Cynandione A Alleviates Neuropathic Pain Through α7-nAChR-Dependent IL-10/β-Endorphin Signaling Complexes

Abstract

Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, exhibits antineuropathic pain effect. This study further explored the target molecule and signaling mechanisms underlying cynandione-A-induced antineuropathic pain. Intrathecal injection of cynandione A significantly attenuated mechanical allodynia in neuropathic rats and substantially increased spinal expression of IL-10 and β-endorphin but not dynorphin A. Cynandione A treatment also enhanced expression of IL-10 and β-endorphin but not α7 nicotinic acetylcholine receptors (nAChRs) in cultured microglia. The IL-10 antibody attenuated cynandione-A-induced spinal or microglial gene expression of β-endorphin and mechanical allodynia, whereas the β-endorphin antiserum blocked cynandione-A-induced mechanical antiallodynia but not spinal or microglial IL-10 gene expression. The α7 nAChR antagonist methyllycaconitine significantly reduced cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and β-endorphin. Furthermore, cynandione A stimulated microglial phosphorylation of PKA, p38, and CREB in an α7-nAChR-dependent manner, and treatment with their inhibitors attenuated cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and β-endorphin. In addition, cynandione A stimulated spinal phosphorylation of the transcription factor STAT3, which was inhibited by methyllycaconitine, the PKA activation inhibitor or IL-10 antibody. The STAT3 inhibitor NSC74859 also abolished cynandione-A-induced mechanical antiallodynia and spinal expression of β-endorphin. These findings suggest that cynandione A suppresses neuropathic pain through α7-nAChR-dependent IL-10/β-endorphin signaling pathway in spinal microglia.

Keywords: cynandione A; interleukin-10; microglia; neuropathic pain; α7 nicotinic acetylcholine receptor; β-endorphin.

FIGURE 1

Effects of cynandione A, given intrathecally, on mechanical allodynia (A), spinal mRNA expression of IL-10 (B), the β-endorphin precursor proopiomelanocortin (POMC (C)) and dynorphin precursor prodynorphin (PDYN (D)), and spinal protein expression of IL-10 (E) and β-endorphin (F) in neuropathic rats induced by L5/L6 spinal nerve ligation. Spinal lumbar enlargements (L3-L5) were obtained from the sacrificed rats 1 h after injection of cynandione A or the vehicle. The gene and protein expression was determined by using qRT-PCR and enzyme-linked immunosorbent fluorescent assays, respectively. Data are shown as means ± SEM (n = 6 per group). ^* p < 0.05 compared with the vehicle group, analyzed by two-tailed and unpaired Student t-test or repeated measures two-way ANOVA followed by the post-hoc Student-Newman-Keuls test.

FIGURE 2

Specific stimulatory effects of cynandione A, given intrathecally, on spinal microglial expression of IL-10 in neuropathic rats induced by tight ligation of L5/L6 spinal nerves. Neuropathic rats that received intrathecal injection of cynandione A or the vehicle were sacrificed 1 h after injection and the spinal lumbar enlargements (L3–L5) were obtained. The specific expression of IL-10 was double fluorescence immunolabeled with the microglial cellular biomarker Iba-1 (A–F), astrocytic cellular biomarker GFAP (G-L), and neuronal cellular biomarker NeuN (M–R) in the contralateral and ipsilateral spinal cord and dorsal horn I-Ⅲ laminate. Arrows indicate yellow double labeling of IL-10 with the corresponding cellular biomarkers. The immunolabeled surface areas of IL-10/Iba-1 (S), IL-10/GFAP (T), and IL-10/NeuN (U) from the indicated spinal dorsal horn laminae I-Ⅲ were quantified using the Image J program. Data are presented as means ± SEM (n = 5 per group). ^* P < 0.05 compared with the vehicle group, analyzed by one-way ANOVA followed by the post-hoc Student-Newman-Keuls test.

FIGURE 3

Specific stimulatory effects of cynandione A, given intrathecally, on spinal microglial expression of β-endorphin in neuropathic rats induced by tight ligation of L5/L6 spinal nerves. Neuropathic rats that received intrathecal injection of cynandione A or the vehicle were sacrificed 1 h after injection and the spinal lumbar enlargements (L3–L5) were obtained. Expression of β-endorphin was double fluorescence immunolabeled with the microglial cellular biomarker Iba-1 (A–F), astrocytic cellular biomarker GFAP (G-L), and neuronal cellular biomarker NeuN (M–R) in the contralateral and ipsilateral spinal cord and dorsal horn I-Ⅲ laminate. Arrows indicate yellow double labeling of β-endorphin with the corresponding cellular biomarkers. The immunolabeled surface areas of β-endorphin/Iba-1 (S), β-endorphin/GFAP (T), and β-endorphin/NeuN (U) from the indicated spinal dorsal horn laminae I-Ⅲ were quantified using the Image J program. Data are presented as means ± SEM (n = 5 per group). ^* P < 0.05 compared with the vehicle group, analyzed by one-way ANOVA followed by the post-hoc Student-Newman-Keuls test.

FIGURE 4

Effects of cynandione A on gene (A–C) expression, protein secretion (D, E), and fluorescent immunostaining (F–S) of IL-10, β-endorphin, and dynorphin A in primary cultures of spinal microglia collected from 1-day-old neonatal rats. Primary microglial cells were incubated with cynandione A for 2 h. The qRT-PCR and enzyme-linked immunosorbent fluorescent kits were used to detect gene expression and protein secretion in the cells and culture medium, respectively. Single and triple immunofluorescence staining was used to detect I-10 (red), Iba-1 (green), and IL-10 (red)/Iba-1 (green)/DAPI (blue) or β-endorphin (red)/Iba-1 (green)/DAPI (blue). The immunolabeled surface areas of IL-10/Iba-1 (R) and β-endorphin/Iba-1 (S) were quantified by using the Image J program. The data are presented as means ± SEM (n = 3 per group with two independent repeats). ^* p < 0.05 compared with the vehicle group, by two-tailed and unpaired Student t-test.

FIGURE 5

Blockade effects of the IL-10 antibody and β-endorphin antiserum on cynandione-A-induced spinal mechanical antiallodynia (A), spinal gene expression of IL-10 (B) and the β-endorphin precursor proopiomelanocortin (POMC (C)), and spinal protein expression of IL-10 (D) and β-endorphin (E) in neuropathic rats induced by L5/L6 spinal nerve ligation. Neuropathic rats received intrathecal injection of saline, the IL-10 antibody or β-endorphin antiserum followed by intrathecal injection of the vehicle or cynandione A 30 min later. The spinal cords were obtained immediately after the completion of behavioral test (1 h after the last injection) for detection of IL-10 and β-endorphin by using qRT-PCR and enzyme-linked immunosorbent fluorescent assays, respectively. Blockade effects of the IL-10 antibody and β-endorphin antiserum on cynandione-A-stimulated gene expression of POMC (F) and IL-10 (G) in cultured microglial cells originated from 1-day-old neonatal rats. Data are shown as means ± SEM (n = 6 per group in neuropathic rats or n = 3 per group with two independent repeats in cultured cells). *, ^# p < 0.05 compared with the control and cynandione A treatment groups, respectively, analyzed by one-way ANOVA followed by the post-hoc Student-Newman-Keuls test.

FIGURE 6

Blockade effects of the α7 nicotinic acetylcholine receptor (α7 nAChR) antagonist methyllycaconitine, given before or after cynandione A injection, on cynandione-A-induced spinal mechanical antiallodynia in the ipsilateral hindpaws (A,B), spinal gene expression of IL-10 (C) and the β-endorphin precursor proopiomelanocortin (POMC (D)), and protein expression of IL-10 (E) and β-endorphin (F) in neuropathic rats induced by spinal nerve ligation. Neuropathic rats received intrathecal injection of the vehicle or methyllycaconitine followed by intrathecal saline or cynandione A 30 min later. The spinal cords were obtained 1 h after the last intrathecal injection for detection of IL-10 and β-endorphin by using qRT-PCR and enzyme-linked immunosorbent fluorescent assays, respectively. For the postinjection study, neuropathic rats received intrathecal injection of cynandione A followed by methyllycaconitine 30 min later. Data are shown as means ± SEM (n = 6 per group in neuropathic rats). *, ^# p < 0.05 compared with the control and cynandione A treatment groups, respectively, analyzed by one-way or measures-repeated two-way ANOVA followed by the post-hoc Student-Newman-Keuls test.

FIGURE 7

Stimulatory effects of cynandione A on expression of IL-10 colocalized with the α7 nicotinic acetylcholine receptor (α7 nAChR) (A–I) and blockade effects of α7 nAChR antagonist methyllycaconitine on cynandione A-stimulated expression of IL-10 and β-endorphin (J–M) in cultured microglial cells originated from 1-day-old neonatal rats. Cultured primary microglial cells were collected 2 h after cynandione A incubation. The single and triple immunofluorescence staining of IL-10 (red), α7 nAChR (green), and IL-10 (red)/α7 nAChR (green)/DAPI (blue) was performed. The immunolabeled surface areas of IL-10 (G), α7 nAChR (H), and IL-10/α7 nAChR/DAPI (I) were quantified by using the Image J program. The qRT-PCR and enzyme-linked immunosorbent fluorescent kits were used to detect gene expression and protein secretion in the cells and culture medium, respectively. Data are presented as means ± SEM (n = 3 per group with two independent repeats). ^* P < 0.05 compared with the control group, analyzed by two-tailed and unpaired Student t-test or one-way followed by the post-hoc Student-Newman-Keuls test.

FIGURE 8

Blockade effects of the adenylyl cyclase inhibitor DDA, PKA activation inhibitor H-89, p38 activation inhibitor SB203580, and CREB activation inhibitor KG501 on cynandione-A-induced spinal mechanical antiallodynia and mRNA expression of IL-10 and the β-endorphin precursor proopiomelanocortin (POMC (C)) in the spinal cords of neuropathic rats induced by spinal nerve ligation (A–C) and cultured primary microglial cells originated from 1-day-old neonatal rats (D, E). Neuropathic rats received intrathecally the vehicle or DDA, H-89, SB203580 and KG501 30 min followed by cynandione A. The spinal cords and cultured microglial cells were obtained 1 h after the last intrathecal injection or 2 h after incubation to detect IL-10 and POMC gene expression by using qRT-PCR. Data are shown as means ± SEM (n = 6 per group in neuropathic rats or n = 3 per group with two independent repeats in cultured cells). *, ^# p < 0.05 compared with the control and cynandione A treatment groups, respectively, analyzed by one-way or two-way ANOVA followed by the post-hoc Student-Newman-Keuls test.

FIGURE 9

Blockade effects of the PKA activation inhibitor H-89 (A,B) and IL-10 antibody (C,D) on cynandione-A-stimulated spinal STAT3 phosphorylation in neuropathic rats induced by L5/L6 spinal nerve ligation. Blockade effects of the STAT3 activation inhibitor NSC74859 on cynandione-A-induced spinal mechanical antiallodynia (E) and gene expression of the β-endorphin precursor proopiomelanocortin (POMC) in the spinal cords of neuropathic rats (F) and cultured primary microglial cells originated from 1-day-old neonatal rats (G). Neuropathic rats received intrathecal H-89, the IL-10 antibody or NSC74859 30 min later followed by intrathecal cynandione A. The spinal cords were obtained 1 h after the last intrathecal injection for detection of STAT3 phosphorylation and POMC expression by using western blot and qRT-PCR, respectively. Data are shown as means ± SEM (n = 6 per group in neuropathic rats or n = 3 per group with two independent repeats in cultured cells). *, ^# p < 0.05 compared with the control and cynandione A treatment groups, respectively, analyzed by one-way or measures-repeated two-way ANOVA followed by the post-hoc Student-Newman-Keuls test.

FIGURE 10

Blockade effects of the α7 nicotinic acetylcholine receptor (α7 nAChR) antagonist methyllycaconitine on cynandione-A-induced phosphorylation of spinal PKA (A,B), p38 (C,D), CREB (E,F), and STAT3 (G,H) in neuropathic rats induced by L5/L6 spinal nerve ligation. Neuropathic rats received intrathecal injection of the vehicle or methyllycaconitine 30 min later followed by intrathecal cynandione A. The spinal cords were obtained 1 h after the last intrathecal injection for the phosphorylation detection by using western blot. Data are shown as means ± SEM (n = 6 per group). *, ^# p < 0.05 compared with the control and cynandione A treatment groups, respectively, analyzed by one-way ANOVA followed by the post-hoc Student-Newman-Keuls test.