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Abstract Midbrain dopamine (mDA) neurons participate in a wide range of brain functions through an intricate regulation of DA biosynthesis. The epigenetic factors and mechanisms in this process are not well understood. Here we report that histone demethylase JMJD3 is a critical regulator for DA biosynthesis in adult mouse mDA neurons. Mice carrying Jmjd3 conditional knockout or undergoing pharmaceutical inhibition of JMJD3 showed consistent reduction of DA content in midbrain and striatum. Histological examination of both mice confirmed that TH and NURR1, two key molecules in DA biosynthesis pathway, were decreased in mDA neurons. Mechanistic experiments in vivo and in vitro further demonstrated that the transcriptions of Th and Nurr1 in mDA neurons were suppressed by JMJD3 deficiency, because of increased repressive H3K27me3 and attenuated bindings of JMJD3 and NURR1 on the promoters of both genes. On behavioral level, a significant prolonged inflammation-induced mechanical hyperalgesia was found in conditional knockout mice regardless of sex and age, whereas motor function appeared to be intact. Our findings establish a novel link between DA level in mDA neurons with intrinsic JMJD3 activity, and suggest prolonged chronic inflammatory pain as a major loss-of-function consequence.

Background/Objectives: Several studies suggest gut microbiota metabolites as important immuno-modulators in inflammatory pain. We aimed to investigate the relationship between vitamin D status and gut dysbiosis markers in fibromyalgia (FM)-associated chronic inflammation. Methods: Blood samples were collected from sixty-eight female FM patients (49.9 ± 12.35 years). Pain intensity was assessed by FIQ-R. The serum levels of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-17, IFN-γ, as well as those of vitamin D (25(OH)D3) and the kynurenine/tryptophan ratio (Kyn/Trp) were determined by ELISA and HPLC, respectively. The plasma levels of the SCFAs acetate, butyrate, and propionate were detected by GC-MS. Results: A mean FIQ-R score indicated that the patients could be classified as having moderate FM. The mean levels of all cytokines, but IL-6 and IL-1β, were higher than the normal reference values. The highest concentrations of cytokines were observed in patients showing the highest FIQ-R scores and the lowest 25(OH)D3 levels. Deficient levels of acetate were found paralleled by an increase in Kyn/Trp. The highest acetate concentrations were detected in patients with the lowest FIQ-R scores and 25(OH)D3 levels. Significantly negative correlations were found between 25(OH)D3 concentrations and FIQ-R scores (p = 0.007) as well as IL-17 levels (p = 0.002) and between acetate and TNF-α (p = 0.040) as well as FIQ-R scores (p = 0.028), while significantly positive correlations were observed between Kyn/Trp and IL-17 (p = 0.027) as well as IFN-γ (p = 0.003). Conclusions: Our preliminary data suggest that the vitamin D status along with altered gut microbiota metabolism plays a major role in FM-related inflammatory pain. Replication of these findings in a larger cohort is required to provide additional insights.

Solanesol, an aliphatic terpene alcohol predominantly found in solanaceous plants, has gained recognition for its anti-inflammatory, antibacterial, and neuroprotective properties. This study investigates the potential efficacy of solanesol in alleviating chronic inflammatory pain induced by injection of complete Freund's adjuvant (CFA) into the left hind paw. Behavioral assessments revealed a significant reduction in mechanical and thermal hypersensitivity following solanesol administration, accompanied by a partial alleviation of concomitant anxiety-like behaviors. Mechanistically, Western blot analysis demonstrated a substantial decrease in the levels of TNF-α and IL-1β after solanesol administration. Immunohistochemical staining further revealed a notable suppression of microglial and astrocytic activation induced by CFA injection. These findings collectively suggest that solanesol holds promise as a latent therapeutic agent for the treatment of chronic inflammatory pain.

Yang Chen, Xiaoen Bian, Junnan Wang, Fang Yan, Jing Gao, Tao Sun Department of Pain Management, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250021, People’s Republic of ChinaCorrespondence: Tao Sun, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 324 Jingwu Road, Jinan, Shandong, 250021, People’s Republic of China, Email suntaosdph@163.comPurpose: Chronic inflammatory pain is usually treated with oral non-steroidal anti-inflammatory drugs (NSAIDs). However, oral NSAIDs can cause some adverse events, and local preparation is an important alternative drug. Currently, small sample clinical studies show that loxoprofen sodium hydrogel patch (LX-P) has good analgesic and anti-inflammatory effects; however, there is a lack of real-world clinical research data.Patients and Methods: This study included 60 patients with chronic inflammatory pain. They were treated with LX-P without affecting their real-world treatment for two weeks.Results: After 2 weeks of continuous medication, 93.33% of the patients stated that the treatment was effective. Only 3.33% of the patients had a relapse after 4 weeks. Moreover, the swelling range and degree of swelling decreased markedly and the dysfunction of the pain site was markedly alleviated. The total satisfaction of patients after treatment reached 90.00%.Conclusion: In this real-world observational study, LX-P showed good efficacy and safety in patients with chronic inflammatory pain.Keywords: loxoprofen sodium hydrogel patch, real-world study, chronic inflammatory pain, non-steroidal anti-inflammatory drugs

Objective: The purpose of this study was to analyze the impact of Shaoyao-Gancao decoction (SGD) on proteins with significant changes in the dorsal root ganglion (DRG) in rats and to explore the role of the Semaphorin 3G (Sema3G) protein in the DRG and its downstream factors, interleukin-6 (IL-6) and CC-motif chemokine ligand 2(CCL2), in the treatment of chronic inflammatory pain (CIP). Methods: We created a CIP rat model using 100 μL of complete Freund's adjuvant (CFA) that was injected into the left posterior plantar of rats. Then, we administered SGD intragastrically. We tested the animals for behavioral changes and protein expression levels in DRG pre- and post-drug intervention. Results: Rats in the SGD group showed significantly increased paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and relative expression levels of the Sema3G protein in the DRG (all P

Abstract Background Chronic inflammatory pain (CIP) frequently coincides with depression among patients. The onset and development of pain and depression are associated with altered neural synaptic plasticity. Electroacupuncture (EA) can effectively relieve CIP and depression. However, the underlying mechanisms have not been fully illustrated. Objective To explore whether EA can relieve CIP and depression by regulating hippocampal synaptic plasticity, and the present study offers foundational evidence for the efficacy of EA in treating CIP‐related depression (CIPD). Methods Rats were divided into four groups: 0.9% normal saline group, complete Freund's adjuvant (CFA) group, CFA + duloxetine group, and CFA + EA group. Pain hypersensitivity was detected by mechanical withdrawal threshold and thermal paw withdrawal latency, and the depression level was gauged using the open field test, the sucrose preference test, and the forced swimming test. The morphology of the hippocampal neurons was observed using Nissl staining. The protein expression levels of synuclein (Syn), postsynaptic density protein‐95 (PSD‐95), brain‐derived neurotrophic factors (BDNFs), tyrosine‐protein kinase B (TrKB), p‐TrkB, cAMP response element binding protein (CREB), and p‐CREB were measured by western blotting and immunofluorescence staining. BDNF and TrkB mRNA expression were detected using quantitative real‐time polymerase chain reaction (PCR) (qRT‐PCR). The content of 5‐hydroxytryptamine (5‐HT) and γ‐aminobutyric acid (GABA) was detected using enzyme‐linked immunosorbent assay, and the glutamic acid (Glu) content was determined using the ultraviolet colorimetry method. The hippocampal neuron ultrastructure was observed using transmission electron microscopy. Results EA could alleviate CIP and related depressive behaviors as well as protect the hippocampal neuronal structure from damage and regulate 5‐HT/GABA/Glu levels in the hippocampus. Additionally, EA could significantly increase the expression of synapse‐associated proteins such as PSD‐95 and Syn by activating the BDNF/TrKB/CREB signaling pathway. Conclusion EA improves pain and depressive behaviors in CIPD rats, and the mechanism may be related to synaptic plasticity mediated by the BDNF/TrKB/CREB signaling pathway.

Traditional drug solutions or suspensions, have been shown to treat pain in complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain in rats, with or without combination with magnetic therapy. In this study, we aimed to prepare, characterize, and evaluate the therapeutic effects of microparticles containing dexamethasone for local administration and treatment of chronic inflammatory pain. The results showed the following; a) Preparation and characterization: two ratios of poly(lactic-co-glycolic acid) (PLGA)/poly(lactic acid) (PLA) were used. The prepared batches were similar in size and magnetic responsiveness. The microparticle size distribution assessed via electron microscopy suggested a homogeneous distribution and absence of aggregates. Dexamethasone release profiles (microparticles synthesized with a feed ratio of 1:4) showed a sustained release in vitro and good biocompatibility with tissues. b) Therapeutic effect: the treatment effect of dexamethasone-PLGA magnetic microspheres + magnetic therapy was substantially better than that observed for other groups on day 4, as monitored by appearance, mechanical pain threshold, and histological analysis. This type of carrier could be a suitable magnetically retainable local drug delivery system for treating chronic pain.

Ling-Ling Xu,1– 4,* Yao Yan,1– 3,* Yu-Min Yuan,1– 3 Ying Li,1– 3 Jun Jiang,1– 3 Li-Cai Zhang1– 3 1Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, People’s Republic of China; 3NMPA Key Laboratory for Research and Evaluation of Narcotic and Psychotropic Drugs, Xuzhou, People’s Republic of China; 4Department of Anesthesiology, Nanjing Gaochun People’s Hospital, Nanjing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li-Cai Zhang, Email licaizhang001@163.comObjective: To assess the receptors of TRPV1 and GABAB1 receptors that were colocalized in cerebrospinal fluid contacting nucleus (CSF-contact nucleus) of chronic inflammatory pain (CIP) rats bringing inspiration for reducing chronic pain.Methods: A rat model of CIP was constructed by plantar injection of complete Freund’s adjuvant (CFA), and the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured 1, 3, 5, 7, 10, and 14 days after plantar injection. In the first part of the experiment, rats with CIP were divided into the immunofluorescence group and the coimmunoprecipitation (Co-IP) group (n = 6). Rats in the immunofluorescence group were injected with the retrograde tracer CB conjugated with Alexa Fluor 594 into the lateral ventricle two days before the injection of CFA into the plantar surface of the left paw. Three days later, rats that exhibited hyperalgesia were perfused, and their brains were extracted and used for double immunofluorescence staining of the CSF-contacting nucleus. Rats in the Co-IP group were anesthetized and dissected 3 days after CFA injection, and fresh brain segments containing the CSF-contacting nucleus were collected for Co-IP to assess the colocalization of TRPV1 and GABAB1 in the CSF-contacting nucleus (n = 6). In the second part of the experiment, SD rats were divided into the normal saline group (control group) and the CFA group. Fresh CSF-contacting nucleus-containing tissues were collected for Western blot analysis 3 days after plantar injection to observe the changes in TRPV1 and GABAB1 expression in the CSF-contacting nucleus.Results: TRPV1 and GABAB1 were co-expressed in the CSF-contacting nucleus in rats with CIP, and their expression was upregulated.Conclusion: TRPV1 and GABAB1 in the CSF-contacting nucleus are jointly involved in CIP in rats, and there is a direct or indirect link between TRPV1 and GABAB1.Keywords: cerebrospinal fluid-contacting nucleus, TRPV1 receptor, GABAB1 receptor, chronic inflammatory pain

Objective(s): Tissue injury in peripheral sites can result in long-term potentiation in nociceptive neurons and surrounding glial cells, potentially resulting in the development of chronic inflammatory pain (CIP). Acupoint injection (AI) is similar to Western phototherapy, which injects solutions at specific sites to mitigate chronic pain. AI has shown greater benefits compared with acupuncture. In this study, we examined the therapeutic effect and explored the underlying mechanisms of AI in mice CIP model. Materials and Methods: We injected thrice complete Freund’s adjuvant (CFA) into the mouse’s hind paw to induce CIP. Results: We found that, after two weeks, CFA injection significantly induced mechanical and thermal hyperalgesia which were attenuated by AI treatment. Transient receptor potential V1 (TRPV1) channels and associated molecules were all increased in CIP in mice dorsal root ganglion (DRG), spinal cord (SC), thalamus, and somatosensory cortex (SSC). The aforementioned molecules were mitigated in AI and Trpv1 knockout mice. Furthermore, Iba1-positive cells (microglial marker) were also potentiated and shared a similar tendency with TRPV1.Conclusion: These findings suggest that AI can alleviate chronic pain by reducing TRPV1 overexpression in both neuronal and microglial cells. Our results suggest new potential therapeutic targets for AI in chronic pain.

Abstract Chronic pain easily leads to concomitant mood disorders, and the excitability of anterior cingulate cortex (ACC) pyramidal neurons (PNs) is involved in chronic pain-related anxiety. However, the mechanism by which PNs regulate pain-related anxiety is still unknown. The GABAergic system plays an important role in modulating neuronal activity. In this paper, we aimed to study how the GABAergic system participates in regulating the excitability of ACC PNs, consequently affecting chronic inflammatory pain-related anxiety. A rat model of CFA-induced chronic inflammatory pain displayed anxiety-like behaviors, increased the excitability of ACC PNs, and reduced inhibitory presynaptic transmission; however, the number of GAD65/67 was not altered. Interestingly, intra-ACC injection of the GABAAR agonist muscimol relieved anxiety-like behaviors but had no effect on chronic inflammatory pain. Intra-ACC injection of the GABAAR antagonist picrotoxin induced anxiety-like behaviors but had no effect on pain in normal rats. Notably, chemogenetic activation of GABAergic neurons in the ACC alleviated chronic inflammatory pain and pain-induced anxiety-like behaviors, enhanced inhibitory presynaptic transmission, and reduced the excitability of ACC PNs. Chemogenetic inhibition of GABAergic neurons in the ACC led to pain-induced anxiety-like behaviors, reduced inhibitory presynaptic transmission, and enhanced the excitability of ACC PNs but had no effect on pain in normal rats. We demonstrate that the GABAergic system mediates a reduction in inhibitory presynaptic transmission in the ACC, which leads to enhanced excitability of pyramidal neurons in the ACC and is associated with chronic inflammatory pain-related anxiety.

Jian-hua He,1,* Ling Yu,2,* Zhi-yong Wang,1 Qiang Wang,3 Jun-Li Cao,4 Lian-bing Gu1 1Department of Anesthesiology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research and The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, People’s Republic of China; 2Department of Ultrasound, Affiliated Hospital of Integrate Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing,People’s Republic of China; 3Department of Anesthesiology, Nanjing Meishan Hospital, Nanjing, People’s Republic of China; 4Jiangsu Key Laboratory of Anesthesiology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lian-bing GuDepartment of Anesthesiology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research and The Affiliated Cancer Hospital of Nanjing Medical University, Baiziting 42#, Nanjing, Jiangsu 210009, People’s Republic of ChinaTel/fax +86 25 8328 4765Email lbg6519@163.comPurpose: Chronic inflammatory pain is a common condition in the clinic, and the underlying mechanism is not being completely understood. Various studies have demonstrated that central and peripheral sensitization and synaptic plasticity could play crucial functions in chronic inflammatory pain. Moreover, families of monocarboxylate transporters (MCTs) are closely related to cellular metabolism and synaptic plasticity, and it is also reported that MCTs participate in chronic inflammatory pain. Nevertheless, there is a probability of the engaging role of MCT 1 is in chronic inflammatory pain, but its specific cellular level mechanism is yet to be investigated. In our study, we hypothesized that MCT 1 in the spinal dorsal horn plays an important part in chronic inflammatory pain.Methods: In experiment A, rats were gone through nociceptive behavioral testing at 1 d day before and 1 d, 3 d, and 7 d after completing complete Freund’s adjuvant (CFA) injection. The specimens collected for detecting MCT 1 by Western blotting. In experiment B, rats were randomly divided into four groups. Intrathecal injection of MCT 1 inhibitor and nociceptive behavioral tests were performed 1 d day before and 1 d, 3 d, 7 d, 14 d, and 21 d after CFA injection. MCT 1 and p-ERK levels in spinal dorsal horn were measured by Western blotting, and GFAP in spinal dorsal horn was detected by immunofluorescence.Results: The expression of MCT 1 in the spinal dorsal horn was increased during chronic inflammatory pain in rats. The intrathecal injection of an MCT 1 inhibitor evidently diminished the expression of MCT 1 and GFAP in the spinal dorsal horn, and the behavioral nociceptive responses were also attenuated. Meanwhile, the expression of p-ERK was also decreased by the intrathecal injection of an MCT 1 inhibitor.Conclusion: Our results indicate that MCT 1 very likely play a critical role in regulating chronic inflammatory pain and may influence the regulation of synaptic plasticity via ERK in the spinal dorsal horn of rats.Keywords: chronic inflammatory pain, monocarboxylate transporter 1, spinal dorsal horn, astrocyte, synaptic plasticity

Abstract Background Liver X receptors (LXRs), including LXRα and LXRβ, are key regulators of transcriptional programs for both cholesterol homeostasis and inflammation in the brain. Here, the modes of action of LXRs and the epigenetic mechanisms regulating LXRβ expression in anterior cingulate cortex (ACC) of chronic inflammatory pain (CIP) are investigated. Methods The deficit of LXR isoform and analgesic effect of LXR activation by GW3965 were evaluated using the mouse model of CIP induced by hindpaw injection of complete Freund’s adjuvant (CFA). The mechanisms involved in GW-mediated analgesic effects were analyzed with immunohistochemical methods, ELISA, co-immunoprecipitation (Co-IP), Western blot, and electrophysiological recording. The epigenetic regulation of LXRβ expression was investigated by chromatin immunoprecipitation, quantitative real-time PCR, and sequencing. Results We revealed that CFA insult led to LXRβ reduction in ACC, which was associated with upregulated expression of histone deacetylase 5 (HDAC5), and knockdown of LXRβ by shRNA led to thermal hyperalgesia. Co-IP showed that LXRβ interacted with NF-κB p65 physically. LXRβ activation by GW3965 exerted analgesic effects by inhibiting the nuclear translocation of NF-κB, reducing the phosphorylation of mitogen-activated protein kinases (MAPKs) in ACC, and decreasing the promoted input-output and enhanced mEPSC frequency in ACC neurons after CFA exposure. In vitro experiments confirmed that HDAC5 triggered histone deacetylation on the promoter region of Lxrβ, resulting in downregulation of Lxrβ transcription. Conclusion These findings highlight an epigenetic mechanism underlying LXRβ deficits linked to CIP, and LXRβ activation may represent a potential novel target for the treatment of CIP with an alteration in inflammation responses and synaptic transmission in ACC.

Chronic inflammatory pain is a common clinical disease that tends to be associated with negative emotions such as anxiety and depression. The anterior cingulate cortex (ACC) is involved in pain and pain-related anxiety, and γ-aminobutyric acid (GABA)-ergic interneurons play an important role in chronic pain and anxiety. Electroacupuncture (EA) has good analgesic and antianxiety effect, but the underlying mechanisms have not yet been fully elucidated. In this study, we established a chronic inflammatory pain model and observed that this model induced anxiety-like behaviors and decreased the numbers of parvalbumin (PV) and somatostatin (SOM) positive cells. Activation of PV but not SOM interneurons by chemogenetic techniques alleviated anxiety-like behaviors and pain sensation. EA treatment improved pain sensation, anxiety-like behaviors and increased the number of PV- positive cells in the ACC, but did not affect on the number of SOM-positive cells in the ACC. Moreover, specific inhibition of PV interneurons by chemogenetic methods reversed the analgesic and antianxiety effects of EA. These results suggest that EA ameliorates chronic inflammatory pain and pain-related anxiety by upregulating PV but not SOM interneurons in the ACC.

Yuanyuan Wu,1 Xinmiao Yao,2 Yongliang Jiang,1 Xiaofen He,1 Xiaomei Shao,1 Junying Du,1 Zui Shen,1 Qiaoying He,1 Jianqiao Fang1 1Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, 2The Third Affiliated Hospital of Zhejiang Chinese Medical University, Zhejiang, China Abstract: Pain is considered a multidimensional conscious experience that includes a sensory component and a negative affective-motivational component. The negative affective-motivational component of pain is different from the sensory component and amplifies the pain experience. Nowadays, a significant number of preclinical research groups have focused their attention on the affective symptoms of pain. In the present study, we investigated the pain aversion and anxiety-like behavior of the complete Freund’s adjuvant (CFA)-induced chronic pain model. CFA rats experienced spontaneous pain during pain-paired conditioning (pain aversion) and spontaneous pain produces an affective response (anxiety-like behavior). Moreover, pain aversion was gradually attenuated, while the anxiety-like behavior increased in 4 weeks. Therefore, although the negative effect (including pain aversion and anxiety) is always associated with hyperalgesia, the manifestations of negative effect may follow different time courses, which may influence the progress of primary disease. The findings illustrate that targeted therapy should focus on a specific aspect in different stages of pain. Our study emphasizes the necessity of using multiple tests to study pain comorbidities. Keywords: pain, pain aversion, pain affection, anxiety-like behavior, chronic inflammatory pain, rat

Chronic pain is a pathological developing course of pain. In clinic, an objective indicator is needed for diagnosing and better controlling chronic pain. The abnormal neural responses in chronic pain are reflected by multiple event-related potentials (ERPs) in time, frequency, and location domain, respectively. However, multiple changes in ERPs are not applicable in clinic. So, the principal feature covered the most informative changes extracted from these three domains of ERP during the development of chronic pain is needed. In the present study, a parallel factor analysis method was employed to extract time–frequency–channel features of laser-evoked potential (LEP) simultaneously from rats with chronic inflammatory pain. Results showed that the main feature of LEP in channel domain locates in the frontal brain region in rats with chronic inflammatory pain while in the parietal brain region in control rats. In the frequency domain, the main frequency of LEP was significantly higher in chronic inflammatory pain rats than that in control rats. These findings indicate that the frontal region with higher frequency response to nociceptive information is the principal feature in the chronic pain state. Our study provided not only a principal feature of LEP but also a promising strategy for chronic pain, which is potential for clinic application.