The Antinociceptive Effect of Nicorandil in Neuropathic and Nociceptive Pain is Partially Mediated via TRPV1/Opioidergic Signaling

Abstract

Neuropathic pain is a serious neurological disorder caused by lesioned somatosensory neurons characterized by multiple pathologies. Transient receptor potential vanilloid 1 (TRPV1) channels and opioid receptors are co-expressed in dorsal root ganglia (DRG) and play a crucial role in the development of neuropathic pain. Here, we investigated the possible involvement of TRPV1 channels and µ-opioid receptors in mediating the antinociception of the K_ATP opener, nicorandil, in neuropathic pain in four nociceptive models: chronic constriction injury of the sciatic nerve (CCI), formalin, capsaicin, and acetic acid writhing tests. Nicorandil (150 mg/kg, twice, 2 h apart, PO) administered to male rats (i) reversed the effects of CCI on nociceptive threshold and cumulative scores assessed by von Frey and acetone test, respectively; (ii) reduced licking time and number of flinches in biphasic formalin and capsaicin tests, and (iii) reduced the number of writhes in the acetic acid test; and (iv) combined nicorandil-capsaicin abolished acetic acid induced writhing response. Similarly, ipsilateral intraplantar injection of nicorandil (37.5 mg/paw, twice, ipl) inhibited nociceptive responses induced by capsaicin, formalin, and acetic acid. Immunohistochemical analysis revealed that nicorandil blunted the CCI-induced elevation of TRPV1 protein expression in DRG. The beneficial effects of nicorandil in all models were attenuated by naloxone. Molecular docking supported the interaction between nicorandil and TRPV1. Histologically, nicorandil improved the pathological changes induced by CCI in the sciatic nerve and DRG. Collectively, these results demonstrate that nicorandil exhibits antinociceptive effects in neuropathic and nociceptive pain via mechanisms involving TRPV1 modulation and opioid receptor signaling. Further investigation is warranted to explore the mechanism of action of nicorandil as an alternative treatment option for neuropathic pain.

Keywords: Neuropathic pain; Nicorandil; Opioidergic; TRPV1.

Fig. 1

Experimental protocol and time schedule for drug administration in neuropathic pain (A) and nociceptive pain models (B). IHC, immunohistochemistry; DRG, dorsal root ganglia

Fig. 2

Mechanical (A) and cold allodynia (B) induced by chronic constriction injury of sciatic nerve (CCI) in male rats. Mechanical nociceptive threshold and cumulative scores were assessed by von Frey and acetone tests, respectively, on alternate days from day 2 after surgery to day 14. All results were expressed as mean ± SEM (n = 5–8). Results were analyzed by multiple unpaired t-test. *P < 0.05 vs sham

Fig. 3

Effect of nicorandil (150 mg/kg, twice, 2 h apart, PO) on the nociceptive threshold (A), the cumulative score (B), and the corresponding area under the curves (C and D) assessed by the von Frey and acetone test, respectively, in chronic constriction injury (CCI) rats. Naloxone (1 mg/kg, twice, 2 h apart, I.P) was used as a standard opioid blocker in nicorandil-treated rats and was administered simultaneously with each dose of nicorandil. All results were expressed as mean ± SEM (n = 5–8). Results were analyzed by two-way ANOVA in temporal course figures (A–B) and one-way ANOVA in AUC figures (C–D) followed by Tukey’s post hoc test. *P < 0.05 vs sham; + P < 0.05 vs CCI; #P < 0.05 vs CCI-nicorandil

Fig. 4

Effect of nicorandil (150 mg/kg, twice, 2 h apart, PO) in absence and presence of naloxone (1 mg/kg, twice, 2 h apart, I.P) on licking time (A, B), number of flinches (C, D) induced by ipl injection of formalin (50 µl, 1%) measured in first and second phases, number of flinches over 1 h (E) and total area under the curve for flinches (F). All results were expressed as mean ± SEM (n = 5–8). Results were analyzed by one-way ANOVA followed by Tukey’s post hoc test; *P < 0.05 vs control; + P < 0.05 vs nicorandil

Fig. 5

Pearson correlation between the number of flinches of the second phase of the formalin test and cold allodynia induced by CCI in rats evaluated at 1.5, 3.5, 5.5, and 7.5 h (A, B, C, and D, respectively), after treatment with vehicle (CMC), nicorandil (150 mg/kg, twice, 2 h apart, PO) and nicorandil + naloxone (1 mg/kg, twice, 2 h apart, I.P) and between AUC of the number of flinches over 1 h and AUC of cumulative score in the cold allodynia test (E)

Fig. 6

Pearson correlation between licking time of the second phase of the formalin test and the cumulative score of cold allodynia induced by CCI in rats evaluated at 1.5, 3.5, 5.5, and 7.5 h (A, B, C, and D, respectively), after treatment with vehicle (CMC), nicorandil (150 mg/kg, twice, 2 h apart, PO) and nicorandil + naloxone (1 mg/kg, twice, 2 h apart, I.P)

Fig. 7

Effect of nicorandil (150 mg/kg, twice, 2 h apart, PO) and morphine (5 mg/kg, I.P) on licking time (A) and number of flinches (B) induced by ipl injection of capsaicin (2 µg/paw). Naloxone (1 mg/kg, twice, 2 h apart, I.P) was used as a standard opioid blocker in nicorandil-treated rats. All results were expressed as mean ± SEM (n = 5–8). Results were analyzed by one-way ANOVA followed by Tukey’s post hoc test; *P < 0.05 VS capsaicin vehicle; + P < 0.05 vs capsaicin, # P < 0.05 vs nicorandil

Fig. 8

Effect of oral nicorandil (100, 150, 150 mg/kg, twice, 2 h apart, PO) alone and in presence of naloxone (1 mg/kg, twice, 2 h apart, I.P) on number of writhes induced by acetic acid (0.6%, 10 ml/kg, I.P). All results were expressed as mean ± SEM (n = 5–8). Results were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 vs control; + P < 0.05 vs nicorandil-treated groups

Fig. 9

Effect of oral nicorandil (100, 150 mg/kg) in the presence of capsaicin (4 µg/paw, ipl) on the number of writhes induced by acetic acid (0.6%, 10 ml/kg, I.P). All results were expressed as mean ± SEM (n = 3–4). Results were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 vs control acetic acid; + P < 0.05 vs acetic acid + capsaicin; #P < 0.05 vs nicorandil 100 mg/kg

Fig. 10

Effect of intraplantar injection of nicorandil (37.5 mg/paw, twice, 2 h interval) and intraplantar injection of capsazepine (16 µg/paw) on licking time (A–B), number of flinches (C–D) of first and second phases, respectively, total number of flinches over 1 h (E) and area under the curve of flinches (F) induced by formalin injection (50 µl, 1%) and licking time (G) and number of flinches (H) induced by capsaicin test (2 µg/paw) and number of writhes (I) induced by acetic acid injection (0.6%, 10 ml/kg, I.P). Naloxone (40 µg/paw, twice, ipl) was administered as a standard opioid antagonist. All results were expressed as mean ± SEM (n = 5–8). Results were analyzed by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 vs control or capsaicin

Fig. 11

Effect of nicorandil (150 mg/kg, twice, 2 h apart, PO) on immunohistochemical protein expression of TRPV1 channels in DRG of CCI rats. Naloxone (1 mg/kg, twice, 2 h apart, I.P) was used as a standard opioid blocker in nicorandil-treated rats. Representative images for immuno-stained sections are also shown. All results were expressed as mean ± SEM (n = 5). Results were analyzed by one-way ANOVA followed by Tukey’s post hoc test; *P < 0.05 vs sham; + P < 0.05 vs CCI-nicorandil

Fig. 12

Docking and binding pattern of (A) capsazepine and (B) nicorandil into TRPV1 active site (PDB ID: 8GFA) in 2D (upper panels) and 3D (lower panels)

Fig. 13

Effect of nicorandil (150 mg/kg, twice, 2 h apart, PO) on CCI-induced histopathological changes in longitudinal sections of sciatic nerve (A–D) and transverse section of dorsal root ganglia (E–H) of sham (A and E), CCI (B and F), nicorandil (150 mg/kg, twice, 2 h apart, PO)-treated CCI rats (C and G) and naloxone (1 mg/kg, twice, 2 h apart, I.P) pretreated CCI rats (D and H), respectively. All images were captured under magnification power × 400 (scale bar, 50 µm) (n = 5). Sciatic nerve: normal nerve fibers (black arrow), myelin sheets degeneration (yellow arrow), areas of edema (red arrow), Schwann cells nuclei (arrow heads), mononuclear cells infiltration (blue arrow), and increase in capillary size (*). Dorsal root ganglia: normal nerve cells (black arrow), Nissl body in the cytoplasm of nerve cells (Nb), satellite cells (blue arrow), nucleus with prominent nucleolus (green arrow), degenerated neurons with absence of Nissl bodies (yellow arrow), areas of edema (red arrow), hypertrophy of satellite cells (arrow heads), and the neuronal cells showed loss of the nuclei and showed a ghost cell-like morphology (orange arrow)