05 compared with the Con group. ^#p < 0.05 compared with the FM group. The western blot bands at the top show the cropped target protein. The lower bands are cropped internal controls (β-actin or α-tubulin).

Figure 4. Expression levels of ASIC3-associated signaling pathway proteins in L3–5 DRG.

(A) ASIC3, (B) pPKA, (C) pPI3K, (D) pPKC, (E) pERK, (F) Nav1.7, and (G) Nav1.8 expression levels in tissues from the Con, FM, EA, Nal, Rol, and ASIC3 null groups (from left to right). Con = Control; FM = Fibromyalgia group; EA = Electroacupuncture; Nal = Naloxone group; Rol = Rolofyllin group. ASIC3 null = ASIC3 gene deletion group. *p < 0.05 compared with the Con group. ^#p < 0.05 compared with the FM group. The western blot bands at the top show the cropped target protein. The lower bands are cropped internal controls (β-actin or α-tubulin).

Figure 5. Expression levels of ASIC3-associated signaling pathway proteins in lumbar SC.

(A) ASIC3, (B) pPKA, (C) pPI3K, (D) pPKC, (E) pERK, (F) Nav1.7, and (G) Nav1.8 expression levels in tissues from the Con, FM, EA, Nal, Rol, and ASIC3 null groups (from left to right). Con = Control; FM = Fibromyalgia group; EA = Electroacupuncture; Nal = Naloxone group; Rol = Rolofyllin group. ASIC3 null = ASIC3 gene deletion group (from left to right). Con = Control; FM = Fibromyalgia group; EA = Electroacupuncture; Nal = Naloxone group; Rol = Rolofyllin group. *p < 0.05 compared with the Con group. ^#p < 0.05 compared with the FM group. The western blot bands at the top show the cropped target protein. The lower bands are cropped internal controls (β-actin or α-tubulin).

Figure 6. Expression levels of ASIC3-associated signaling pathway proteins in thalamus.

(A) ASIC3, (B) pPKA, (C) pPI3K, (D) pPKC, (E) pERK, (F) Nav1.7, and (G) Nav1.8 expression levels in tissues from the Con, FM, EA, Nal, Rol, and ASIC3 null groups (from left to right). Con = Control; FM = Fibromyalgia group; EA = Electroacupuncture; Nal = Naloxone group; Rol = Rolofyllin group. ASIC3 null = ASIC3 gene deletion group (from left to right). Con = Control; FM = Fibromyalgia group; EA = Electroacupuncture; Nal = Naloxone group; Rol = Rolofyllin group. *p < 0.05 compared with the Con group. ^#p < 0.05 compared with the FM group. The western blot bands at the top show the cropped target protein. The lower bands are cropped internal controls (β-actin or α-tubulin).

Figure 7. Expressions of ASIC3, Nav1.7, and Nav1.8 in the DRG of Con, FM, EA, Nal, and Rol mice.

ASIC3-positive neurons (green) in the DRG of (A) Con, (B) FM, (C) EA, (D) Nal, and (E) Rol mice. Nav1.7-positive neurons (green) in the DRG of (F) Con, (G) FM, (H) EA, (I) Nal, and (J) Rol mice. Nav1.8-positive neurons (green) in the DRG of (K) Con, (L) FM, (M) EA, (N) Nal, and (O) Rol mice. Scale bar means 100 μm. Arrows identify immunopositive neurons.

Figure 8. Acid-sensing ion channel 3 or voltage-gated sodium currents in L3-L5 DRG neurons.

(A) Representative acid-sensing ion channel 3 currents traces in Con, FM, EA, and ASIC3^−/− groups. The acid-sensing ion channel 3 currents were induced by injection of pH5.0 saline directly to DRG neurons. (B) Representative voltage-gated sodium currents traces in Con, FM, EA, and ASIC3^−/− groups. The voltage-gated sodium currents were induced by membrane depolarization from −70 to 0 mV. (C) Representative voltage-gated sodium currents traces in pH 7.4, pH 5.0, pH 5.0 + SA, and pH 5.0 + U0126 groups. (A) Mean peak amplitudes of acid-sensing ion channel 3 currents in Con, FM, EA, and ASIC3^−/− groups. (B) Mean peak amplitudes of voltage-gated sodium currents in Con, FM, EA, and ASIC3^−/− groups. (C) Mean peak amplitudes of voltage-gated sodium currents traces in pH 7.4, pH 5.0, pH 5.0 + SA, and pH 5.0 + U0126 groups. *p < 0.05 compared with Con or pH 7.4 groups. ^#p < 0.05 compared with FM or pH 5.0 groups.