Throughout the current study, we demonstrated that melatonin has potent anti-inflammatory and analgesic properties in a rat NP model. Additionally, we identified the critical contribution of the NLPR3 inflammasome to the development of SNL-induced NP, which was associated with the production of the pro-inflammatory cytokines. These results explained our previous findings that the inflammatory response triggered by the NLPR3 inflammasome critically participated in NP (Wang et al., 2020). Furthermore, we found that the close connection between NLRP3 inflammasome-mediated pyroptosis and NF-κB signaling is an underlying mechanism for the analgesic effect of melatonin. Thus, the NF-κB/NLRP3/pyroptosis signaling pathway is an attractive drug for the clinical application of melatonin for the prevention of NP.
NP is a complex chronic inflammatory disease, and there are no available therapeutic strategies to completely control the chronic pain. Although the inflammatory nature of NP has already been well studied, the molecular signaling mechanisms behind the inflammatory response leading to NP are still incompletely clarified. In keeping with previous studies, we have successfully established a rat SNL pain model which produced persistent pain hypersensitivity (Yang et al., 2019). Previous research has shown that NP is immunologically active and involved in the activation of various inflammasomes during the process of chronic inflammation (Tonkin et al., 2018). Consistent with these results, our present study demonstrated that the behavioral changes were correlated with elevated production of IL-18 and IL-βin the spinal cord.
The NLRP3 inflammasome is one of the indispensable multimeric protein complexes that regulate the autoimmune system, which then leads to the secretion of pro-inflammatory molecules, for example IL-1β and IL-18 (Christ et al., 2018). Grace et al. (Grace et al., 2016) reported that the initiation and continuation of NP were mediated by morphine-induced NLRP3 inflammasome activation in the spinal cord and the associated release of IL-1β in microglia. The relationship between inflammatory pain and the NLRP3 inflammasome has been well studied, including during chemotherapy and peripheral nerve injury-induced NP (Tonkin et al., 2018; Jia et al., 2017). Previous reports have demonstrated that melatonin is able to suppress the inflammatory process and improve NP (Tonkin et al., 2018; Lin et al., 2017; Hsieh et al., 2017). In the current study, we found that intraperitoneal injection of melatonin prevented the upregulation of the NLRP3 inflammasome and the subsequent activation of pro-inflammatory cytokines. Intriguingly, in parallel with the downregulation of IL-18 and IL-1βupon administration of melatonin, our behavioral data also showed that melatonin treatment markedly reversed the paw withdrawal threshold in rats with SNL-induced NP. These data demonstrated SNL evokes an immune system response resulting in a persistent inflammatory cascade and sensitization of nociceptive receptors in models of NP, and that melatonin administration may be used as a practical treatment to regulate the immune reaction and ease the pain.
Recent work has shown that NLRP3 inflammasome activation engages a kind of inflammatory cell death, named pyroptosis, in immune cells (He et al., 2016), typically characterized by the release of pro-inflammatory molecules (Shi et al., 2017). Gasdermin D (GSDMD), a reliable marker of pyroptosis, is involved in various inflammatory disease processes (Ding et al., 2016). However, the mechanism underlying NP-induced pyroptosis remained obscure. Our experiments demonstrated that SNL-induced NP increased caspase-1 P10, NLRP3, ASC, and GSDMD expression and resulted in pyroptosis in contrast to the sham group. This increase was suppressed by the administration of melatonin. Therefore, we speculate that melatonin alleviated the neuronal inflammatory response by suppressing the expression of pyroptosis. Taken together, these results imply that pyroptosis, an inflammatory form of regulated cell death, could present a target for melatonin-mediated inhibition of neuronal inflammation.
Caspase-1, executing the central role in the NLRP3 pathway, is responsible for the active process of inflammatory factors, which are pivotal mediators of innate immunity and critically take part in various inflammatory diseases (Van Opdenbosch & Lamkanfi, 2019). Therefore, we next performed an inhibitory experiment with a specific caspase-1 inhibitor (VX-765) to determine whether a similar mechanism applies to caspase-1-dependent pyroptosis activation that occurs during the progression of NLRP3 inflammasome-related NP. Here, we validated that NLRP3, ASC, and caspase-1 synthesis and the activation of pyroptosis were promoted in an SNL-induced NP model and that these effects were neutralized by the caspase-1 inhibitor, suggesting that SNL-induced pyroptosis was dependent on the activation of the NLRP3 inflammasome. Moreover, the mechanical sensitivity threshold had improved following the application of VX-765 similar to the effect of melatonin. Thus, our results showed that the NLRP3 inflammasome-activated pyroptosis is a key target for the alleviation of NP using melatonin.
Although it is well known that inflammatory caspase-1 is associated with pyroptosis, the mechanism of the NLRP3 inflammasome-induced pyroptosis in NP is largely unclear. TheNF-κB transcription factor is involved in the cellular signaling pathway in inflammation-related disease. Several reports have suggested that the NF-κB pathway directly relates to the development of NP. Zhang et al (Zhang et al., 2013) showed that NF-κB activation was ascended in rat lumbar DRG neurons following type 2 diabetes mellitus-induced NP. Zhong et al (Zhong et al., 2020) have shown that bone marrow mesenchymal stem cells down-regulate the NF-κB pathway to alleviate deafferentation pain in rats. Recently, a revolutionary idea has been put forward suggesting that induction of pyroptosis is accompanied by NF-κB activation during inflammation (Yao & Sun, 2019). Therefore, we examined the role of the NF-κB signaling pathway in the inflammatory process in an SNL rat model. We found an increase inNF-κB phosphorylation in the spinal cord in the rat model of SNL-induced NP and a down-regulation of NF-κB expression upon melatonin treatment. BAY 11-7085 (Juliana et al., 2010), as a potent and specific NF-κB signaling pathway inhibitor, has been shown to possess anti-inflammatory and neuroprotective activities. Interestingly, a key finding of the current study demonstrated that the increased biosynthesis of pyroptosis-related proteins, including NLRP3, ASC, caspase-1, and GSDMD were in each case reversed or blunted in the presence of BAY 11-7085 and also reversed pain-related behavior in the rat pain models of SNL, demonstrating that SNL activated the NF-κB signaling pathway to accelerate one or more of the signaling molecules upstream of the NLRP3 inflammasome-mediated pyroptosis cascade in the SNL-induced NP model. Thus, these data revealed the possibility that melatonin alleviated NP, in part, due to the suppression of inflammasome-mediated pyroptosis which may be regulated by the NF-κB signaling pathway.
In conclusion, the purpose of our study was to elucidate the analgesic effect of melatonin in the regulatory molecular signaling involved in the inflammatory reaction. Our results showed that the NF-κB pathway and NLRP3 inflammasome-mediated pyroptosis played key roles in the onset and perpetuation of SNL-induced NP. Furthermore, melatonin and an NF-κB inhibitor attenuated the NLRP3 inflammasome- and pyroptosis activation and promoted pain-relief in NP by inactivating the NF-κB signaling pathway. A mechanism targeting the NF-κB pathway and the NLRP3 inflammasome-mediated pyroptosis might be a prospective therapeutic avenue for the prevention and treatment of NP. However, this being one of the mechanisms towards the generation of NP, further studies are needed to uncover the specific pathogenesis processes.