Protectin D1 ameliorates non-compressive lumbar disc herniation through SIRT1-mediated CGRP signaling.

Background. Neuro-inflammatory response promotes the initiation and sustenance of lumbar disc herniation (LDH). Protectin D1 (PD1), as a new type of specialized pro-resolving mediator (SPM), can improve the prognosis of various inflammatory diseases. Recent studies have shown that over representation of calcitonin gene-related peptides (CGRP) may activate nociceptive signaling following nerve injury. Silent information regulator 1 (SIRT1) is ubiquitously expressed in the dorsal horn of the spinal cord and plays a role in the pathogenesis of LDH. In this study, we investigated the analgesic effects of PD1 and elucidated the impact of neurogenic inflammation in the pathogenesis of neuropathic pain induced by non-compressive lumbar disc herniation (NCLDH) in a rat model. Methods. NCLDH models were established by applying protruding autologous nucleus pulposus to the L5 Dorsal root ganglion (DRG). PD1, SIRT1 antagonist or agonist, CGRP or antagonist were administered as daily intrathecal injections for three consecutive days postoperatively. Behavioral tests were conducted to assess mechanical and thermal hyperalgesia. The ipsilateral lumbar (L4-6) segment of the spinal dorsal horn was isolated for further analysis. Alterations in the release of SIRT1 and CGRP were explored using western blot and immunofluorescence. Results. Application of protruded nucleus (NP) materials to the DRG induced mechanical and thermal allodynia symptoms, and deregulated the expression of pro-inflammatory and anti-inflammatory cytokines in rats. Intrathecal delivery of PD1 significantly reversed the NCLDH-induced imbalance in neuro-inflammatory response and alleviated the symptoms of mechanical and thermal hyperalgesia. In addition, NP application to the DGRs resulted the spinal upregulation of CGRP and SIRT1 expression, which was almost restored by intrathecal injection of PD1 in a dose-dependent manner. SIRT1 antagonist or agonist and CGRP or antagonist treatment further confirmed the result. Conclusion. Our findings indicate PD1 has a potent analgesic effect, and can modulate neuro-inflammation by regulating SIRT1-mediated CGRP signaling in NCLDH.

Aspirin-triggered Resolvin D1 ameliorates activation of the NLRP3 inflammasome via induction of autophagy in a rat model of neuropathic pain.

Background: Several studies performed thus far indicate that neuroinflammation may be one of the mechanisms underlying the pathogenesis of neuropathic pain (NP). Autophagy, as an adaptive response, has been regarded as an active process of removing the inflammatory stimulus and restoring homeostatic balance. Resolution of inflammation is a biochemical process mediated by the so-called aspirin-triggered specialized proresolving lipid mediators (AT-SPMs), which are thought to exert protective effects in NP. Recent studies have proposed mechanisms in models of inflammatory disorders and showed a relationship between resolution of inflammation and autophagy. This study aimed to validate the functional effects of Aspirin-triggered Resolvin D1 (AT-RvD1) on in vitro and in vivo models of inflammation and to determine their roles in the regulation of autophagy and activation of the Nod-like receptor protein 3 (NLRP3) inflammasome signaling pathway. Methods: An NP model was established using L5-6 spinal nerve ligation (SNL) and a model of tumor necrosis factor alpha (TNF-α)-stimulated primary microglia was established to evaluate the effect of SPMs. Western blotting was used to detect the level of NLRP3 inflammasomes complexes proteins (NLRP3, ASC, and Caspase-1) and autophagy-related proteins (LC3B, and Beclin1). Immunofluorescence staining was used to understand the autophagy and NLRP3 inflammasome activation process. The behavioral changes in rats were analyzed using paw withdrawal thresholds (PWT) and paw withdrawal latency (PWL) test. Results: Our results showed that AT-SPMs significantly upregulated the activation of autophagy, which was characterized by an increase in the ratio of LC3B-II/I and accumulation of ATG5 and Beclin1. AT-RvD1 showed a dose-dependent decrease in the upregulated PWT and PWL induced by SNL and suppressed the expression of the NLRP3 inflammasome protein and the production of its corresponding downstream proinflammatory factors. Additionally, AT-RvD1 induced the activation of autophagy of the microglia and decreased the expression of the NLRP3 inflammasome protein and the accumulation of proinflammatory factors in TNF-ɑ-challenged microglia. Conclusion: Thus, these results showed that AT-RvD1 may be a potential alternative therapeutic strategy for the prevention or treatment of NP by inhibition of the NLRP3 inflammasome signaling pathway by targeting the induction of autophagy.

Resolvin D1 Alleviates Mechanical Allodynia via ALX/FPR2 Receptor Targeted Nod-like Receptor Protein 3/Extracellular Signal-Related Kinase Signaling in a Neuropathic Pain Model.

The current study aimed to investigate the role and underlying mechanism of Resolvin D1 (RvD1) alleviating spinal nerve ligation (SNL)-induced neuropathic pain (NP) and its interplay with regulatory cascades of Nod-like Receptor Protein 3 (NLRP3) inflammasome. Sprague-Dawley male rat models of SNL-stimulated NP were established, which were pre-treated with different doses of RvD1, WRW4 (ALX/FPR2 inhibitor) or U0126 (ERK inhibitor) for three successive days following the operation. Pain behavior was assessed by measuring changes in the mechanical sensitivity of the hind paws during an observation period of seven consecutive days. The spinal cord (SC) and dorsal root ganglions (DRGs) tissues were collected on postoperative day 7. Immunohistochemistry (IHC) and Western blot were performed to determine the expression levels of NLRP3 inflammasome complex, ALX/FPR2 receptor and extracellular signal-related kinase (ERK). The pro-inflammatory mediators (IL-1ß and IL-18) were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that RvD1 could alleviate mechanical allodynia significantly in the SNL-induced NP rat models. Also, RvD1 inhibited the expression of p-ERK, the NLRP3 inflammasomes complex and its corresponding downstream pro-inflammatory mediators which were significantly enhanced in the SC and DRGs of the rat SNL models. While these changes were partially reversed by pre-administration of WRW4 and further strengthened by co-treated with U0126. Our results suggest that RvD1 dependent on ALX/FPR2 may have an analgesic and anti-inflammatory influence on SNL-induced NP driven by inhibiting NLRP3 inflammasome via ERK signaling pathway. These data also provide strong support for the recent modulation of neuro-inflammatory priming and highlight the potential for specialized pro-resolving mediators (SPMs) as novel therapeutic avenues for NP.

The Role of NF-κB/NLRP3 Inflammasome Signaling Pathway in Attenuating Pyroptosis by Melatonin Upon Spinal Nerve Ligation Models.

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

The anti-inflammatory and analgesic effects of intraperitoneal melatonin after spinal nerve ligation are mediated by inhibition of the NF-κB/NLRP3 inflammasome signaling pathway.

OBJECTIVE: To explore the potential analgesic effect of melatonin and its underlying molecular mechanisms in a neuropathic pain model induced by spinal nerve ligation (SNL). METHODS: The experimental animals were divided into different groups including sham, vehicle, melatonin (MT) treatment, caspase-1 inhibitor (VX-765) treatment and MT2 antagonist (4P-PDOT) treatment. On the first three successive postoperative days, rats were intraperitoneally administered with MT, VX-765 or combination of MT and 4P-PDOT. Hyperalgesic behavior after SNL was evaluated using the paw withdrawal threshold (PWT). We then assessed expression of tumor necrosis factor-α (TNF-α), IL-18, interleukin-1ß (IL-1ß), NLRP3 inflammasome components, and nuclear factor-κB (NF-κB) activation using enzyme-linked immunosorbent assay kits (ELISA), real-time PCR, immunohistochemistry, and western blot, respectively, in spinal cord horn tissues extracted on postoperative day 7. RESULTS: The results showed that melatonin treatment alleviated SNL-induced allodynia. We observed an SNL-induced upregulation of TNF-α, IL-18, IL-1ß, NLRP3, ASC, cleaved caspase-1, and NF-κB in the lumbar spinal cord horn of rats, which was significantly attenuated by intraperitoneal injection of melatonin or VX-765. Additionally, co-treatment of melatonin and 4P-PDOT abrogated the analgesic and anti-inflammatory effect of melatonin. CONCLUSION: Melatonin had potent analgesic and anti-inflammatory effects in SNL-induced neuropathic pain via NF-κB/NLRP3 inflammasome signaling pathway. Our results therefore suggested that this pathway could represent a novel therapeutic target for the management of neuropathic pain.