BackgroundVisceral pain in inflammatory bowel disease (IBD) lacks effective therapies. Whether the TREM1/DAP12 pathway is associated with electroacupuncture (EA) analgesia and cortical EEG modulation remains unclear.ObjectiveTo determine if spinal TREM1/DAP12 is associated with EA's analgesic and neuromodulatory effects in TNBS-induced visceral pain.MethodsMale Sprague-Dawley rats with TNBS-induced colitis received EA (ST36/ST37), the TREM1 inhibitor Nangibotide, or their combination. Behaviors (PWT, CRD, DAI), spinal TREM1/DAP12, downstream inflammatory mediators, and microglial colocalization were evaluated. Cortical EEG was analyzed using normalized power spectral density (Normalized PSD, %).ResultsEA significantly alleviated visceral/referred hyperalgesia and reduced DAI scores (P < 0.05). TNBS upregulated spinal TREM1, DAP12, TNF-α, NLRP3, and TLR4 and increased TREM1/DAP12 microglial colocalization (P < 0.0001), all reversed by EA (P < 0.001). Nangibotide alone partially mimicked EA's effects, and their combination yielded greater analgesia and molecular suppression (P < 0.05). Normalized EEG analysis revealed that TNBS increased δ power (P < 0.0001) and reduced θ and β power (P < 0.001). Notably, EA and Nangibotide specifically reversed δ elevation and restored θ power (P<0.05) without altering β power (P > 0.05), with greater δ suppression than either treatment alone (P < 0.0001).ConclusionEA alleviates TNBS visceral pain and normalizes cortical δ/θ dysrhythmia, with these effects associated with suppression of microglial TREM1/DAP12 neuroinflammation. Pharmacological TREM1 blockade mimics and enhances EA's effects, supporting this pathway as a promising therapeutic target for visceral pain.

ObjectiveTo evaluate whether genetically proxied leukocyte telomere length (TL), a systemic marker of biological aging and cumulative cellular stress, is associated with trigeminal neuralgia (TN) risk.BackgroundTN is a severe craniofacial pain disorder classically attributed to neurovascular compression and focal trigeminal root demyelination. However, comparable neurovascular anatomy does not always produce symptoms, suggesting that host-level biological vulnerability may influence susceptibility.MethodsWe performed a two-sample Mendelian randomization study using genome-wide significant single nucleotide polymorphisms for leukocyte TL as genetic instruments. The outcome was FinnGen ICD-10 G50.0 trigeminal neuralgia. Primary analysis used inverse variance weighting (IVW), with MR-Egger, weighted median, weighted mode, and MR-PRESSO as complementary estimators. Sensitivity analyses included Steiger filtering, pleiotropy testing, heterogeneity testing, and leave-one-out analysis. Reverse MR, exploratory multivariable MR, and comparator analyses for atypical facial pain, migraine, and polyneuropathy were performed.ResultsLonger genetically proxied TL was associated with lower TN risk using IVW MR (OR=0.732, p=0.008). Results were concordant across weighted median (OR=0.606, p=0.011), weighted mode (OR=0.577, p=0.017), and MR-PRESSO (OR=0.732, p=0.008), while MR-Egger was directionally consistent but nonsignificant (OR=0.671, p=0.055). Steiger-filtered IVW remained directionally consistent but attenuated to non-significance while Reverse MR was non-significant. The TL-TN association remained directionally consistent after exploratory multivariable MR conditioning and was not observed for comparator phenotypes.ConclusionLonger genetically proxied leukocyte TL was associated with lower TN risk. These findings identify leukocyte telomere length as a candidate systemic aging-related susceptibility marker for trigeminal neuralgia and support further studies integrating telomere biology with neurovascular compression grading, trigeminal nerve morphometry, and clinical outcomes.

Background: Both depressive and pain symptoms are highly prevalent and relevant comorbid conditions in Veterans with Gulf War Illness (GWI). This study assessed how the severity of depressive symptoms may affect supraspinal pain modulatory functional connectivity (FC) in Veterans with GWI-related chronic Headache And body, muscle, and joint Pain conditions (GWI-HAP) and how depressive symptoms alone or paired with pain symptomology or other neuropsychological and functional assessments were able to predict supraspinal functional connectivity (FC) relevant to pain modulation. Methods: Resting state functional MRI (rs-fMRI) conducted in Veterans with GWI-HAP and moderate to severe depressive symptoms reflected by the Hamilton Rating Scale for Depression (HRSD) score <14(GWI ≥ 14) and their counterparts with no or mild depressive symptoms (GWI<14) were compared along with pain symptomology, functional, quality of life, and Post-Traumatic Stress Disorder assessments. Results: Region of interest (ROI)-to-ROI rs-fMRI FC analyses indicated the GWI ≥ 14 group (N=33) showed significantly enhanced FC of the left dorsolateral prefrontal cortex (DLPFC) with the contralateral Insula (BA 13; P < 0.001; T= 7.14), visuomotor cortex (BA7, P<0.01, T=4.82) and two regions of contralateral thalamus (P < 0.01, P < 0.001; T= 5.32, T= 5.25, respectively); and decreased FC with the ipsilateral premotor cortex (BA 6; P < 0.01; t = -3.34) and contralateral anterior cingulate cortex (BA 32; P < 0.001; T= -3.64) than the GWI<14 group (N=33). Independent T-tests also indicated that the GWI ≥ 14 group demonstrated a significantly (P<0.05) higher level of pain severity, functional, and quality of life impairments, and PTSD symptomology in comparison with the GWI<14 group. Stepwise regression analyses of the combined group indicated that despite the detected significant between-group difference in supraspinal FC of the left DLPFC to other pain-relevant supraspinal regions, either the degree of depressive symptoms or any other study assessment was able to reliably predict the FC of the left DLPFC to the right ACC. In addition, other assessments alone did not reliably predict the left DLPFC to other pain relevant ROI. However, adding other pain symptomology, neuropsychological, and functional assessments to depressive symptom severity assessment can enhance the prediction of the supraspinal FC relevant to pain modulation. Conclusions: While significant between-group differences in supraspinal modulatory FC were found based on the severity of depressive symptoms, pain symptomology, functional, and quality of life impairments, these assessments alone did not predict the FC correlation of left the DLPFC in most detected ROI differences. However, individually pairing mood with other pain and functional assessments can enhance the prediction of the underlying supraspinal pain modulatory FC. Potential non-invasive brain stimulation interventions such as TMS with various treatment targeting options, which are applicable for addressing these highly prevalent co-morbid conditions and functional impairments, may incorporate this mechanistic correlation and prediction in formulating a corresponding therapeutic strategy.

The most burdensome symptom of temporomandibular joint disorders (TMJDs) is mechanically evoked pain. To identify the trigeminal ganglion (TG) neuronal subtypes mediating mechanical sensitivity, we combined whole-cell patch-clamp electrophysiology with immunohistochemistry (IHC) to characterize mouse TMJ-innervating TG neurons retrogradely labeled from the TMJ. Electrophysiological analysis identified five distinct neuronal groups, only one of which responded to piezo actuator-induced mechanical stimulation. TMJ innervation was dominated by C- and A-nociceptors, comprising four of the five identified groups. The fifth, a minor population of small-diameter neurons (S1), displayed properties consistent with either vGlut3-positive, C-low-threshold mechanoreceptors (C-LTMRs) or Sst-positive neurons. Two mechanically insensitive C-nociceptor groups, distinguished by their action potential firing patterns, likely corresponded to CGRP-positive and non-Mrgpr neuronal populations. One mechanically insensitive A-nociceptor group likely represented Smr2-positive Aδ high-threshold mechanoreceptors (Aδ-HTMRs). The only mechanically responsive neurons were large-diameter A-nociceptors, which likely correspond to the weakly TrkC- expressing neuronal population. No recorded neurons displayed electrophysiological characteristics of classical A-LTMRs. Together, these findings demonstrate that the TMJ is innervated by a distinct repertoire of TG sensory neurons compared with the skin and masseter muscle, with mechanically responsive afferents restricted to a single A-nociceptor subtype rather than classical low-threshold mechanoreceptors.

Orofacial neuropathic pain is a debilitating pain condition. It is poorly treated, partially due to an incomplete understanding of its underlying neurophysiological mechanisms. Orofacial neuropathic pain is often caused by injury or dysfunction of trigeminal nerve branches, including the infraorbital nerve (IoN). Clinically, a main symptom of orofacial neuropathic pain is mechanical allodynia, excruciating pain induced by gentle touch. While previous studies on neuropathic pain have mainly focused on the involvement of C- and Aδ-afferents, the potential contribution of Aβ-afferents to orofacial neuropathic pain has not been well explored. In the present study, we have established a mouse model of chronic constrictive injury of the IoN (IoN-CCI) and assessed orofacial pain behaviors and excitability of Aβ-afferent trigeminal ganglion (TG) neurons. Behavioral assessment using the von Frey test has revealed significantly increased mechanical sensitivity 3-4 weeks post-surgery in IoN-CCI mice compared with the sham group. Machine-learning-based eyelid distance measurement has demonstrated orbital tightening in IoN-CCI mice, suggesting spontaneous pain. Using ex vivo TG preparations and patch-clamp recordings, we have found that action potential (AP) thresholds are significantly reduced in both non-nociceptive-like and nociceptive-like Aβ-afferent TG neurons of IoN-CCI mice. Consistently, there is a significant increase in voltage-gated Na[+] currents in both non-nociceptive-like and nociceptive-like Aβ-afferent TG neurons following IoN-CCI. Our findings demonstrate that both non-nociceptive-like and nociceptive-like Aβ-afferent TG neurons undergo hyperexcitability following infraorbital nerve injury, raising the possibility that both types of Aβ-afferent TG neurons may contribute to orofacial neuropathic pain following infraorbital nerve injury.

ObjectiveThe effect of Gelsemine on neuropathic pain was investigated using a sciatic nerve (SN) chronic constriction injury (CCI) rat model.MethodsNetwork pharmacology analysis was performed to identify potential molecular targets of Gelsemine associated with neuropathic pain, followed by molecular docking analysis of the identified targets. Neuropathic pain was induced in male rats by SN-CCI. The rats were divided into four groups: sham, CCI, Gelsemine 2 mg/kg, and Gelsemine 4 mg/kg. Gelsemine (2 or 4 mg/kg, i.p.) was administered to the treatment groups for 30 consecutive days. The effect of Gelsemine on mechanical hypersensitivity was evaluated, and the BBB score was used as a secondary assessment of general locomotor function following CCI. The levels and expression of inflammatory mediators were analyzed by ELISA and qRT-PCR, respectively, and the expression of THIK1, caspase-8, and cytosolic phospholipase A2 (cPLA2) in nerve tissue was analyzed by western blot.ResultsNetwork pharmacology analysis identified ASK1 and TrkB (NTRK2) as potential molecular targets of Gelsemine in neuropathic pain, and molecular docking predicted favorable binding interactions with these proteins. Gelsemine significantly attenuated mechanical hypersensitivity and improved BBB locomotor scores in CCI rats. It also reduced the altered serum levels of IL-4, IL-6, IL-1β, and TNF-α, as well as the mRNA expression of NF-κB, IL-6, and TNF-α, compared with the CCI group. Furthermore, Gelsemine was associated with significantly reduced mRNA expression of ASK1, TrkB (NTRK2), and MAPK1 in SN tissue and normalization of the altered expression of THIK1, caspase-8, and cPLA2 in male CCI rats.ConclusionGelsemine alleviated mechanical hypersensitivity and improved locomotor function, while ameliorating inflammatory and oxidative stress-related changes in male CCI rats. Computational analyses identified ASK1 and TrkB (NTRK2) as potential molecular targets, while Gelsemine treatment was associated with reduced expression of ASK1, TrkB, and MAPK1. These findings suggest a possible association between Gelsemine treatment and ASK1/TrkB/MAPK-related signaling; however, further functional studies are required to establish their causal involvement in the analgesic effects of Gelsemine. Only male rats were included in this study; therefore, the findings are limited to male physiology and cannot be generalized to female animals.

High-impact musculoskeletal pain (i.e., pain that limits daily activities and function) affects a substantial proportion of older adults and is associated with accelerated mobility decline. While skeletal muscle is central to movement and functional capacity, its molecular contribution to pain-related disability in aging remains poorly understood. We conducted a cross-sectional analysis of baseline data from 742 community-dwelling adults aged 70 years or older from the Study of Muscle, Mobility and Aging (SOMMA). Bulk RNA sequencing was performed on vastus lateralis muscle biopsies. Pain impact was classified using self-reported pain and limitations in activities of daily living. Differential gene expression was examined comparing high-impact pain, low-impact pain, and no pain, with sex-stratified analyses. Pathway enrichment analyses were conducted to identify biological processes associated with high-impact pain. High-impact musculoskeletal pain was associated with a distinct skeletal muscle transcriptomic profile, whereas minimal differences were observed between low-impact pain and no pain. Sex-stratified analyses revealed substantial differences in pain-related gene expression for men and women. In men, high-impact pain was characterized by enrichment of musculoskeletal structural and extracellular matrix pathways. In women, high-impact pain was associated with broader transcriptional alterations involving immune, inflammatory, metabolic, and cytoskeletal processes. High-impact musculoskeletal pain in older adults was associated with sex-dependent skeletal muscle transcriptomic signatures distinct from those observed in low-impact pain. These findings implicate muscle-specific molecular pathways in pain-related functional limitations and highlight the importance of sex-informed approaches to understanding and treating chronic pain in aging.

Chronic itch, affecting 8-10% of the global population, is a debilitating condition linked to dermatoses, systemic diseases, and neurological disorders, with limited therapeutic efficacy due to unclear central mechanisms. Spinal microglia, central nervous system-resident immune cells, regulate neuroinflammation and synaptic plasticity in chronic pain and neurodegeneration, but their role in chronic itch remains unknown. This study investigated the role of spinal microglia in chronic itch. Using an allergic contact dermatitis (ACD) mouse chronic itch model, we found robust microglial activation, accompanied by c-Fos-positive neuronal activation, in the spinal dorsal horn of a chronic itch model. Pharmacological inhibition (minocycline) or genetic ablation of microglia significantly reduced mechanical itch hypersensitivity and spontaneous scratching, alongside decreased microglial/neuronal activation. Morphological and molecular analyses revealed downregulated antioxidants (glutathione peroxidase 4 [GPX4], superoxide dismutase [SOD], glutathione [GSH],) and upregulated lipid peroxidation (malondialdehyde [MDA], acyl-CoA synthetase long-chain family member 4 [ACSL4]) in the spinal cord, indicating ferroptosis. Intrathecal liproxstatin-1 (ferroptosis inhibitor) alleviated itch behaviors and restored GPX4, SOD, GSH and MDA level, without affecting microglial activation. Our findings demonstrate that spinal microglia promote chronic itch by triggering ferroptosis in spinal dorsal horn. These findings provide the first evidence of microglia-ferroptosis crosstalk in chronic itch, providing a mechanistic basis for targeting glial activity or ferroptosis to develop novel therapies.

Secondary plant metabolites are widely used in traditional and naturopathic medicine to treat pain, infection, and inflammation. The sesquiterpenoid zerumbone, derived from the edible ginger Zingiber zerumbet, has been shown to exert analgesic, antioxidant, antitumoral, and antimicrobial effects in preclinical models, acting through multiple receptor systems including cannabinoid (CB) receptors, peroxisome proliferator-activated receptors, N-methyl-D-aspartate (NMDA) receptors, and the serotonergic system. Zerumbone also modulates inflammatory and nitric oxide-cGMP-K[+]ATP signaling pathways implicated in antiallodynic responses, yet its direct impact on nociceptor-associated ion channels like the Transient receptor potential vanilloid 1 (TRPV1) and the Twik-related spinal cord potassium channel (TRESK) in human cellular systems remains poorly characterized. In the present study, the antinociceptive properties of zerumbone were investigated in human HEK293-based and induced pluripotent stem cell (iPSC)-derived nociceptor models. Here, the zerumbone treatment reduced cell viability and proliferation in a concentration-dependent manner without altering TRPV1 or TRESK protein expression. Whole-cell patch-clamp recordings revealed that zerumbone reversibly inhibited TRESK-mediated outward potassium currents upon direct application, whereas bath-applied zerumbone increased outward currents, suggesting context-dependent modulation. In TRPV1-expressing HEK293 cells, SH-SY5Y neuroblastoma cells, and human iPSC-derived nociceptors, zerumbone reduced TRPV1-mediated ion currents and capsaicin-evoked calcium signals, indicating a direct or competitive inhibitory effect on TRPV1 activity. In iPSC-derived nociceptors, zerumbone decreased action potential firing at moderate depolarizing currents, shifted the resting membrane potential, and increased rheobase, consistent with reduced nociceptor excitability, while no significant changes in TRESK-mediated currents were detected. These data suggest that zerumbone attenuates human iPSC-derived nociceptor-like neuron excitability largely via TRPV1 inhibition, with a more complex, concentration- and application-method-dependent modulation of TRESK. The findings provide a mechanistic basis for the antiallodynic effects of zerumbone observed in rodent models and highlight the need for further studies in human-derived systems to clarify the precise channel-level interactions and their relevance to inflammatory and neuropathic pain.

Inflammatory pain represents a significant detriment to patient quality of life and currently lacks adequate treatment options. Conditions associated with inflammatory pain can occur across the lifespan but are most common in aged individuals. There has been increasing public interest in the analgesic potential of cannabinoids, and CB1 agonists have been preclinically tested for several conditions. However, their use is potentially limited by the development of tolerance. The S426A/S430A transgenic mouse line was previously generated to explore this tolerance, with two point mutations preventing the phosphorylation that represents the first step in CB1 desensitization. Therefore, we investigated the antinociceptive efficacy of CB1-selective agonism (ACEA) in the formalin model of inflammatory pain, using mice that varied in sex (female vs male), age (3- vs 12-month-old [MO], and genotype (C57BL/6J vs S426A/S430A). ACEA provided significant antinociception that was equivalent across all tested group. The S426A/S430A mutation significantly delayed tolerance at both ages tested. Additionally, the S426A/S430A mutation was associated with increased body weight in 3MO, 12MO female, and 12MO male mice. Mice that were 12MO had accelerated tolerance relative to 3MO mice. No significant differences in rotarod (motor coordination), open field (locomotion and center duration), or rectal temperature were noted. These results demonstrate the antinociceptive benefit of CB1 agonists and the significant limitation in their use (tolerance). Further studies are needed to investigate the mechanisms of CB1 tolerance development, the increase in body weight with the S426A/S430A mutation, and the potential clinical significance of these findings.

Electroacupuncture (EA) effectively alleviates inflammatory pain, yet its underlying mechanisms remain incompletely understood. Emerging evidence implicates AMP-activated protein kinase (AMPK) and autophagy in modulating inflammation, but whether they mediate EA's analgesic action is unknown. This study tests the hypothesis that EA relieves inflammatory pain by activating AMPK in macrophages, thereby triggering autophagy and suppressing pro-inflammatory mediators (iNOS and IL-1β) in inflamed tissues. In a mouse model of complete Freund's adjuvant (CFA)-induced inflammatory pain, we assessed pain behavior and inflammatory markers following EA. Pharmacological modulators- AMPK antagonist Compound C, AMPK activator AICAR, and autophagy inhibitor 3-methyladenine (3-MA) - were applied to dissect the AMPK- autophagy-cytokine cascade. Complementary in vitro experiments using NR8383 macrophages with a autophagy reporter (stubRFP-sensGFP-LC3 Lentivirus) further validated the pathway. EA not only reduced CFA-induced hyperalgesia but also concurrently augmented AMPK phosphorylation, enhanced autophagic flux, and diminished iNOS and IL-1β levels in injured skin. Mirroring these effects, AICAR alone produced analgesia, increased autophagy, and lowered cytokine expression. Conversely, Compound C blocked EA's actions on autophagy and inflammatory markers, while 3-MA abrogated EA's analgesic and anti-inflammatory effects. Macrophage experiments corroborated the in vivo results, confirming that AMPK activation drives autophagy and subsequent suppression of iNOS/IL-1β. Our findings establish a mechanistic axis whereby EA engages AMPK in macrophages to upregulate autophagy, which in turn curtails iNOS and IL-1β production, ultimately mitigating inflammatory pain. This work unveils a novel cellular pathway for EA's anti-nociceptive effect and highlights AMPK-autophagy as a potential therapeutic target in pain management.

Small diameter unmyelinated (group IV) and myelinated (group III) muscle afferents transmit action potentials to signal muscle pain and the exercise pressor reflex (EPR). The EPR helps to drive the cardiovascular response to exercise. µ- (µOR), δ- (δOR), and κ- (κOR) opioid receptors (OR) have been shown to be expressed by dorsal root ganglia (DRG) neurons, but only peripheral µOR activation has been shown to inhibit the EPR mediated by group IV afferents. To determine if this results from differential OR expression, we identified group IV neurons in DRG and axons in gastrocnemius muscles from both male and female rats, and used immunocytochemistry to compare the expression of µOR, δOR, and κOR. We show that µOR labeling was more frequently observed in group IV somata and axons vs. δOR or κOR. OR axonal labeling was lower than somata labeling, particularly for δOR & κOR. There were no significant differences in OR staining between male and female animals. The relatively high µOR axonal expression suggests that µOR are selectively transported to the peripheral group IV axons and can explain the selective effect of µOR agonists to inhibit group IV afferent activity that generates the EPR and muscle pain.

ObjectiveChronic migraine (CM) is characterized by central sensitization (CS), in which microglial activation plays a key role. The neuron-glia communication axis CD200/CD200R1 is a critical immunomodulatory pathway. This study investigated whether and how CD200/CD200R1 signaling modulates CS in a CM model.MethodsA CM mouse model was established by repeated nitroglycerin (NTG) injections. Pain thresholds were assessed. Protein expression in the trigeminal nucleus caudalis (TNC) was analyzed. The role of STAT6 was examined using inhibitor AS1517499.ResultsNTG treatment induced sustained hyperalgesia and downregulated CD200 and CD200R1 expression in the TNC. Administration of the CD200R1 agonist CD200Fc reversed pain hypersensitivity, suppressed the overexpression of CGRP and c-Fos, and reduced pro-inflammatory cytokines. CD200Fc promoted STAT6 phosphorylation and increased expression of anti-inflammatory markers (Arg-1, YM-1, YM-2). Inhibition of STAT6 phosphorylation abolished the therapeutic effects of CD200Fc on pain biomarkers without altering CD200R1 expression.ConclusionOur findings demonstrate that enhancing the CD200/CD200R1 pathway alleviates central sensitization and pain hypersensitivity in CM, likely by promoting an anti-inflammatory microglial phenotype via STAT6 phosphorylation. The CD200/CD200R1/STAT6 axis represents a novel therapeutic target for chronic migraine.

Chemotherapy-induced peripheral neuropathy (CIPN) is a prevalent and debilitating consequence of cancer treatment with limited effective therapeutic options. While peripheral nerve injury is a key driver, emerging evidence suggests that maladaptive plasticity within central pain circuits, including the anterior cingulate cortex (ACC), contributes to the maintenance of neuropathic pain. Here, we tested the hypothesis that the ACC is a critical substrate for cisplatin-induced mechanical allodynia and a target for cannabigerol (CBG)-mediated antinociception. Adult male C57BL/6 mice received cisplatin (5 mg/kg, i.p., once weekly for four weeks) to induce CIPN. Mechanical allodynia was assessed using electronic von Frey testing. Systemic administration of CBG (20 mg/kg, i.p.) significantly reversed mechanical allodynia in CIPN mice without affecting baseline thresholds in non-neuropathic animals, indicating a state-dependent effect. Chemogenetic inhibition of ACC neurons using hM4Di DREADDs similarly attenuated mechanical allodynia, identifying the ACC as a functionally relevant component of the CIPN pain state. To determine whether CBG acts within this circuit, bilateral intra-ACC microinjections of CBG (20 nM and 20 µM) were performed, both of which produced transient antinociceptive effects. These findings demonstrate that the ACC contributes to the maintenance of mechanical allodynia in CIPN and establish this region as a site of action for CBG. Together, our results support a model in which the ACC represents a convergent cortical mechanism underlying pathological pain and highlight the potential for centrally targeted, non-euphoriant cannabinoid-based therapies.

BackgroundChronic inflammatory pain has a high incidence, and existing drugs have side effects. The natural product Cimifugin has anti-inflammatory and analgesic potential, but its role and mechanism in treating chronic inflammatory pain remain unclear. This study aims to explore the potential targets, signaling pathways, and in vivo mechanism of Cimifugin in the treatment of chronic inflammatory pain.MethodsNetwork pharmacology was used to predict the potential targets and related pathways of Cimifugin. A mouse model of chronic inflammatory pain induced by complete Freund's adjuvant (CFA) was established. Behavioral tests (mechanical/thermal hyperalgesia, elevated plus maze) were conducted to evaluate pain sensation and anxiety-like behaviors. ELISA was used to detect inflammatory factors (IL-1β, IL-6, TNF-α). Western blot was employed to analyze synaptic functional proteins. HPLC-MS/MS was used to verify Cimifugin's ability to cross the blood-brain barrier.ResultsNetwork pharmacology analysis predicted that the 64 cross-targets of Cimifugin and chronic inflammatory pain are enriched in pathways such as MAPK and PI3K-Akt. Animal experiments showed that Cimifugin dose-dependently increased pain thresholds, reduced paw swelling, improved anxiety-like behaviors, decreased inflammatory factor levels, reversed abnormal expression of synaptic functional proteins, and could cross the blood-brain barrier.ConclusionCimifugin alleviates chronic inflammatory pain and related behavioral abnormalities, an effect associated with inhibition of inflammatory responses and restoration of synaptic protein expression. These findings suggest a multi-target, multi-pathway mechanism of action.