SIRT1 mediates the excitability of spinal CaMKIIα-positive neurons and participates in neuropathic pain by controlling Nav1.3.

AIMS: Neuropathic pain is a common chronic pain disorder, which is largely attributed to spinal central sensitization. Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) activation in the spinal dorsal horn (SDH) is a major contributor to spinal sensitization. However, the exact way that CaMKIIα-positive (CaMKIIα+) neurons in the SDH induce neuropathic pain is still unclear. This study aimed to explore the role of spinal CaMKIIα+ neurons in neuropathic pain caused by chronic constriction injury (CCI) and investigate the potential epigenetic mechanisms involved in CaMKIIα+ neuron activation. METHODS: CCI-induced neuropathic pain mice model, Sirt1loxP/loxP mice, and chemogenetic virus were used to investigate whether the activation of spinal CaMKIIα+ neurons is involved in neuropathic pain and its involved mechanism. Transcriptome sequence, western blotting, qRT-PCR, and immunofluorescence analysis were performed to assay the expression of related molecules and activation of neurons. Co-immunoprecipitation was used to observe the binding relationship of protein. Chromatin immunoprecipitation (ChIP)-PCR was applied to analyze the acetylation of histone H3 in the Scn3a promoter region. RESULTS: The expression of sodium channel Nav1.3 was increased and the expression of SIRT1 was decreased in the spinal CaMKIIα+ neurons of CCI mice. CaMKIIα neurons became overactive after CCI, and inhibiting their activation relieved CCI-induced pain. Overexpression of SIRT1 reversed the increase of Nav1.3 and alleviated pain, while knockdown of SIRT1 or overexpression of Nav1.3 promoted CaMKIIα+ neuron activation and induced pain. By knocking down spinal SIRT1, the acetylation of histone H3 in the Scn3a (encoding Nav1.3) promoter region was increased, leading to an increased expression of Nav1.3. CONCLUSION: The findings suggest that an aberrant reduction of spinal SIRT1 after nerve injury epigenetically increases Nav1.3, subsequently activating CaMKIIα+ neurons and causing neuropathic pain.

Spinal Sirtuin 3 Contributes to Electroacupuncture Analgesia in Mice With Chronic Constriction Injury-Induced Neuropathic Pain.

BACKGROUND: Electroacupuncture (EA) is a traditional Chinese therapeutic technique that has a beneficial effect on neuropathic pain; however, the specific mechanism remains unclear. In this study, we investigated whether EA inhibits spinal Ca/calmodulin-dependent protein kinase II (CaMKIIα) phosphorylation through Sirtuin 3 (SIRT3) protein, thus relieving neuropathic pain. MATERIALS AND METHODS: We used wild-type and SIRT3 knockout (SIRT3-/-) mice and used chronic constriction injury (CCI) as a pain model. We performed Western blotting, immunostaining, von Frey, and Hargreaves tests to gather biochemical and behavioral data. Downregulation and overexpression and spinal SIRT3 protein were achieved by intraspinal injection of SIRT3 small interfering RNA and intraspinal injection of lentivirus-SIRT3. To test the hypothesis that CaMKIIα signaling was involved in the analgesic effects of EA, we expressed CaMKIIα-specific designer receptors exclusively activated by designer drugs (DREADDs) in the spinal dorsal horn (SDH) of mice. RESULTS: These results showed that the mechanical and thermal hyperalgesia induced by CCI was related to the decreased spinal SIRT3 expression in the SDH of mice. A significant reduction of mechanical and thermal thresholds was found in the SIRT3-/- mice. SIRT3 overexpression or EA treatment alleviated CCI-induced neuropathic pain and prevented the spinal CaMKIIα phosphorylation. Most importantly, EA increased the expression of spinal SIRT3 protein in the SDH. Downregulation of spinal SIRT3 or CaMKIIα Gq-DREADD activation inhibited the regulatory effect of EA on neuropathic pain. CONCLUSION: Our results showed that CaMKIIα phosphorylation was inhibited by spinal SIRT3 in neuropathic pain and that EA attenuated CCI-induced neuropathic pain mainly by upregulating spinal SIRT3 expression in the SDH of mice.

Role of spinal RIP3 in inflammatory pain and electroacupuncture-mediated analgesic effect in mice.

AIMS: Electroacupuncture (EA) is a potentially useful treatment for inflammatory pain. Receptor-interacting protein 3 (RIP3) triggers the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome; activation independent of necroptosis has been reported. However, the role of RIP3 in inflammatory pain and its EA-induced analgesic effects remains unclear. MAIN METHODS: Mice were treated with EA (2 Hz, 2 mA) after complete Freund's adjuvant (CFA) pain models were established. Inhibition or activation of spinal RIP3 was achieved by intrathecal administration of GSK-843 (a specific RIP3 inhibitor) or microinjection of lentivirus-RIP3, respectively. Mechanical analgesiometry and thermal analgesiometry were used to assess paw withdrawal threshold and paw withdrawal latency in mice. Quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to evaluate the expression of RIP3 and NLPR3 in spinal dorsal horn (SDH) of mice. KEY FINDINGS: The expression of spinal RIP3 and NLPR3 increased significantly after CFA injection. Both intrathecal administration of GSK-843 and EA alleviated mechanical and thermal pain behaviors induced by CFA and inhibited the expression of RIP3 and NLRP3 in the SDH of CFA mice. Over-expression of RIP3 induces pain-like symptoms in mice and inhibits the regulatory effects of EA on inflammatory pain. SIGNIFICANCE: Our results indicate that the EA analgesia effect may be related to suppression of RIP3 and NLRP3 expression in the SDH. This study could provide potential insights into the underlying spinal mechanisms involved in the analgesic effect of EA.