The spinal microglial IL-10/β-endorphin pathway accounts for cinobufagin-induced mechanical antiallodynia in bone cancer pain following activation of α7-nicotinic acetylcholine receptors

Abstract

Background: Cinobufagin is the major bufadienolide of Bufonis venenum (Chansu), which has been traditionally used for the treatment of chronic pain especially cancer pain. The current study aimed to evaluate its antinociceptive effects in bone cancer pain and explore the underlying mechanisms. Methods: Rat bone cancer model was used in this study. The withdrawal threshold evoked by stimulation of the hindpaw was determined using a 2290 CE electrical von Frey hair. The β-endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. Results: Cinobufagin, given intrathecally, dose-dependently attenuated mechanical allodynia in bone cancer pain rats, with the projected E max of 90% MPE and ED 50 of 6.4 μg. Intrathecal cinobufagin also stimulated the gene and protein expression of IL-10 and β-endorphin (but not dynorphin A) in the spinal cords of bone cancer pain rats. In addition, treatment with cinobufagin in cultured primary spinal microglia but not astrocytes or neurons stimulated the mRNA and protein expression of IL-10 and β-endorphin, which was prevented by the pretreatment with the IL-10 antibody but not β-endorphin antiserum. Furthermore, spinal cinobufagin-induced mechanical antiallodynia was inhibited by the pretreatment with intrathecal injection of the microglial inhibitor minocycline, IL-10 antibody, βendorphin antiserum and specific μ-opioid receptor antagonist CTAP. Lastly, cinobufagin-and the specific α-7 nicotinic acetylcholine receptor (α7-nAChR) agonist PHA-543613-induced microglial gene expression of IL-10/βendorphin and mechanical antiallodynia in bone cancer pain were blocked by the pretreatment with the specific α7-nAChR antagonist methyllycaconitine. Conclusions: Our results illustrate that cinobufagin produces mechanical antiallodynia in bone cancer pain through spinal microglial expression of IL-10 and subsequent β-endorphin following activation of α7-nAChRs. Our results also highlight the broad significance of the recently uncovered spinal microglial IL-10/β-endorphin pathway in antinociception.

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

“…More directly, cultured microglial cells coexpressed IL-10 and α7 nAChRs and cynandione A incubation stimulated expression of IL-10 but not α7 nAChRs, which was totally blocked by methyllycaconitine. It was also recently reported that the specific α7 nAChR agonist PHA-543163 stimulated methyllycaconitine-reversible IL-10/β-endorphin expression in cultured microglial cells ( Apryani et al, 2020 ). These results together suggest that cynandione A induces spinal microglial expression of IL-10/β-endorphin and produces antinociception through α7 nAChR activation.…”

“…However, this conclusion is compromised by lacking elucidated interactions of cynandione A with α7 nAChRs at the molecular level. Particularly, our previous studies also revealed that the spinal microglial IL-10/β-endorphin pathway in bone cancer pain and neuropathic pain mediated the α7 nAChR agonists PHA-543613-, cinobufagin- and lemairamin-induced antinociception, although their chemical structures were different ( Apryani et al, 2020 ; Wang et al, 2020 ). Further studies are needed to assess the activities of cynandione A, as well as PHA-543613, cinobufagin, and lemairamin, on α7 nAChRs by using radioligand analysis, electrophysiology, or calcium imaging FLIPR assays.…”

“…It was suggested that activation of α7 nAChRs blocked neuropathic pain through promoting expression of the anti-inflammatory cytokine IL-10 and inhibiting expression of proinflammatory cytokines ( Arredondo et al, 2006 ; Zhang et al, 2017 ). It was also recently revealed that activation of α7 nAChRs produced antinociception in rat models of neuropathic pain and bone cancer pain via spinal microglial expression of IL-10 and β-endorphin ( Apryani et al, 2020 ; Wang et al, 2020 ).…”

Cynandione A alleviates neuropathic pain through spinal microglial interleukin-10/β-endorphin expression following α7 nicotinic acetylcholine receptor activation

“…It has been extensively reported that 7 nAChRs are colocalized on microglial cells as determined by qRT-PCR, western blot, immuno uorescent, and immunohistochemistry analyses and their activation is associated with antinociception and neuroprotection [29,[53][54][55]. More speci cally, it was recently identi ed that the spinal microglial IL-10/β-endorphin pathway mediated the α7 nAChR agonists PHA-543613-, cinobufaginand lemairamin-induced antinociception in bone cancer pain and neuropathic pain [31,32]. Our current study demonstrated that methyllycaconitine signi cantly blocked cynandione A-induced mechanical antiallodynia in neuroapthic rats, and the expression of IL-10 and β-endorphin in the spinal cords and cultured primary microglial cells.…”

“…It was suggested that activation of α7 nAChRs blocked neuropathic pain through promoting expression of the antiin ammatory cytokine IL-10 and inhibiting the expression of proin ammatory cytokines such as IL-1β, IL-6, and TNF-α [29,30]. It was also recently revealed that activation of α7 nAChRs produced antinociception in rat models of neuropathic pain and bone cancer pain via spinal microglial pathway of IL-10 and β-endorphin [31,32].…”