Electroacupuncture suppresses neuronal ferroptosis to relieve chronic neuropathic pain.

Growing evidence supports the analgesic efficacy of electroacupuncture (EA) in managing chronic neuropathic pain (NP) in both patients and NP models induced by peripheral nerve injury. However, the underlying mechanisms remain incompletely understood. Ferroptosis, a novel form of programmed cell death, has been found to be activated during NP development, while EA has shown potential in promoting neurological recovery following acute cerebral injury by targeting ferroptosis. In this study, to investigate the detailed mechanism underlying EA intervention on NP, male Sprague-Dawley rats with chronic constriction injury (CCI)-induced NP model received EA treatment at acupoints ST36 and GV20 for 14 days. Results demonstrated that EA effectively attenuated CCI-induced pain hypersensitivity and mitigated neuron damage and loss in the spinal cord of NP rats. Moreover, EA reversed the oxidative stress-mediated spinal ferroptosis phenotype by upregulating reduced expression of xCT, glutathione peroxidase 4 (GPX4), ferritin heavy chain (FTH1) and superoxide dismutase (SOD) levels, and downregulating increased expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), malondialdehyde levels and iron overload. Furthermore, EA increased the immunofluorescence co-staining of GPX4 in neurons cells of the spinal cord of CCI rats. Mechanistic analysis unveiled that the inhibition of antioxidant pathway of Nrf2 signalling via its specific inhibitor, ML385, significantly countered EA's protective effect against neuronal ferroptosis in NP rats while marginally diminishing its analgesic effect. These findings suggest that EA treatment at acupoints ST36 and GV20 may protect against NP by inhibiting neuronal ferroptosis in the spinal cord, partially through the activation of Nrf2 signalling.

Chemogenetics Modulation of Electroacupuncture Analgesia in Mice Spared Nerve Injury-Induced Neuropathic Pain through TRPV1 Signaling Pathway.

Neuropathic pain, which is initiated by a malfunction of the somatosensory cortex system, elicits inflammation and simultaneously activates glial cells that initiate neuroinflammation. Electroacupuncture (EA) has been shown to have therapeutic effects for neuropathic pain, although with uncertain mechanisms. We suggest that EA can reliably cure neuropathic disease through anti-inflammation and transient receptor potential V1 (TRPV1) signaling pathways from the peripheral to the central nervous system. To explore this, we used EA to treat the mice spared nerve injury (SNI) model and explore the underlying molecular mechanisms through novel chemogenetics techniques. Both mechanical and thermal pain were found in SNI mice at four weeks (mechanical: 3.23 ± 0.29 g; thermal: 4.9 ± 0.14 s). Mechanical hyperalgesia was partially attenuated by 2 Hz EA (mechanical: 4.05 ± 0.19 g), and thermal hyperalgesia was fully reduced (thermal: 6.22 ± 0.26 s) but not with sham EA (mechanical: 3.13 ± 0.23 g; thermal: 4.58 ± 0.37 s), suggesting EA's specificity. In addition, animals with Trpv1 deletion showed partial mechanical hyperalgesia and no significant induction of thermal hyperalgesia in neuropathic pain mice (mechanical: 4.43 ± 0.26 g; thermal: 6.24 ± 0.09 s). Moreover, we found increased levels of inflammatory factors such as interleukin-1 beta (IL1-ß), IL-3, IL-6, IL-12, IL-17, tumor necrosis factor alpha, and interferon gamma after SNI modeling, which decreased in the EA and Trpv1-/- groups rather than the sham group. Western blot and immunofluorescence analysis showed similar tendencies in the dorsal root ganglion, spinal cord dorsal horn, somatosensory cortex (SSC), and anterior cingulate cortex (ACC). In addition, a novel chemogenetics method was used to precisely inhibit SSC to ACC activity, which showed an analgesic effect through the TRPV1 pathway. In summary, our findings indicate a novel mechanism underlying neuropathic pain as a beneficial target for neuropathic pain.

Electroacupuncture alleviates streptozotocin-induced diabetic neuropathic pain via suppressing phosphorylated CaMKIIα in rats.

Diabetic neuropathic pain (DNP) is a frequent complication of diabetes. Calcium/calmodulin-dependent protein kinase II α (CaMKIIα), a multi-functional serine/threonine kinase subunit, is mainly located in the surface layer of the spinal cord dorsal horn (SCDH) and the primary sensory neurons in dorsal root ganglion (DRG). Numerous studies have indicated electroacupuncture (EA) takes effect in various kinds of pain. In this research, we explored whether CaMKIIα on rats' SCDH and DRG participated in DNP and further explored the mechanisms underlying the analgesic effects of EA. The DNP model in rats was successfully established by intraperitoneal injection of streptozotocin. Certain DNP rats were treated with intrathecal injections of KN93, a CaMKII antagonist, and some of the DNP rats received EA intervention. The general conditions, behaviors, the expressions of CaMKIIα and phosphorylated CaMKIIα (p-CaMKIIα) were evaluated. DNP rats' paw withdrawal threshold was reduced and the expressions of p-CaMKIIα in SCDH and DRG were upregulated compared with the Normal group, while the level of CaMKIIα showed no significance. KN93 attenuated DNP rats' hyperalgesia and reduced the expressions of p-CaMKIIα. We also found EA attenuated the hyperalgesia of DNP rats and reduced the expressions of p-CaMKIIα. The above findings suggest that p-CaMKIIα in SCDH and DRG is involved in DNP. The analgesic effect of EA in DNP might be related to the downregulation of p-CaMKIIα expression level. Our study further supports that EA can be an effective clinical treatment for DNP.

PolyphyllinVI alleviates the spared nerve injury-induced neuropathic pain based on P2X3 receptor-mediated the release of inflammatory mediators.

ETHNOPHARMACOLOGICAL RELEVANCE: PolyphyllinVI (PPⅥ) is the main bioactive component of Chonglou which is a traditional Chinese herbal with various effects, including antitumor, anti-inflammatory, and analgesia. AIM OF THE STUDY: This study aimed to investigate the properties and mechanisms of the analgesia of PPⅥ by using neuropathic pain (NPP) mice. MATERIALS AND METHODS: The potential targets and mechanisms of PPⅥ in alleviating NPP were excavated based on the network pharmacology. Subsequently, the construction of a spared nerve injury (SNI) mice model was used to evaluate the effect of PPⅥ on NPP and the expression of the P2X3 receptor. We identified the signaling pathways of PPⅥ analgesia by RNA sequencing. RESULTS: The results of network pharmacology showed that BCL2, CASP3, JUN, STAT3, and TNF were the key targets of the analgesic effect of PPⅥ. PPⅥ increased the MWT and TWL of SNI mice and decreased the level of P2X3 receptors in the dorsal root ganglion (DRG) and spinal cord (SC). Additionally, PPⅥ reduced the release of pro-inflammatory mediators (TNF-α, IL-1ß, and IL-6) in the DRG, SC, and serum. Based on the KEGG enrichment of differentially expressed genes (DEGs) identified by RNA-Seq, PPVI may relieve NPP by regulating the AMPK/NF-κB signaling pathway. Western blotting results showed that the AMPK signaling pathway was activated, followed by inhibition of the NF-κB signaling pathway. CONCLUSION: PPⅥ increased the MWT and TWL of SNI mice maybe by inhibiting the expression of the P2X3 receptor and the release of inflammatory mediators. The properties of the analgesia of PPⅥ may be based on the AMPK/NF-κB pathway.

Tibetan medicine Ruyi Zhenbao Pill ameliorates neuropathic pain by inhibiting the CXCL10-CXCR3 pathway in spinal cord of spinal nerve ligation model.

ETHNOPHARMACOLOGICAL RELEVANCE: Ruyi Zhenbao Pill (RYZBP) is a traditional Tibetan medicine that has been used for over 300 years in China to treat neurological diseases, specifically neuropathic pain (NP). However, its characteristics and mechanism of action in treating NP remains unclear. AIM OF THE STUDY: Based on animal experiments and transcriptomics to evaluate the characteristics and mechanism of RYZBP in treating NP. METHODS: Mice were divided into six groups using random assignment: sham-operation group, spinal nerve ligation (SNL) group, RYZBP low (0.65 g kg-1), medium (1.30 g kg-1), high (2.60 g kg-1) doses groups, and positive drug pregabalin (PGB, 0.05 g kg-1) group. Mice received intragastrical administered for 14 consecutive days. SNL and intrathecal injection models were employed. The analgesic effects were assessed using the Von Frey test, Acetone test, and Hot Plate test. L5 spinal dorsal horns were collected for transcriptomics on day 15. The potential signaling pathways and Hub genes of RYZBP to ameliorate NP were obtained through transcriptomics and network pharmacology. Molecular docking was utilized to evaluate the binding ability of candidate active ingredients with the Hub genes. Finally, western blot (WB) and immunofluorescence (IF) were used to validate the predicted targets. RESULTS: RYZBP demonstrated a dose-dependent alleviation of mechanical allodynia, cold and heat stimulus-induced pain in SNL mice. Transcriptomics analysis identified 24 differentially expressed genes, and pathway enrichment analysis revealed that the CXCL10-CXCR3 signal axis may be the primary biological pathway through which RYZBP relieve NP. Molecular docking test indicated that the active ingredient in RYZBP exhibit a strong affinity for the target protein CXCL10. WB and IF tests showed that RYZBP can significantly inhibit CXCL10 and CXCR3 and its downstream molecules expression in the spinal dorsal horn of SNL mice. Additionally, intrathecal injection of rmCXCL10 worsened pain hypersensitivity, while RYZBP was able to suppress the pain hypersensitivity response induced by rmCXCL10 and reduce the expression levels of CXCL10 and CXCR3 and its downstream molecules. CONCLUSION: RYZBP had a significant analgesic effect on NP model, and this effect may be related to inhibiting the CXCL10-CXCR3 pathway in the spinal dorsal horn.

Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate neuropathic pain.

(Switching from the microglial M1 phenotype to the M2 phenotype is a promising therapeutic strategy for neuropathic pain (NP). This study aimed to investigate the potential use of stigmasterol for treating NP. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, chronic constriction injury (CCI) group, CCI + ibuprofen group, and CCI + stigmasterol group. We performed behavioral tests, enzyme-linked immunosorbent assay, hematoxylin-esoin staining (H&E) staining and immunohistochemistry, immunofluorescence, and Western blotting. In cell experiments, we performed flow cytometry, immunofluorescence, Western blotting, and qRT-PCR. Stigmasterol reduced thermal and mechanical hyperalgesia and serum IL-1ß and IL-8 levels and increased serum IL-4 and TGF-ß levels in CCI rats. Stigmasterol reduced IL-1ß, COX-2, and TLR4 expression in the right sciatic nerve and IL-1ß expression in the spinal cord. Stigmasterol reduced the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, COX-2, IL-1ß, and CD32 in the spinal cord of CCI rats while increasing the expression of IL-10 and CD206. Stigmasterol decreased M1 polarization markers and increased M2 polarization markers in lipopolysaccharide (LPS)-induced microglia and decreased the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, iNOS, COX-2, and IL-1ß in LPS-treated microglia while increasing the expression of Arg-1 and IL-10. Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate NP.

RTA-408 Regulates p-NF-κB/TSLP/STAT5 Signaling to Ameliorate Nociceptive Hypersensitivity in Chronic Constriction Injury Rats.

Neuropathic pain following nerve injury is a complex condition, which often puts a negative impact on life and remains a sustained problem. To make pain management better is of great significance and unmet need. RTA 408 (Omaveloxone) is a traditional Asian medicine with a valid anti-inflammatory property. Thus, we aim to investigate the therapeutic effect of RTA-408 on mechanical allodynia in chronic constriction injury (CCI) rats as well as the underlying mechanisms. Neuropathic pain was induced by using CCI of the rats' sciatic nerve (SN) and the behavior testing was measured by calibrated forceps testing. Activation of Nrf-2, the phosphorylation of nuclear factor-κB (NF-κB), and the inflammatory response were assessed by western blots. The number of apoptotic neurons and degree of glial cell reaction were examined by immunofluorescence assay. RTA-408 exerts an analgesic effect on CCI rats. RTA-408 reduces neuronal apoptosis and glial cell activation by increasing Nrf-2 expression and decreasing the inflammatory response (TNF-α/ p-NF-κB/ TSLP/ STAT5). These data suggest that RTA-408 is a candidate with potential to reduce nociceptive hypersensitivity after CCI by targeting TSLP/STAT5 signaling.

Metformin inhibits paclitaxel-induced mechanical allodynia by activating opioidergic pathways and reducing cytokines production in the dorsal root ganglia and thalamus.

It has been shown that AMP-activated protein kinase (AMPK) is involved in the nociceptive processing. This observation has prompted us to investigate the effects of the AMPK activator metformin on the paclitaxel-induced mechanical allodynia, a well-established model of neuropathic pain. Mechanical allodynia was induced by four intraperitoneal (i.p) injections of paclitaxel (2 mg/kg.day) in mice. Metformin was administered per os (p.o.). Naltrexoneandglibenclamide were used to investigate mechanisms mediating metformin activity. Concentrations of cytokines in the dorsal root ganglia (DRG) and thalamus were determined. After a single p.o. administration, the two highest doses of metformin (500 and 1000 mg/kg) attenuated the mechanical allodynia. This response was attenuated by all doses of metformin (250, 500 and 1000 mg/kg) when two administrations, 2 h apart, were carried out. Naltrexone (5 and 10 mg/kg, i.p.), but not glibenclamide (20 and 40 mg/kg, p.o.), attenuated metformin activity. Concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and CXCL-1 in the DRG were increased after administration of paclitaxel. Metformin (1000 mg/kg) reduced concentrations of TNF-α, IL-1ß and CXCL-1 in the DRG. Concentration of IL-6, but not TNF-α, in the thalamus was increased after administration of paclitaxel. Metformin (1000 mg/kg) reduced concentration of IL-6 in the thalamus. In summary, metformin exhibits activity in the model of neuropathic pain induced by paclitaxel. This activity may be mediated by activation of opioidergic pathways and reduced production of TNF-α, IL-1ß and CXCL-1 in the DRG and IL-6 in the thalamus.

Traditional Chinese medicine alleviating neuropathic pain targeting purinergic receptor P2 in purinergic signaling: A review.

Past studies have suggested that Chinese herbal may alleviate neuropathic pain, and the mechanism might target the inhibition of purinergic receptor P2. This review discusses whether traditional Chinese medicine target P2 receptors in neuropathic pain and its mechanism in order to provide references for future clinical drug development. The related literatures were searched from Pubmed, Embase, Sinomed, and CNKI databases before June 2023. The search terms included"neuropathic pain", "purinergic receptor P2", "P2", "traditional Chinese medicine", "Chinese herbal medicine", and "herb". We described the traditional Chinese medicine alleviating neuropathic pain via purinergic receptor P2 signaling pathway including P2X2/3 R, P2X3R, P2X4R, P2X7R, P2Y1R. Inhibition of activating glial cells, changing synaptic transmission, increasing painful postsynaptic potential, and activating inflammatory signaling pathways maybe the mechanism. Purine receptor P2 can mediate the occurrence of neuropathic pain. And many of traditional Chinese medicines can target P2 receptors to relieve neuropathic pain, which provides reasonable evidences for the future development of drugs. Also, the safety and efficacy and mechanism need more in-depth experimental research.

Electroacupuncture exerts prolonged analgesic and neuroprotective effects in a persistent dental pain model induced by multiple dental pulp injuries: GABAergic interneurons-astrocytes interaction.

Pain within the trigeminal system, particularly dental pain, is poorly understood. This study aimed to determine whether single or multiple dental pulp injuries induce persistent pain, its association with trigeminal central nociceptive pathways and whether electroacupuncture (EA) provides prolonged analgesic and neuroprotective effects in a persistent dental pain model. Models of single dental pulp injury (SDPI) and multiple dental pulp injuries (MDPI) were used to induce trigeminal neuropathic pain. The signs of dental pain-related behavior were assessed using the mechanical head withdrawal threshold (HWT). Immunofluorescence and western blot protocols were used to monitor astrocyte activation, changes in apoptosis-related proteins, and GABAergic interneuron plasticity. SDPI mice exhibited an initial marked decrease in HWT from days one to 14, followed by progressive recovery from days 21 to 42. From days 49 to 70, the HWT increased and returned to the control values. In contrast, MDPI mice showed a persistent decrease in HWT from days one to 70. MDPI increased glial fibrillary acidic protein (GFAP) and decreased glutamine synthetase (GS) and glutamate transporter-1 (GLT1) expression in the Vi/Vc transition zone of the brainstem on day 70, whereas no changes in astrocytic markers were observed on day 70 after SDPI. Increased expression of cleaved cysteine-aspartic protease-3 (cleaved caspase-3) and Bcl-2-associated X protein (Bax), along with decreased B-cell lymphoma/leukemia 2 (Bcl-2), were observed at day 70 after MDPI but not after SDPI. The downregulation of glutamic acid decarboxylase (GAD65) expression was observed on day 70 only after MDPI. The effects of MDPI-induced lower HWT from days one to 70 were attenuated by 12 sessions of EA treatment (days one to 21 after MDPI). Changes in astrocytic GFAP, GS, and GLT-1, along with cleaved caspase-3, Bax, Bcl-2, and GAD65 expression observed 70 days after MDPI, were reversed by EA treatment. The results suggest that persistent dental pain in mice was induced by MDPI but not by SDPI. This effect was associated with trigeminal GABAergic interneuron plasticity along with morphological and functional changes in astrocytes. EA exerts prolonged analgesic and neuroprotective effects that might be associated with the modulation of neuron-glia crosstalk mechanisms.

[Mechanism of Mongolian drug Naru-3 in initiation of neuroinflammation of neuropathic pain from MMP9/IL-1ß signaling pathway].

Neuropathic pain(NP) has similar phenotypes but different sequential neuroinflammatory mechanisms in the pathological process. It is of great significance to inhibit the initiation of neuroinflammation, which has become a new direction of NP treatment and drug development in recent years. Mongolian drug Naru-3 is clinically effective in the treatment of trigeminal neuralgia, sciatica, and other NPs in a short time, but its pharmacodynamic characteristics and mechanism of analgesia are still unclear. In this study, a spinal nerve ligation(SNL) model simulating clinical peripheral nerve injury was established and the efficacy and mechanism of Naru-3 in the treatment of NPs was discussed by means of behavioral detection, side effect evaluation, network analysis, and experimental verification. Pharmacodynamic results showed that Naru-3 increased the basic pain sensitivity threshold(mechanical hyperalgesia and thermal radiation hyperalgesia) in the initiation of SNL in animals and relieved spontaneous pain, however, there was no significant effect on the basic pain sensitivity threshold and motor coordination function of normal animals under physiological and pathological conditions. Meanwhile, the results of primary screening of target tissues showed that Naru-3 inhibited the second phase of injury-induced nociceptive response of formalin test in mice and reduced the expression of inflammatory factors in the spinal cord. Network analysis discovered that Naru-3 had synergy in the treatment of NP, and its mechanism was associated with core targets such as matrix metalloproteinase-9(MMP9) and interleukin-1ß(IL-1ß). The experiment further took the dorsal root ganglion(DRG) and the stage of patho-logical spinal cord as the research objects, focusing on the core targets of inducing microglial neuroinflammation. By means of Western blot, immunofluorescence, agonists, antagonists, behavior, etc., the mechanism of Naru-3 in exerting NP analgesia may be related to the negative regulation of the MMP9/IL-1ß signaling pathway-mediated microglia p38/IL-1ß inflammatory loop in the activation phase. The relevant research enriches the biological connotation of Naru-3 in the treatment of NP and provides references for clinical rational drug use.

A solid lipid particle formulation of long pepper extract reduces pain and astrocyte activation in a rat model of neuropathic pain.

OBJECTIVES: To investigate the effects of solid lipid microparticle (SLM) creams containing a long pepper extract (LPE) or piperine on neuropathy-related pain and the expression of glial fibrillary acidic protein (GFAP) as a measure of astrogliosis. METHODS: Neuropathic pain in male Spraque Dawley rats was induced by sciatic nerve ligation (SNL) and followed by treatment with LPE-SLM, piperine-SLM, capsaicin or vehicle creams. The pain score was assessed by thermal hyperalgesia test. The GFAP expression in the spinal cord was determined by immunohistochemistry. RESULTS: Pain scores were significantly increased after SNL and decreased when treated by LPE-SLM. The number of GFAP immunopositive cells was significantly increased in the SNL rats. Treated by LPE-SLM and capsaicin creams resulted in a significant reduction of the number of GFAP immunopositive cells. The LPE-SLM treated rats showed greater effects than the piperine and capsaicin preparations. CONCLUSIONS: The LPE-SLM cream has a potential effect on pain attenuation via a decrease of spinal astrocyte activation-related mechanism. The LPE in SLM preparation could provide an alternative therapeutic strategy for treating neuropathic pain.

Levo-tetrahydropalmatine ameliorates neuropathic pain by inhibiting the activation of the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis.

BACKGROUND: Because of the complex pathogenesis of neuropathic pain (NP), the therapeutic efficacy of existing drugs is not satisfactory. Accumulating studies have indicated that neuroinflammation may play a key role in NP onset and progression. Levo-tetrahydropalmatine (l-THP) has been extensively used for relieving chronic pain for decades. However, its potential mechanisms against NP have not yet been fully elucidated. PURPOSE: Exploring and elucidating the therapeutic effect and pharmacological mechanism of l-THP in treating NP. METHODS: RNA-seq and bioinformatics analyses were carried out to identify effective target profiling of I-THP in chronic constrictive injury (CCI) rats. The I-THP related hub targets and signaling pathways were obtained via bioinformatics analysis, then subjected to in-depth analyses through experiments in vivo. A gain-of-function study further confirmed the role of Clec7a in l-THP-mediated pain relief. Finally, the interaction between l-THP and Clec7a was verified through molecular docking and surface plasmon resonance (SPR). RESULTS: l-THP treatment effectively alleviated mechanical and thermal allodynia in NP model rats. Functionally, the I-THP effective targets were mainly enriched in inflammatory response-related pathways. Furthermore, Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis was selected as one of the potential pathways of l-THP against NP. Mechanically, l-THP markedly reduced CCI-induced Clec7a overexpression, significantly inhibited the Clec7a-triggered phosphorylation of MAPK and NF-κB-p65, and decreased the expression of pyroptosis-related protein NLRP3 and Caspase-1-p20. The analgesic effect of l-THP on NP was partly eliminated when transfecting the overexpression vector virus pLVSO5Clec7a. Importantly, molecular docking and SPR data revealed that l-THP directly binds with the Clec7a protein. CONCLUSION: This study is the first to indicate that l-THP may exert an analgesic effect through inhibiting neuroinflammation via the Clec7a-MAPK/NF-κB-NLRP3 inflammasome axis, supporting the clinical utility of l-THP in NP therapy.

Electroacupuncture alleviates neuropathic pain caused by SNL by promoting M2 microglia polarization through PD-L1.

As a common clinical disease, neuropathic pain is difficult to be cured with drugs. The occurrence and progression of pain is closely related to the response of spinal microglia. Aspartof the regulation of microglialactivity,PD-L1 playsacriticalrole. Loss of PD-L1 promoted the polarization of M1-like microglia. Increased expression of PD-L1 promoted M2-like polarization. Electroacupuncture has a significant analgesic effect in clinical practice, but its specific mechanism remains to be further explored. In this study, we verified the role of PD-L1 in EA analgesia and the underlying molecular mechanism through spinal nerve ligation (SNL) in rats and lipopolysaccharide (LPS)-treated BV2 microglial cells. Forbehavioralstudiesofrats,mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured, and spinal cord neuros were examined under transmission electron microscopyto determine changes to their myelin structure. The expression levels of PD-L1 and M1/M2-specific markers in rat spinal cord and BV2 microglial cells were measured by enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence staining and Western blot analysis. Our study showed that EA increased the pain threshold, reduced the destruction of myelin structure, promoted the expression of PD-L1 and PD-1, inhibited the MAPK signaling pathway, and promoted the conversion of microglial polarization from the M1 phenotype to the M2 phenotype in SNL rats. PD-L1 knockdown reversed these effects of EA. In addition, PD-L1 knockdown activated the MAPK signaling pathway, promoted microglial polarization to the M1 phenotype, decreased the expression of anti-inflammatory mediators and increased the expression of proinflammatory factors in LPS-stimulated BV2 microglial cells. Our results showed that EA may regulate the excitability of primary afferent neurons through PD-L1 and then inhibit the MAPK signaling pathway to promote the transformation of activated M1 microglia into M2 microglia, reduce inflammatory reactions, and finally achieve analgesic effects. A therapy targeting PD-L1 may be an effective strategy for treating neuropathic pain.

Chinese Tuina remodels the synaptic structure in neuropathic pain rats by downregulating the expression of N-methyl D-aspartate receptor subtype 2B and postsynaptic density protein-95 in the spinal cord dorsal horn.

OBJECTIVE: To investigate whether the Chinese massage system, Tuina, exerts analgesic effects in a rat model of chronic constriction injury (CCI) by remodeling the synaptic structure in the spinal cord dorsal horn (SCDH). METHODS: Sixty-nine male Sprague-Dawley rats were randomly and evenly divided into the normal group, sham group, CCI group, CCI + Tuina group, CCI + MK-801 [an -methyl D-aspartate receptor subtype 2B (NR2B) antagonist] group, and CCI + MK-801 + Tuina group. The neuropathic pain model was established using CCI with right sciatic nerve ligation. Tuina was administered 4 d after CCI surgery, using pressing manipulation for 10 min, once daily. Motor function was observed with the inclined plate test, and pain behaviors were observed by the Von Frey test and acetone spray test. At 19 d after surgery, the L3-L5 spinal cord segments were removed. Glutamate, interleukin 1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) levels were detected by enzyme-linked immunosorbent assay. The protein expression levels of NR2B and postsynaptic density protein-95 (PSD-95) were detected by Western blot, and the synaptic structure was observed by transmission electron microscopy (TEM). RESULTS: CCI reduced motor function and caused mechanical and cold allodynia in rats, increased glutamate concentration and TNF-α and IL-1ß levels, and increased expression of synapse-related proteins NR2B and PSD-95 in the SCDH. TEM revealed that the synaptic structure of SCDH neurons was altered. Most of these disease-induced changes were reversed by Tuina and intrathecal injection of MK-801 ( < 0.05 or < 0.01). For the majority of experiments, no significant differences were found between the CCI + MK-801 and CCI + MK-801 + Tuina groups. CONCLUSIONS: Chinese Tuina can alleviate pain by remodeling the synaptic structure, and NR2B and PSD-95 receptors in the SCDH may be among its targets.

Electroacupuncture Alleviates Neuropathic Pain by Suppressing Ferroptosis in Dorsal Root Ganglion via SAT1/ALOX15 Signaling.

Neuropathic pain affects globally about 7-10% of the general population. Electroacupuncture (EA) effectively relieves neuropathic pain symptoms without causing any side effects; however, the underlying molecular mechanisms remain unclear. We established a chronic constriction injury (CCI)-induced rat model of neuropathic pain. RNA sequencing was used to screen for differentially expressed genes in the dorsal root ganglion after CCI and EA treatment. We identified gene markers of ferroptosis spermidine/spermine N1-acetyltransferase 1 (Sat1) and arachidonate 15-lipoxygenase (Alox15) to be dysregulated in the CCI-induced neuropathic pain model. Furthermore, EA relieved CCI-induced pain as well as ferroptosis-related symptoms in the dorsal root ganglion, including lipid peroxidation and iron overload. Finally, SAT1 knockdown also alleviated mechanical and thermal pain hypersensitivity and reversed ferroptosis damage. In conclusion, we showed that EA inhibited ferroptosis by regulating the SAT1/ALOX15 pathway to treat neuropathic pain. Our findings provide insight into the mechanisms of EA and suggest a novel therapeutic target for neuropathic pain.

Electroacupuncture alleviates mechanical allodynia and anxiety-like behaviors induced by chronic neuropathic pain via regulating rostral anterior cingulate cortex-dorsal raphe nucleus neural circuit.

AIMS: Epidemiological studies in patients with neuropathic pain have demonstrated a strong association between neuropathic pain and psychiatric conditions such as anxiety. Preclinical and clinical work has demonstrated that electroacupuncture (EA) effectively alleviates anxiety-like behaviors induced by chronic neuropathic pain. In this study, a potential neural circuitry underlying the therapeutic action of EA was investigated. METHODS: The effects of EA stimulation on mechanical allodynia and anxiety-like behaviors in animal models of spared nerve injury (SNI) were examined. EA plus chemogenetic manipulation of glutamatergic (Glu) neurons projecting from the rostral anterior cingulate cortex (rACCGlu ) to the dorsal raphe nucleus (DRN) was used to explore the changes of mechanical allodynia and anxiety-like behaviors in SNI mice. RESULTS: Electroacupuncture significantly alleviated both mechanical allodynia and anxiety-like behaviors with increased activities of glutamatergic neurons in the rACC and serotoninergic neurons in the DRN. Chemogenetic activation of the rACCGlu -DRN projections attenuated both mechanical allodynia and anxiety-like behaviors in mice at day 14 after SNI. Chemogenetic inhibition of the rACCGlu -DRN pathway did not induce mechanical allodynia and anxiety-like behaviors under physiological conditions, but inhibiting this pathway produced anxiety-like behaviors in mice at day 7 after SNI; this effect was reversed by EA. EA plus activation of the rACCGlu -DRN circuit did not produce a synergistic effect on mechanical allodynia and anxiety-like behaviors. The analgesic and anxiolytic effects of EA could be blocked by inhibiting the rACCGlu -DRN pathway. CONCLUSIONS: The role of rACCGlu -DRN circuit may be different during the progression of chronic neuropathic pain and these changes may be related to the serotoninergic neurons in the DRN. These findings describe a novel rACCGlu -DRN pathway through which EA exerts analgesic and anxiolytic effects in SNI mice exhibiting anxiety-like behaviors.

Parthenolide as a potential analgesic in the treatment of paclitaxel-induced neuropathic pain: the rat modeling.

In this study, we determined the therapeutic effect of parthenolide (PTL), the active component of Tanacetum parthenium, on neuropathic pain caused by paclitaxel (PTX), a chemotherapeutic drug frequently used in cancer treatment, at the gene and protein levels. To this end, 6 groups were formed: control, PTX, sham, 1 mg/PTL, 2 mg/kg PTL, and 4 mg/kg PTL. Pain formation was tested by Randall-Selitto analgesiometry and locomotor activity behavioral analysis. Then, PTL treatment was performed for 14 days. After the last dose of PTL was taken, Hcn2, Trpa1, Scn9a, and Kcns1 gene expressions were measured in rat brain (cerebral cortex/CTX) tissues. In addition, changes in the levels of SCN9A and KCNS1 proteins were determined by immunohistochemical analysis. Histopathological hematoxylin-eosin staining was also performed to investigate the effect of PTL in treating tissue damage on neuropathic pain caused by PTX treatment. When the obtained data were analyzed, pain threshold and locomotor activity decreased in PTX and sham groups and increased with PTL treatment. In addition, it was observed that the expression of the Hcn2, Trpa1, and Scn9a genes decreased while the Kcns1 gene expression increased. When protein levels were examined, it was determined that SCN9A protein expression decreased and the KCNS1 protein level increased. It was determined that PTL treatment also improved PTX-induced tissue damage. The results of this study demonstrate that non-opioid PTL is an effective therapeutic agent in the treatment of chemotherapy-induced neuropathic pain, especially when used at a dose of 4 mg/kg acting on sodium and potassium channels.

Folic acid relieves bone cancer pain by downregulating P2X2/3 receptors in rats.

BACKGROUND: Bone cancer pain (BCP) remains a clinical challenge due to the limited and side effects of therapeutic methods. Folic acid has been known as an FDA approved dietary supplement and proved to have an analgesic effect in neuropathic pain. Here we investigate the role and mechanism of folic acid in bone cancer pain of a rat model. METHODS: Walker 256 tumor cells were inoculated into the left tibia of rats to induce bone cancer pain model. Pain reflex were assessed by paw withdrawal threshold (PWT) response to Von Frey filaments and paw withdrawal latency (PWL) response to thermal stimulation. Folic acid was injected intraperitoneally to evaluate its analgesic effect in rats with bone cancer pain. Western blotting and qPCR were used to determine P2X2/3 receptor protein and mRNA levels in ipsilateral L4-6 dorsal root ganglion (DRG) and spinal dorsal horn (SDH). RESULTS: The PWT and PWL of rats with bone cancer pain were obviously decreased compared to the naïve and sham rats. Interestingly, continuous folic acid treatment significantly increased the PWT and PWL of rats with bone cancer pain. P2X2 and P2X3 receptors were clearly upregulated at both mRNA and protein expression in L4-6 DRG and SDH of rats with bone cancer pain. P2X2 and P2X3 receptors were mainly localized with CGRP (calcitonin gene-related peptide) or IB4 (isolectin B4) positive neurons in L4-6 DRG of rats with bone cancer pain. Notably, continuous folic acid treatment significantly reduced the expression of P2X2 and P2X3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Finally, intrathecal injection of A317491 (a selective antagonist of P2X2/3 receptors) markedly elevated the PWT and PWL of rats with bone cancer pain. CONCLUSION: These results suggest that folic acid has an effective antinociceptive effect on bone cancer pain, which is mediated by downregulating P2X2/3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Folic acid may be a novel therapeutic strategy in cancer patients for pain relief.

Disinhibition of Mesolimbic Dopamine Circuit by the Lateral Hypothalamus Regulates Pain Sensation.

Our recent study demonstrated the critical role of the mesolimbic dopamine (DA) circuit and its brain-derived neurotropic factor (BDNF) signaling in mediating neuropathic pain. The present study aims to investigate the functional role of GABAergic inputs from the lateral hypothalamus (LH) to the ventral tegmental area (VTA; LHGABA→VTA) in regulating the mesolimbic DA circuit and its BDNF signaling underlying physiological and pathologic pain. We demonstrated that optogenetic manipulation of the LHGABA→VTA projection bidirectionally regulated pain sensation in naive male mice. Optogenetic inhibition of this projection generated an analgesic effect in mice with pathologic pain induced by chronic constrictive injury (CCI) of the sciatic nerve and persistent inflammatory pain by complete Freund's adjuvant (CFA). Trans-synaptic viral tracing revealed a monosynaptic connection between LH GABAergic neurons and VTA GABAergic neurons. Functionally, in vivo calcium/neurotransmitter imaging showed an increased DA neuronal activity, decreased GABAergic neuronal activity in the VTA, and increased dopamine release in the NAc, in response to optogenetic activation of the LHGABA→VTA projection. Furthermore, repeated activation of the LHGABA→VTA projection was sufficient to increase the expression of mesolimbic BDNF protein, an effect seen in mice with neuropathic pain. Inhibition of this circuit induced a decrease in mesolimbic BDNF expression in CCI mice. Interestingly, the pain behaviors induced by activation of the LHGABA→VTA projection could be prevented by pretreatment with intra-NAc administration of ANA-12, a TrkB receptor antagonist. These results demonstrated that LHGABA→VTA projection regulated pain sensation by targeting local GABAergic interneurons to disinhibit the mesolimbic DA circuit and regulating accumbal BDNF release.SIGNIFICANCE STATEMENT The mesolimbic dopamine (DA) system and its brain-derived neurotropic factor (BDNF) signaling have been implicated in pain regulation, however, underlying mechanisms remain poorly understood. The lateral hypothalamus (LH) sends different afferent fibers into and strongly influences the function of mesolimbic DA system. Here, utilizing cell type- and projection-specific viral tracing, optogenetics, in vivo calcium and neurotransmitter imaging, our current study identified the LHGABA→VTA projection as a novel neural circuit for pain regulation, possibly by targeting the VTA GABA-ergic neurons to disinhibit mesolimbic pathway-specific DA release and BDNF signaling. This study provides a better understanding of the role of the LH and mesolimbic DA system in physiological and pathological pain.