Melatonin Attenuates Pyroptosis Upon Spinal Nerve Ligation in Rats via the NF-κB/NLRP3 Inammasome Signaling Pathway

Accumulated evidences have demonstrated causative links between neuropathic pain (NP) and immune-mediated inammatory disorders. However, the role of inammasome-induced pyroptosis in NP remains elusive. Melatonin possesses a well-documented analgesic action in various pain models. A rat model of spinal nerve ligation was established to explore the potential mechanism of melatonin in pyroptosis. The current study aimed to test our hypothesis that melatonin regulated pyroptosis to alleviate NP by inhibiting NF-κB/NLRP3-dependent signaling. Behavioral experiments revealed that SNL provoked severe allodynia which were suppressed by the administration of melatonin, caspase-1 inhibitor (VX-765) or NF-κB inhibitor (BAY 11-7085). SNL signicantly up-regulated the inammatory cytokines associated with the excessive activation of NLRP3 components and NF-κB signaling, as well as the marked pyroptosis activation which were partially inhibited by melatonin, VX-765 or BAY 11-7085. Collectively, Melatonin has potent analgesic and anti-inammatory effects in SNL models through preventing pyroptosis via the NF-κB/NLRP3 inammasome signaling pathway.


Introduction
Neuropathic pain (NP), characterized by di cult treatment decisions and poor outcomes, has become a public health epidemic all over the world, with its long-term, chronic discomfort and pain seriously affecting patients' quality of life (Gierthmühlen & Baron, 2016). To date, the accumulated evidence has demonstrated the critical role of neuroin ammation in the pathogenesis of NP, which leads to enhanced pain sensitivity and is characterized by hyperalgesia and allodynia (Jiang et al., 2016;Huang et al., 2018). In view of the complexity of neuropathic symptoms and the resistance to treatment of NP, these published studies provide crucial insights for this eld.
The nucleotide-binding oligomerization domain-like receptors family pyrin domain containing 3 (NLRP3) in ammasome is a multimeric protein complex, which includes the adaptor apoptosis-associated specklike protein containing a CARD (ASC) and the effector pro-caspase-1 (Elliott & Sutterwala, 2015). When cells are exposed to danger signals, the NLRP3 in ammasome drives caspase-1 activation, which leads to the upregulation of mature IL-1β and an extended immune response Dong et al., 2019). Although several studies Pan et al., 2018) have demonstrated that NLRP3 participates in the on-set and continuation of NP, the molecular biological frame mechanisms regarding upstream and downstream effectors of NP remain to be elucidated.
Pyroptosis is emerging as a unique in ammatory-regulated cell death mechanism that is dependent on caspase-1 activity and has been reported to be involved in various chronic in ammatory disease (Zhang et al., 2018;McKenzie et al., 2018). Gasdermin D (GSDMD) triggers pyroptosome formation and caspase-1-mediated pyroptosis, forming membrane pores to induce the release of proin ammatory cytokines (Sborgi et al., 2016;Xu et al., 2018). However, the underlying mechanisms by which caspase-1-mediated pyroptosis participates in spinal nerve ligation (SNL)-induced NP remain unclear.
Nuclear factor-kappa B (NF-κB) is a transcription factor that takes a pivotal part in the onset of in ammation (Mitchell & Carmody, 2018). The NLRP3 in ammasome increases the production of in ammatory mediators by activating NF-κB signaling during chronic in ammation (Fann et al., 2018).
Recent studies have reported that GSDMD-related pyroptosis mediated by TLR4/NF-κB signaling may be associated with the onset and progression of chronic in ammatory diseases . Therefore, in the present study, we hypothesized that cell pyroptosis is induced in an NLRP3-dependent manner during the progression of SNL which is mediated by the NF-κB signaling pathway.
Melatonin (N-acetyl-5-methoxytryptamine), an endogenous and synthetic hormone, plays a protective role in various in ammatory disorders owing to its powerful anti-in ammatory properties (Hardeland, 2019;Ping et al., 2017). It has been demonstrated that melatonin alleviated in ammasome-triggered pyroptosis through the GSDMD-dependent NF-κB signaling pathway in adipose cells (Liu et al., 2017). Moreover, Galley et al. have shown that oral administration of melatonin alleviates pain behaviors in a rat model of paclitaxel-induced peripheral neuropathy (Galley et al., 2017). These studies implied that melatonin might play a prominent role in the development of NP by regulating cell pyroptosis, which is mediated by the NF-κB signaling pathway. Thus, this is the rst time that such a concept has been experimentally proposed.
In this study, we explored the effects of melatonin on cell pyroptosis in a rat model of SNL-induced NP.
We also aimed to get insight into how the potential NF-κB/NLRP3 in ammasome signaling mechanisms are affected upon treatment of NP with melatonin.

Experimental Animals and Experimental Groups
Sprague-Dawley (SD) rats (male, weighing 200-250g) used in this study were supplied by Shandong University Experimental Animal Center (Shandong, China). The rats were reared in groups of about 3-5 rats. Each group was subjected to a 12-hour light/dark cycle and bred with standard rodent chow and water. The ambient temperature was controlled at (25±1°C) and humidity at (45±5%), respectively. The entire animal trial was approved by the Qingdao University Animal Care and Use Committee.

Spinal nerve ligation Model
The well-established L 5-6 SNL rat model of neuropathy was used in this study. The procedure was performed as follows: the experimental animals were placed on a sterile operating table in a prone position and anesthetized by sodium pentobarbital (50 mg/kg, intraperitoneally). The skin of the back was incised longitudinally and the left paraspinal muscles were segregated from spinous processes. The left L 5-6 inter-laminar space was exposed, and the spinal nerves were tightly ligated with 6-0 silk sutures after carefully separating them from the surrounding tissues. The sham group was subjected to all surgical procedures, except the nerve ligation. All rats were placed post-surgery in a warming chamber with individual cages and body temperatures were maintained at approximately 38℃ until they were completely awake. Then, on the 7th postoperative day rats from different groups were sacri ced and the spinal cord tissues from the ipsilateral lumbar enlargement were dissected and cryo-preserved at -80℃. All surgical procedures were performed aseptically and carried out in the morning to avoid diurnal variations on the immune response.

Assessment of mechanical hyperalgesia
All rats were assessed for mechanical hyperalgesia one day before surgery and on 7 consecutive days post-surgery. Before the assessment, all experimental rats were individually kept for an hour in a quiet room to acclimatize to the environment. We used the 50% paw withdrawal latency (PWL) to assess mechanical sensitivity as described previously (Chaplan et al., 1994). Von Frey laments (Stoelting, USA) were utilized to stimulate the fourth or fth metatarsal surface of the right posterior hind paw following the "up-down" calculating approach. The stimulus was applied for 6-8 seconds and this was repeated ve times in 10-15-min intervals to avoid sensitization. A positive response included the rapid withdrawal or lapping of the paw and the lowest stimulus that evoked a clear positive reaction was accepted as the threshold. Testing was performed by a single experienced investigator, who was blinded to treatment the rats received, at approximately the same time of the day.
Protein levels of in ammatory factors in the ipsilateral spinal cord horns were assessed by ELISA. The spinal cord samples were homogenized in 150 μl of RIPA lysis buffer and centrifuged at 20,000 r/min for 30 min at 4℃. Interleukin (IL)-1β and IL-18 secretion in each group was determined from the supernatant using ELISA kits (RD Systems, USA; ExCell, China) following the manufacturer's instructions. Absorbance measurements were read at 450 nm wavelength using a microtiter plate reader. We used a standard calibration curve from standards provided by the manufacturer to calculate the antigenic values for the cytokines.

Analysis of cell death
Pyroptosis was assessed using propidium iodide (PI) uorescent staining. The spinal cord tissues were collected and cryo-preserved at -80℃ on the 7 th -day post-SNL. The PI dye (1 mg/kg, 100ul) was perfused intraperitoneally one hour before the rats were killed. The frozen spinal cord tissues (7μm) were cut into sections on a freezing microtome. Sections were blocked with 5% fetal bovine serum for 1h in PBS containing 0.1% Triton X-100 (Sigma) in the dark at room temperature. Subsequently, the sections were incubated in the presence of the primary antibody (anti-caspase-1, 1:100) at 4℃ overnight. Real-time quantitative polymerase chain reaction (RT-qPCR) Total mRNA was extracted from spinal cord tissues with TRIzol Reagent (Life Technologies, USA). We used a Prime Script RT reagent kit (Takara, Dalian, China) to performed PCR and the SYBR-Green Premix Ex Taq (TaKaRa, Dalian, China) on a Light Cycler® 480 Instrument II (Roche, Switzerland) to detect the relative mRNA levels. The expression of the target gene sequences was normalized toβ-actin. The data were quanti ed using the 2 -ΔΔ method. Table 1 shows the sequences of gene-speci c primers used in this study.

Statistical analysis
All data were presented as mean ± standard deviation (SD). SPSS statistical software system (version 20.0 for Windows, Chicago, IL) was used for data analysis. Comparisons of the levels of pyroptosis, NF-κB signaling, expression levels of the NLRP3 in ammasome components, and in ammatory cytokines in response to melatonin, VX-76, and BAY treatments were carried out using analysis of variance (ANOVA). P< 0.05 was set as the threshold for statistical signi cance.

Melatonin-promoted pain relief and reduced in ammatory response
The effect of SNL on the mechanical hypersensitive thresholds was examined. The withdrawal thresholds had signi cantly decreased compared with the pre-surgery baseline and the sham group ( Fig.  1 A). We then assessed the mechanical withdrawal thresholds after melatonin administration. Compared with the vehicle group, melatonin injection signi cantly elevated the mechanical withdrawal threshold in the SNL treatment group (Fig. 1 A). However, there was no difference between the sham groups with or without melatonin (Fig. 1 A).
Then, to investigate whether the decreased withdrawal threshold following SNL was due to in ammation and to assess the effect of melatonin, we determined the expression levels of IL-18 and IL-1β by ELISA.
As shown in Fig. 1B and C, the production of both pro-in ammatory factors was signi cantly elevated in the spinal cord following SNL compared to the sham group. Meanwhile, melatonin administration was effective in suppressing the increased production of IL-18 and IL-1βcompared with the vehicle group ( Fig.  1B and C). Moreover, the levels of pro-in ammatory cytokines in the sham group remained unchanged following melatonin administration.

Melatonin attenuates SNL-induced NLRP3 in ammasome activation
Since an important function of the NLRP3 in ammasome activation is to promote the maturation of proin ammatory cytokines, we tested whether melatonin prevented the activation of NLRP3 in ammasomerelated molecules in SNL-induced NP models. Analysis by RT-PCR showed that the mRNA expression levels of NLRP3, ASC, and caspase-1 P10 were signi cantly enhanced in the vehicle group compared with the sham group but were inhibited by melatonin administration as shown in Fig. 2A. Meanwhile, similar results were uncovered using a Western blot assay demonstrating that melatonin administration alleviated the up-regulation of caspase-1 P10, NLRP3, and ASC induced by SNL (Fig. 2B). However, we did not observe any statistically signi cant difference between the sham groups with or without melatonin. Moreover, the expression of caspase-1 P45 was unchanged (Fig. 2B).

Melatonin mitigates SNL-induced GSDMD activation and pyroptosis
As shown in Fig. 3A, Western blotting analysis demonstrated that the protein expression of GSDMD was markedly increased upon SNL but was alleviated after melatonin treatment. However, there was no statistically signi cant difference between the sham groups with or without melatonin treatment.
To further con rm the presence of pyroptosis, we performed immunohistochemistry. The presence of cleaved-caspase-1 and PI-positive cells indicated pyroptosis. Our immunohistochemistry results showed that SNL induced a high cell death rate along with the elevation of cleaved caspase-1, which was reversed upon melatonin treatment ( Fig 3B). Therefore, our results demonstrated that melatonin could regulate the GSDMD-mediated pyroptosis.

Caspase-1 inhibitor blunted spinal cord pyroptosis and alleviated SNL-induced mechanical hyperalgesia and in ammatory response
We next investigated whether injection of the speci c caspase-1 inhibitor VX-765 could also mitigate the key in ammasome and pyroptosis activation in an SNL-treated rat model. Our Western blotting results suggested that, similar to melatonin, VX-765 effectively blocked the induction of high levels of expression of caspase-1 P10, NLRP3, and ASC, and of GSDMD upon SNL treatment (Fig 4A).
We further examined the role of VX-765 in regulating in ammasome-triggered pyroptosis using immunohistochemistry staining. The results we obtained were consistent with those of the Western blotting assay. Administration of VX-765 led to down-regulation of the SNL-induced positive expression of caspase-1 and pyroptosis (Fig 4B). In addition, we found that melatonin displayed a more potent inhibition relative to VX-765.
We further tested the in ammatory reaction upon intraperitoneal injection of VX-765 using ELISA. We found that the expression of the pro-in ammatory cytokines increased markedly following SNL, while administration of either VX-765 or melatonin inhibited the SNL-induced secretion of IL-18 and IL-1β.
Next, we investigated the effect of caspase-1 activation on mechanical pain allodynia and whether VX-765 had analgesic effects similar to melatonin on mechanical hypersensitivity. Consistent with the effects of injection of melatonin, administration of VX-765 reversed the lowering of the mechanical withdrawal threshold that was seen upon SNL-induced in ammation.
Taken together, suppressing the NLRP3-mediated pyroptosis could promote in ammatory reaction resolution and control pain. The addition of melatonin improved radicular pain by inhibiting the activation of pyroptosis.
Melatonin reduces SNL-induced mechanical algesthesia mediated by the NF-κB/NLRP3 signal pathway Taking into consideration the critical role of the NF-κB pathway in NP, we used Western blotting to explore whether NF-κB signaling is over-activated in our SNL-treatment rat model of NP. As seen in Fig. 6A, the phosphorylation of NF-κB was markedly elevated in the spinal cord of rats upon SNL compared with sham-treated rats, whereas melatonin administration suppressed the increase of NF-κB phosphorylation upon SNL.
Similar to the administration of melatonin, we found that BAY treatment dramatically inhibited the components of the NLRP3 in ammasome and its downstream effector GSDMD in the spinal cord of the rat SNL model compared with the control group (Fig. 6B). Similarly, the mRNA levels of caspase-1 P10, NLRP3, ASC, and GSDMD were also markedly elevated upon SNL but were signi cantly reduced following BAY injection compared to the vehicle group (Fig. 6B). However, we did not nd any statistically signi cant difference between the melatonin treatment group and the melatonin and BAY co-treatment group.
Finally, we assessed the effect of treatment with BAY on SNL-induced mechanical hyperalgesia. In the BAY or melatonin-administrated group, mechanical hyperalgesia was signi cantly recovered compared with the SNL-treated control group. Furthermore, co-treatment of BAY and melatonin marked alleviated the reduction of the mechanical withdrawal thresholds (Fig. 6C).

Discussion
Throughout the current study, we demonstrated that melatonin has potent anti-in ammatory and analgesic properties in a rat NP model. Additionally, we identi ed the critical contribution of the NLPR3 in ammasome to the development of SNL-induced NP, which was associated with the production of the pro-in ammatory cytokines. These results explained our previous ndings that the in ammatory response triggered by the NLPR3 in ammasome critically participated in NP (Wang et al., 2020). Furthermore, we found that the close connection between NLRP3 in ammasome-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 in ammatory disease, and there are no available therapeutic strategies to completely control the chronic pain. Although the in ammatory nature of NP has already been well studied, the molecular signaling mechanisms behind the in ammatory response leading to NP are still incompletely clari ed. In keeping with previous studies, we have successfully established a rat SNL pain model which produced persistent pain hypersensitivity . Previous research has shown that NP is immunologically active and involved in the activation of various in ammasomes during the process of chronic in ammation (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 in ammasome is one of the indispensable multimeric protein complexes that regulate the autoimmune system, which then leads to the secretion of pro-in ammatory molecules, for example IL-1β ). In the current study, we found that intraperitoneal injection of melatonin prevented the upregulation of the NLRP3 in ammasome and the subsequent activation of pro-in ammatory 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 in ammatory 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 in ammasome activation engages a kind of in ammatory cell death, named pyroptosis, in immune cells (He et al., 2016), typically characterized by the release of pro-in ammatory molecules . Gasdermin D (GSDMD), a reliable marker of pyroptosis, is involved in various in ammatory 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 in ammatory response by suppressing the expression of pyroptosis. Taken together, these results imply that pyroptosis, an in ammatory form of regulated cell death, could present a target for melatonin-mediated inhibition of neuronal in ammation.
Caspase-1, executing the central role in the NLRP3 pathway, is responsible for the active process of in ammatory factors, which are pivotal mediators of innate immunity and critically take part in various in ammatory diseases (Van Opdenbosch & Lamkan , 2019). Therefore, we next performed an inhibitory experiment with a speci c 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 in ammasome-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 in ammasome. 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 in ammasome-activated pyroptosis is a key target for the alleviation of NP using melatonin.
Although it is well known that in ammatory caspase-1 is associated with pyroptosis, the mechanism of the NLRP3 in ammasome-induced pyroptosis in NP is largely unclear. TheNF-κB transcription factor is involved in the cellular signaling pathway in in ammation-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 in ammation (Yao & Sun, 2019). Therefore, we examined the role of the NF-κB signaling pathway in the in ammatory 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 speci c NF-κB signaling pathway inhibitor, has been shown to possess anti-in ammatory and neuroprotective activities. Interestingly, a key nding 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 in ammasome-mediated pyroptosis cascade in the SNLinduced NP model. Thus, these data revealed the possibility that melatonin alleviated NP, in part, due to the suppression of in ammasome-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 in ammatory reaction. Our results showed that the NF-κB pathway and NLRP3 in ammasome-mediated pyroptosis played key roles in the onset and perpetuation of SNL-induced NP. Furthermore, melatonin and an NF-κB inhibitor attenuated the NLRP3 in ammasomeand 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 in ammasome-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 speci c pathogenesis processes.

Declarations
Ethics approval and consent to participate: The animal experiments were approved by the Qingdao University Animal Care and Use Committee.

Consent for publication:
Not applicable.
Availability of data and material: The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Competing interests:
All the authors declare no nancial or commercial con icts of interest. Funding: The study received no funds.

Authors' contributions:
Drs. Yi-hao Wang and Xiao Gao were responsible for carrying out the major part of the study and writing the manuscript. Dr. Yu-ru Tang carried out the statistical analyses and helped conduct the animal models and western blot study. Dr. Zhao-jun Liang helped carry out the behavioral tests. Drs. Nan-nan Zhang and Juan Liu helped carry out the ELISA study. Dr. Yan Li conceived and designed the study, and revised the manuscript.