Since there is little evidence that the effect of ICTF on chronic inflammatory pain and its underlying mechanisms. For this purpose, our experiment was designed to investigate the potential inhibitory effects of ICTF on CFA-induced inflammatory pain in rats, we then highlighted whether involvement of spinal α7nAChR, HMGB1/NF-κB signaling, and inflammatory cytokines in ICTF alleviating pain-related behaviors in CFA rats. In this study, we found that ICTF exerted anti-nociceptive and anti-inflammatory effects on CFA-induced pain hypersensitivity through inhibiting HMGB1/NF-κB signaling pathway following activation of α7nAChR-dependent in the spinal cord of rats. Further molecular docking assay demonstrated that ICTF targeted α7nAChR for suppressing CFA-induced inflammatory pain. Findings from this experiment might provide new insights into the mechanisms underlying ICTF being acted as a potential therapeutic candidate for CFA-induced inflammatory pain.
It is generally accepted that chronic inflammatory pain is an increasingly severe global public health problem and greatly impacts patient’s life quality(1). Currently available and conventional analgesics with unsatisfactory efficacy for inflammatory pain relief including non-steroidal anti-inflammatory drugs (NSAID) and opioids(20, 37). It is thus critical to uncover and develop novel high-efficacy and low-toxicity interventions against chronic inflammatory pain. More importantly, traditional Chinese medicine is proved to be effective candidates for chronic pain relief(38). We previously confirmed that ICTF exerts a neuroprotective role by suppressing excitotoxicity and cerebral ischemia-reperfusion injury(24). It is known that neuroinflammation is a crucial and inevitable pathogenesis process in mediating inflammatory pain development(6). Thereby, in this study, provoking us to apply a rat model of CFA-induced inflammatory pain to define whether ICTF has an anti-nociceptive effect in CFA rats. Our results showed that both PWT and PWL were significantly reduced on day 1 after injected CFA, indicating that CFA-induced inflammatory pain rat model was successfully established. While ICTF could dose-dependently inhibit CFA-induced mechanical allodynia and thermal hyperalgesia. Further, we explored the time-course effect of ICTF (3.0 mg/kg) in CFA-induced inflammatory pain rats. It is found that ICTF treatment of CFA rats effectively enhanced both PWT and PWL from day 14 and retained until day 21 post-CFA injection. These findings suggested that ICTF exhibited an anti-nociceptive effect on CFA-induced inflammatory pain.
Nowadays, CatWalk gait analysis that obtained an objective and quantitative assessment of gait parameters has been utilized to determine the pain hypersensitivity in neuropathic pain(39). Thus, we hypothesized that CatWalk gait analysis may provide an interesting complementary option to evaluate the alterations of motor function related to CFA-induced pain hypersensitivity. This study was designed to apply CatWalk gait analysis to elucidate the possible effects of ICTF on gait parameters in CFA rats. We currently observed that CFA obviously reduced the gait parameters involving stand time, swing speed, and print area but increased swing time on day 21 post-CFA injection, suggesting that CFA elicited painful sensory and motor function disorder in rats. While ICTF could reverse CFA-induced alterations of gait parameters. In addition, 2D images further exhibited that after ICTF treatment of CFA rats, the stand time was longer but swing time was shorter, as well as paw print area was markedly enhanced. The results from CatWalk gait analysis strongly supported that ICTF indeed had an inhibitory effect of pain-related behaviors induced by CFA in rats.
It is reported that sensitization of peripheral nociceptors by pro-inflammatory mediators is a major pathogenesis of inflammatory pain(40). During inflammation, pro-inflammatory mediators are always released into the damage tissues, resulting in redness and swelling of inflamed areas(41). Injection of CFA into rodent paw plantar is often utilized to establish a chronic inflammatory pain model owing to eliciting inflammatory responses, such as swelling, inflammatory infiltrate formation, and pro-inflammatory mediators release(42), implying that reduction of pro-inflammatory mediators is an effective therapeutic approach for alleviating inflammatory pain. Thereby, our research explored whether ICTF has anti-inflammatory effect on CFA-induced paw edema volume and paw tissue inflammation in rats. CFA-induced paw edema volume markedly enhanced starting from day 1 and retained until day 21 post-CFA injection, while ICTF could reduce the paw edema volume of CFA rats beginning from day 14 post-CFA injection. Further, the photographs exhibited that CFA provoked obvious swelling on the plantar surface of the injected paw starting from day 1 and paw swelling became more severe from day 7, 14, and 21 post-CFA injection, whereas the paw swelling of CFA rats gradually reduced from day 14 following ICTF treatment. Additionally, HE staining showed that CFA produced massive accumulation of inflammatory cells in CFA rats, while the inflammatory cells were markedly reduced by ICTF treatment on day 21 post-CFA injection. This is consistent with other results(43). Altogether, the data supported the notion that ICTF has profound anti-inflammatory and anti-edema effects on CFA-induced inflammation.
Based on the above findings, we postulated that ICTF may be served as a promising therapeutic reagent against CFA-induced inflammatory pain, but the precise mechanism underlying the inhibitory effects of ICTF on CFA rats need further investigation. The nervous system interacting with immune system is crucial for modulating neuroimmune responses and controlling inflammation(44, 45). Within this process, cholinergic anti-inflammatory pathway regulates the inflammatory response through the activation of α7nAChR-dependent(46). Further, α7nAChR was identified to mainly express on pain transmission pathways such as spinal cord, which mediated anti-inflammatory effects by down-regulating pro-inflammatory mediators(47, 48). Intraplantar injection of CFA induces neuroinflammation and inflammatory pain, and exhibits more edema, hyperalgesia and allodynia in the α7nAChR KO mice compared with the wild-type(49). Previously studies have shown that activating spinal α7nAChR exerts anti-inflammatory and analgesic effects through modulation of pro-inflammatory cytokines, and thus alleviates pain hypersensitivity in CFA rats(50). Further, Our recent data have confirmed that 2 Hz EA stimulation can attenuate mechanical hypersensitivity in SNI rats by up-regulating the mRNA and protein expression of spinal α7nAChR, and reducing the pro-inflammatory cytokine IL-1β(33). These results supported that α7nAChR maybe a new target for treating chronic pain in the spinal cord. Thereby, this indeed provides novel option for the utility of activating α7nAChR signal in the chronic pain relief. In our study, the expression of spinal α7nAChR mRNA and protein dramatically decreased on day 21 after given CFA, and immunofluorescence staining also showed that expression of α7nAChR reduced in the spinal dorsal horn of CFA rats, suggesting that spinal α7nAChR participated in the modulation of CFA-induced inflammatory pain. This is consistent with other reports. While following ICTF treatment, the levels of spinal α7nAChR mRNA and protein significantly enhanced on day 21 after CFA injection. These results are consistent with the alleviation of CFA-induced mechanical allodynia and thermal hyperalgesia by ICTF treatment. Further, immunofluorescence results exhibited that TCTF treatment also increased the expression of α7nAChR in the spinal dorsal horn of CFA rats, implying that α7nAChR might mediate the anti-nociception of ICTF in the spinal cord of CFA rats. To further elucidate the effect of spinal α7nAChR on ICTF-mediated anti-nociception and its possible mechanism in CFA rats, intrathecally injected α7nAChR antagonist α-Bgtx was utilized. We found that α-Bgtx successfully reversed the inhibitory effects of ICTF on CFA-induced pain hypersensitivity and the reduction of α7nAChR protein level induced by CFA in the spinal cord of rats, suggesting an suppression of ICTF-provoked anti-nociception to CFA-induced inflammatory pain. Collectively, our research indicates that the spinal α7nAChR is implicated in the attenuating CFA-induced pain hypersensitivity mediated by ICTF treatment in CFA rats.
It is clear that the mechanism underlying inflammatory pain is highly complex, such as neuroinflammation induced by pro-inflammatory mediators(51). As a pro-inflammatory mediator, HMGB1 is highly expressed in the spinal cord and exhibits an important role in the inflammation response(52), suggesting that spinal HMGB1 signal contributes to neuroinflammation and inflammatory pain development. Therefore, whether the anti-inflammatory effect of ICTF through inhibiting HMGB1 signal is an interesting question that remains to be fully defined. In our study, the mRNA and protein expression of spinal HMGB1 was markedly increased in CFA rats, but ICTF treatment of CFA rats obviously reduced spinal HMGB1 mRNA and protein levels, indicating that ICTF exerts anti-inflammatory effect may through inhibiting spinal HMGB1 signal in CFA rats. It is known that α7nAChR-dependent cholinergic signaling is implicated in suppressing the release of HMGB1(16). For example, under inflammatory pain states, the application of agonist triggering α7nAChR signal effectively block the release of HMGB1(53), conversely, administration of α7nAChR inhibitor α-Bgtx can completely reverse this effect(54). Suggesting that α7nAChR-HMGB1 signaling may be involved in modulating chronic inflammatory pain. Further, we determine the effect of α-Bgtx on spinal HMGB1 level by ICTF treatment in CFA rats, after intrathecally injected α-Bgtx to CFA rats, spinal HMGB1 protein expression in CFA rats was significantly enhanced, indicating that α-Bgtx successfully reversed the inhibitory effect of ICTF on spinal HMGB1 in CFA rats. The data supported that ICTF exhibits anti-nociception on CFA-induced inflammatory pain by inhibiting HMGB1 cascade via the activation of α7nAChR.
Evidence showed that blockade of HMGB1/NF-κB signaling could alleviate CFA-induced inflammatory pain(17), implying a promising therapeutic approach for inflammatory pain by targeting HMGB1/NF-κB signaling. Notably, the inflammatory response has been identified as a vital mechanism underlying the pathogenesis of inflammatory pain(55). It is reported that NF-κB, a critical nuclear transcription factor, plays an important role in the modulation of the inflammatory response(56). The activated NF-κB p65 is translocated to the nucleus and promoted the transcription of target genes including pro-inflammatory cytokines(57). It is known that activation of NF-κB signal mediated neuroinflammation by increasing pro-inflammatory cytokines expression. For example, within the inflammatory pain, NF-κB p65 expression was found to be enhanced(30), conversely, inhibition of NF-κB cascade can attenuate inflammatory pain. Suggesting that NF-κB cascade plays an important role in inflammatory pain. Consistent with other reports(17), our present results showed that CFA markedly enhanced mRNA and protein levels of spinal NF-κB p65, while it was restored by ICTF treatment. It is clear that activation of α7nAChR can reduce NF-κB activity and suppress inflammatory response(16). In this study, α-Bgtx effectively reverse the inhibitory effect of ICTF treatment on spinal NF-κB signal in CFA rats, indicating that α7nAChR-mediated suppression of spinal NF-κB activation is implicated in the inhibitory effects of ICTF on CFA-induced inflammatory pain.
It is well known a role of neuroinflammation that closely associated with pro-inflammatory mediators and microglial activation in the pathogenesis of inflammatory pain(7). Spinal microglia is indicated to be a critical resident immune cell and plays an essential role in the inflammatory response(58). The activated microglia triggers pro-inflammatory cytokines release, such as IL-1β, IL-6, and TNF-α(59), conversely, the pro-inflammatory cytokines can also activate microglia(60). Microglia has been acted as a key initiator in inflammatory pain development(61). For instance, the activation of microglia within the spinal cord following inflammatory pain was well recognized to produce pain hypersensitivity(62). Microglial activation leads to an enhancement of pro-inflammatory cytokines to maintain central sensitization and inflammatory pain(63), thereby the blockade of microglial activation maybe a novel management for inflammatory pain. In the spinal cord, we presently applied the microglial marker IBA-1 to assess the activation of microglia in CFA-induced inflammatory pain. A obvious up-regulation of spinal IBA-1 expression was found in CFA rats, indicating that CFA induced the activation of microglia in the spinal cord. Additionally, CFA also produced an enhancement of spinal IL-1β in mRNA and protein expression. These data further supported the notion that microglial activation is the main source of pro-inflammatory cytokines in inflammatory pain(59). While ICTF treatment markedly reduced the enhancement of spinal IBA-1 expression induced by CFA, and decreased the level of spinal IL-1β in CFA rats. Thus, we postulate that the anti-inflammatory effects of ICTF on CFA-induced inflammatory pain may through suppressing both microglial activation and IL-1β in the spinal cord.
As previously reported, the interactions between the nervous and immune systems participate in modulating chronic pain(64). The release of pro-inflammatory mediators and the loss of anti-inflammatory cytokines result in neuroimmune response(65), indicating that an imbalance in the cytokines micro-environment mediates regulating inflammatory pain. It is clear that some well-known pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α are implicated in inflammatory pain(51), conversely, IL-10 is identified as a profound anti-inflammatory cytokine that is greatly expressed on microglia, which exhibits marked anti-nociception in inflammatory pain(66). For example, IL-10 can suppress CFA-induced inflammatory pain by reduction of pro-inflammatory cytokines(66, 67), and treatment of IL-10 to inflammatory pain rats effectively provoked an anti-nociceptive effect in the spinal cord(17). Presently we also noticed that CFA obviously produced an elevation in IL-1β mRNA and protein levels, and together with a significant reduction of IL-10 mRNA and protein expression in the spinal cord, implying that an imbalance between pro-inflammatory cytokines (IL-1β) and anti-inflammatory cytokine (IL-10) in the spinal cord triggered CFA-induced inflammatory pain. We currently found that ICTF treatment markedly reversed the enhancement of IL-1β protein level and the increase of IL-10 protein expression induced by CFA in the spinal cord. Thus, ICTF has anti-inflammatory activity maybe via re-balancing the cytokine micro-environment in the spinal cord of CFA rats. Increasing evidence have reported that α7nAChR inhibits inflammation response by decreasing pro-inflammatory cytokines(68). In the spinal cord, α7nAChR is abundantly expressed on microglia, which is proven to induce and maintain chronic inflammatory pain. More strikingly, we presently discovered that intrathecal injection of α-Bgtx could antagonize the decrease of IL-1β protein level and the elevation of IL-10 protein expression induced by ICTF treatment in the spinal cord of CFA rats. Our current findings further highlighted that ICTF relieves inflammatory pain by reducing IL-1β expression and increasing IL-10 level via activating a7nAChR in the spinal cord of CFA rats.