TET1-mediated epigenetic regulation of tumor necrosis factor-α in trigeminal ganglia contributes to chronic temporomandibular joint pain.

Chronic temporomandibular joint (TMJ) pain profoundly affects patients' quality of life. Trigeminal tumor necrosis factor-α (TNFα) plays a pivotal role in mediating TMJ pain in mice, yet the underlying epigenetic mechanisms remain enigmatic. To unravel these epigenetic intricacies, we employed a multifaceted approach. Hydroxymethylated DNA immunoprecipitation (hMeDIP) and chromatin immunoprecipitation (ChIP) followed by qPCR were employed to investigate the demethylation of TNFα gene (Tnfa) and its regulation by ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in a chronic TMJ pain mouse model. The global levels of 5-hydroxymethylcytosine (5hmc) and percentage of 5hmc at the Tnfa promoter region were measured in the trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) following complete Freund's adjuvant (CFA) or saline treatment. TET1 knockdown and pain behavioral testing were conducted to ascertain the role of TET1-mediated epigenetic regulation of TNFα in the pathogenesis of chronic TMJ pain. Our finding revealed an increase in 5hmc at the Tnfa promoter region in both TG and Sp5C of CFA-treated mice. TET1 was upregulated in the mouse TG, and the ChIP result showed TET1 direct binding to the Tnfa promoter, with higher efficiency in the CFA-treated group. Immunofluorescence revealed the predominant expression of TET1 in trigeminal neurons. TET1 knockdown in the TG significantly reversed CFA-induced TNFα upregulation and alleviated chronic TMJ pain. In conclusion, our study implicates TET1 as a vital epigenetic regulator contributing to chronic inflammatory TMJ pain via trigeminal TNFα signaling. Targeting TET1 holds promise for epigenetic interventions in TMJ pain management.

Excitatory neurons in the lateral parabrachial nucleus mediate the interruptive effect of inflammatory pain on a sustained attention task.

BACKGROUND: Attentional deficits are among the most common pain-induced cognitive disorders. Pain disrupts attention and may excessively occupy attentional resources in pathological states, leading to daily function impairment and increased disability. However, the neural circuit mechanisms by which pain disrupts attention are incompletely understood. METHODS: We used a three-choice serial reaction time task (3CSRTT) to construct a sustained-attention task model in male C57BL/6J mice. Formalin or complete Freund's adjuvant was injected into a paw to establish an inflammatory pain model. We measured changes in 3CSRTT performance in the two inflammatory pain models, and investigated the neural circuit mechanisms of pain-induced attentional deficits. RESULTS: Acute inflammatory pain impaired 3CSRTT performance, while chronic inflammatory pain had no effect. Either inhibition of the ascending pain pathway by blockade of the conduction of nociceptive signals in the sciatic nerve using the local anesthetic lidocaine or chemogenetic inhibition of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) neurons in the lateral parabrachial nucleus (LPBN) attenuated the acute inflammatory pain-induced impairment of 3CSRTT performance, while chemogenetic activation of CaMKIIα neurons in the LPBN disrupted the 3CSRTT. Furthermore, the activity of CaMKIIα neurons in the LPBN was significantly lower on Day 2 after complete Freund's adjuvant injection than on the day of injection, which correlated with the recovery of 3CSRTT performance during chronic inflammatory pain. CONCLUSIONS: Activation of excitatory neurons in the LPBN is a mechanism by which acute inflammatory pain disrupts sustained attention. This finding has implications for the treatment of pain and its cognitive comorbidities.

BMP-induced non-canonical signaling is upregulated during autophagy-mediated regeneration in inflamed mesothelial cells.

Previously, we showed that after Freund's adjuvant-induced peritonitis, rat mesothelial cells regain their epithelial phenotype through mesenchymal-epithelial transition (MET) accompanied by autophagy. Since bone morphogenetic proteins (BMPs) are well-known MET-inducers, we were interested in the potential expression of BMPs and BMP-induced pathways. Although mesothelial cells expressed lower amounts of BMP7, its level in the peritoneal cavity and mesothelial synthesis of BMP4 were significantly increased during inflammation. BMPR1A and BMPR2 were also significantly expressed. Expression of transforming growth factor beta-activated kinase (TAK1) and c-Jun NH2-terminal kinases (JNK1-JNK2) were more intense than that of phosphorylated Mothers Against Decapentaplegic homolog 1/5 (p-SMAD1/5), confirming that the non-canonical pathway of BMPs prevailed in our model. JNK signaling through B-cell lymphoma-2 (Bcl-2) can contribute to Beclin-1 activation. We demonstrated that TAK1-JNK-Bcl-2 signaling was upregulated simultaneously with the autophagy-mediated regeneration. A further goal of our study was to prove the regenerative role of autophagy after inflammation. We used a specific inhibitor, bafilomycin A1 (BafA1), and found that BafA1 treatment decreased the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3B) and resulted in morphological signs of cell death in inflamed mesothelial cells indicating that if autophagy is arrested, regeneration turns into cell death and consequently, mesothelial cells die.

Anti-arthritic effects of geranium essential oil loaded chitosan nanoparticles in Freund's complete adjuvant induced arthritic rats through down-regulation of inflammatory cytokines.

Geranium essential oil (GEO) has been widely used in aromatherapy and traditional medicines. Nanoencapsulation, a novel technique has emerged to overcome the environmental degradation and less oral bioavailability of essential oils. This work was undertaken to encapsulate geranium essential oil in chitosan nanoparticles (GEO-CNPs) by ionic gelation technique and to explore anti-arthritic and anti-inflammatory potential in FCA-induced arthritic model in rats. The GEO was characterized by gas chromatography flame ionization detector (GCFID) and the nanosuspension was characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-rays diffraction (XRD). The Wistar albino rats (n = 32) were separated into four groups; Group 1 and 2 were considered as normal and arthritic controls. Group 3 was positive control that received oral celecoxib for 21 days while Group 4 was treated with oral GEO-CNPs after the induction of arthritis. Hind paw ankle joints diameters were weekly measured throughout the study and significant decrease (5.5 ± 0.5 mm) was observed in GEO-CNPs treatment group in comparison to arthritic group (9.17 ± 0.52 mm). Blood samples were drawn at end for evaluation of hematological, biochemical and inflammatory biomarkers. A significant upregulation of red blood cells and hemoglobin while downregulation of white blood cells, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP) and rheumatoid factor (RF) was observed. Ankles were transected for the histopathological and radiographic examination after animals were sacrificed which confirmed the alleviation of necrosis along cellular infiltration. It was concluded that GEO-CNPs were found to possess excellent therapeutic potential and promising candidates to reduce FCA-induced arthritis.

Cassia absus-mediated upregulation of IL-4, IL-10 and downregulation of IL-1ß, IL-6, TNF- α, NF-κB, IFN-γ in CFA-induced arthritis model.

Traditional use of Cassia absus as an anti-inflammatory in conjunctivitis and bronchitis is well reported. Owing to its anti-inflammatory potential, the current study appraised in vivo anti-arthritic activity of n-hexane and aqueous extracts of Cassia absus seeds (200 mg/kg) using Complete Freund's Adjuvant (CFA) rat model of arthritis. Changes in paw size (mm), joint diameter (mm), and pain response (sec) were recorded at the baseline and then after CFA induction at the interval of 4 days till the 28th day. Blood samples of anesthetized rats were collected for the estimation of hematological, oxidative, and inflammatory biomarkers. Results showed percent inhibition in paw edema (45.09% and 60.79%) with both n-hexane and aqueous extracts, respectively. Significant reduction in paw size and ankle joint diameter (P < 0.01) was seen in extracts treated rats. Erythrocyte Sedimentation rate, C-Reactive Protein, White Blood Cell levels significantly lowered, and Hemoglobin, Platelets and Red Blood Cell count significantly increased post-treatments. Superoxide Dismutase, Catalase, and Glutathione were significantly improved (P < 0.0001) in treated groups as compared to CFA induced arthritic control. Real-time polymerase chain reaction investigation showed significant downregulation (P < 0.05) of Interleukin-1ß, Tumor Necrosis Factor-α, Interleukin-6, Cycloxygenase-2, Nuclear Factor-κB, Prostaglandin E Synthase 2, Interferon Gamma and upregulation of Interleukin-4, Interleukin-10 in both n-hexane and aqueous extract-treated groups. It is thereby concluded that Cassia absus can significantly attenuate CFA-induced arthritis by modulation of oxidative and inflammatory biomarkers.

Bioactive fraction of Tragia involucrata Linn leaves attenuates inflammation in Freund's complete adjuvant-induced arthritis in Wistar albino rats via inhibiting NF-κB.

Tragia involucrata Linn. (T. involucrata) belongs to the family of Euphorbiaceae found in the subtropical regions. Traditionally, the plant parts are used to treat inflammation, wounds and skin infection by people of the Western Ghats, India. Few studies on the acute anti-inflammatory activity of T. involucrata extracts were reported earlier. The present study aims to identify the bioactive fraction of T. involucrata and to evaluate its mechanism in Complete Freund's Adjuvant-induced arthritic rat model. The leaf extract was highly effective among the methanolic leaf and root extracts. The hexane (HF) and a methanolic fraction (MF) of the leaf extract of T involucrata were further identified as a bioactive fraction evaluated through protein denaturation assay. The HF and MF were further studied for their anti-inflammatory potential in a chronic inflammatory model, and their mechanism of action was explored further. Arthritis was induced by administering 0.1 ml of CFA intradermally. The treatment was started the next day with HF (100 and 250 mg/kg/day) and MF (100 and 250 mg/kg/day), while the HF and MF alone group served as the drug control, Indomethacin-treated group served as the positive control. On the 25th day, the animals were euthanized, and their body weight, paw thickness, arthritic score, spleen and thymus weight, haematological parameters, biochemical parameters, radiographs and histopathology were analyzed. Results showed that the MF-treated animals maintained dry weight, reduced paw thickness, arthritic scores, and haematological and biological parameters compared to the HF-treated and CFA-induced arthritic rats. Both radiological and histopathological analyses of the joints revealed that the MF-treated groups restored bone architecture without any erosion and normal tissue architecture with nil signs of active inflammation. Western blot analysis revealed that MF has effectively inhibited the protein expression levels of MMP-3, MMP-9, and NF-κB in the synovial tissues compared to that of CFA-induced arthritic rats. Besides, HPLC analysis revealed the presence of flavonoids, including gallic acid, rutin and Quercetin, in the MF of T. involucrata, which had shown to have potent anti-inflammatory potential. Thus, it can be emphasized that T. involucrata could be a potential therapeutic candidate for treating inflammatory diseases, which needs further experimental studies to confirm its safety and efficacy.

Adjuvant Injections Altered the Ileal and Fecal Microbiota Differently with Changes in Immunoglobulin Isotypes and Antimycobacterial Antibody Responses.

Alterations in the gut microbiota, "dysbiosis," have been reported in autoimmune diseases, including multiple sclerosis (MS), and their animal models. Although the animal models were induced by injections of autoantigens with adjuvants, including complete Freund's adjuvant (CFA) and pertussis toxin (PT), the effects of adjuvant injections on the microbiota are largely unknown. We aimed to clarify whether adjuvant injections could affect the microbiota in the ileum and feces. Using 16S rRNA sequencing, we found decreased alpha diversities of the gut microbiota in mice injected with CFA and PT, compared with naïve mice. Overall, microbial profiles visualized by principal component analysis demonstrated dysbiosis in feces, but not in the ileum, of adjuvant-injected mice, where the genera Lachnospiraceae NK4A136 group and Alistipes contributed to dysbiosis. When we compared the relative abundances of individual bacteria, we found changes in 16 bacterial genera in feces and seven genera in the ileum of adjuvant-injected mice, in which increased serum levels of antibody against mycobacteria (a component of CFA) and total IgG2c were correlated with the genus Facklamia. On the other hand, increased IgG1 and IgA concentrations were correlated with the genus Atopostipes. Therefore, adjuvant injections alone could alter the overall microbial profiles (i.e., microbiota) and individual bacterial abundances with altered antibody responses; dysbiosis in animal models could be partly due to adjuvant injections.

Anti-Arthritic Effect of Edaravone Against Complete Freund Adjuvant Induced Arthritis via Osteoclast Differentiation and HIF-1α-VEGF-ANG-1 Axis.

Background: Bone dysfunction is a crucial problem that occurs during rheumatoid arthritis (RA) disease. Osteoclast plays a significant role in bone resorption and osteoclast differentiation and its enhancement of bone destruction. Edaravone remarkably exhibited free radical scavenging and anti-inflammatory effects. The objective of the current investigation is to comfort the inhibitory effect of Edaravone (ED) against complete Freund adjuvant (CFA) rat model via inhibition of angiogenesis and inflammation. Methods: Subcutaneous injection of CFA (1%) was used to induce arthritis; the rats were divided into different groups and received the oral administration of ED. Paw edema, body weight, and arthritis score were regularly estimated. Biochemical parameters were estimated, respectively. We also estimate the level of hypoxia-inducible factor-1α (HIF-1α), angiopoietin 1 (ANG-1), and vascular endothelial growth factor (VEGF). We also checked into how ED affected the differentiation of osteoclasts utilising a co-culture system with monocytes and synovial fibroblasts in arthritis rats. Results: ED treatment significantly (P<0.001) suppressed the arthritis score and paw edema and improved the body weight. ED treatment significantly (P<0.001) altered the antioxidant parameters and pro-inflammatory cytokines: inflammatory mediator nuclear kappa B factor (NF-κB), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2), respectively. Furthermore, ED treatment significantly (P<0.001) suppressed the level of ANG-1, HIF-1α, and VEGF, respectively. The results suggest that ED suppressed osteoclast differentiation and also decreased the level of cytokines and osteopontin (OPN), receptor activator for nuclear factor-κ B Ligand (RANKL) and macrophage colony stimulating factor (M-CSF) in the co-culture supernatant of monocytes and synovial fibroblasts. Conclusion: Edaravone could mitigate CFA via inhibiting angiogenesis and inflammatory reactions, which may be linked with the HIF-1α-VEGF-ANG-1 axis and also enhance the bone destruction of murine arthritis via suppression of osteoclast differentiation and inflammatory reaction.

Cryoneurolysis with Injectable Ice Slurry Modulates Mechanical Skin Pain.

Cutaneous pain is a common symptom of skin disease, and available therapies are inadequate. We developed a neural selective and injectable method of cryoneurolysis with ice slurry, which leads to a long-lasting decrease in mechanical pain. The aim of this study is to determine whether slurry injection reduces cutaneous pain without inducing the side effects associated with conventional cryoneurolysis. Using the rat sciatic nerve, we examined the effects of slurry on nerve structure and function in comparison with the effects of a Food and Drug Administration‒approved cryoneurolysis device (Iovera). Coherent anti-Stokes Raman scattering microscopy and immunofluorescence staining were used to investigate histological effects on the sciatic nerve and on downstream cutaneous nerve fibers. Complete Freund's Adjuvant model of cutaneous pain was used to study the effect of the slurry on reducing pain. Structural changes in myelin induced by slurry were comparable with those induced by Iovera, which uses much colder temperatures. Compared with that of Iovera, the decrease in mechanical pain due to slurry was less profound but lasted longer without signs of dysesthesia. Slurry did not cause a reduction of epidermal nerve fibers or a change in thermal pain sensitivity. Slurry-treated rats showed reduced cutaneous mechanical pain in response to Complete Freund's Adjuvant. Slurry injection can be used to successfully reduce cutaneous pain without causing dysesthesia.

[Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome].

OBJECTIVE: To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. METHODS: Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups (n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1ß, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB). RESULTS: Compared with the normal group, the circumference of the knee joint was increased (P<0.01), the skin temperature was decreased (P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1ß, IL-6, and CRP were increased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased (P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased (P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased (P<0.01), whlie the skin temperature was increased (P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1ß, IL-6 and CRP were decreased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased (P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased (P<0.01) in the heat-reinforcing needling and agonist groups. CONCLUSION: Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1ß, IL-6 and CRP.

Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway.

HMGB1 is a highly conserved nuclear protein that is rapidly released into the extracellular environment during infection or tissue damage. In osteoarthritis, HMGB1 acts as a pro-inflammatory cytokine inducing a positive feedback loop for synovial inflammation and cartilage degradation. The aim of this study was to explore the role of HMGB1 in inflammation and catabolism of temporomandibular joint osteoarthritis (TMJOA) and whether inhibition of HMGB1 affects TMJOA. Human synovial fibroblasts were incubated with HMGB1, the expression of pro-inflammatory cytokines and catabolic mediators were measured by Western blot and ELISA. NF-κB signaling pathway involvement was studied by the NF-κB inhibitor and detected by Western blotting and immunofluorescence staining. TMJOA was induced by an injection of Complete Freund's adjuvant (CFA) into anterosuperior compartment of rat's joint. An anti-HMGB1 antibody was used to assess the effect to HMGB1 in the synovium and cartilage of the CFA-induced TMJOA rats by H&E, Safranin O, Masson trichrome staining, immunohistochemistry and immunofluorescence. HMGB1 markedly increased the production of MMP13, ADAMTS5, IL-1ß and IL-6 through activating NF-κB signaling pathway in human synovial fibroblasts. In vivo, application of the HMGB1 neutralizing antibody effectively ameliorated the detrimental extent of TMJOA. Furthermore, the HMGB1 neutralizing antibody reduced the expression of NF-κB, pro-inflammatory cytokines and catabolic mediators in the synovium and cartilage of CFA-induced TMJOA rats. HMGB1 inhibition alleviates TMJOA by reducing synovial inflammation and cartilage catabolism possibly through suppressing the NF-κB signaling pathway and may become a therapeutic method against TMJOA.

Jobelyn® extends the life span and improves motor function in Drosophila melanogaster exposed to lipopolysaccharide via augmentation of antioxidant status.

Jobelyn® (JB), a dietary supplement, derived from polyphenol-rich leaf sheath of Sorghum bicolor, has been reported to attenuate sensorimotor deficits and oxidative stress evoked by complete Freund-adjuvant in mice. This present study evaluated its effects on the life span, motor function and changes in oxidative stress parameters as well as acetylcholinesterase activity in Drosophila melanogaster exposed to lipopolysaccharide (LPS). The flies (50 per vial), in 5 replicates were fed with LPS (250 µg/kg diet) alone or in combination with JB (0.25-1.0 mg/kg diet) daily for 7 days. The mortality rate and motor function were evaluated on day 7. The flies were afterwards processed for determination of oxidative stress parameters and acetylcholinesterase activity. The effects of JB (0.25-1.0 mg/g diet) on the longevity of Drosophila was also investigated wherein the flies were monitored daily for mortality throughout their lifespan. The flies exposed to LPS (250 µg/kg diet) had reduced life span and elevated oxidative stress when compared with control. However, JB (0.25 and 1.0 mg/kg diet) improved the motor function and also reduced the mortality rate of the flies exposed to LPS. It also restored the cellular antioxidant status and reduced acetylcholinesterase activity, accumulation of hydrogen peroxide as well as nitric oxide in Drosophila fed with LPS. JB also extended the longevity of the flies relative to control. The findings that JB improves motor function and extended the lifespan of Drosophila flies by boosting the antioxidant status and cholinergic function, suggest it might be helpful in delaying the onset of neuropsychiatric illnesses associated with the aging processes.

Co-treatment of Nimbolide augmented the anti-arthritic effects of methotrexate while protecting against organ toxicities.

Prolonged exposure to the pharmacological doses of disease-modifying anti-rheumatic drugs (DMARDs) often results in major organ toxicities resulting in poor patient compliance. Methotrexate (MTX) is one of the commonly prescribed DMARDs for the treatment of arthritis, which results in vital organ dysfunction. To retain the anti-arthritic activity of MTX with the reduction in toxicities, combination therapies are warranted. Nimbolide (NMB) is a potent anticancer, anti-inflammatory and anti-fibrotic agent whose potential has been demonstrated in various pre-clinical models. Monoarthritis was developed with Complete Freund's Adjuvant in the knees of Wistar rats and treatment was given with either NMB (3 mg/kg/day) or MTX (2 mg/kg/week) alone or combination therapy (NMB + MTX). The anti-arthritic effects were evaluated by arthritic scoring, radiological imaging, synovial tissue proteins analysis, and histopathological staining. While hepato-renal toxicity was assessed in serum by evaluating the kidney and liver functional parameters, in tissues by oxidative-nitrosative stress markers, and pro-inflammatory cytokines levels. Histopathological analysis was performed to study the extent of tissue damage. Molecular studies like immunoblotting and immunohistochemistry were performed to understand the effect of combination therapy. We thereby report that monotherapy with either NMB or MTX exhibited significant anti-arthritic effects, while combination therapy resulted in augmented anti-arthritic effects with significant reduction in hepato-renal toxicity produced by MTX probably through anti-inflammatory and anti-oxidant effects. Therefore, our proposed combination of NMB and MTX may serve as a potential strategy for the effective management of arthritis.

Complete Freund's adjuvant-induced protein dysregulation correlated with mirror image pain as assessed by quantitative proteomics of the mouse spinal cord.

Inflammation or trauma occurring on one side of the body can cause pathological pain on the contralateral noninjured side in a phenomenon called mirror-image pain (MIP). Although some potential mechanisms involved in MIP have been reported, including those involving the immune system and glial cells as well as neural mechanisms, the molecular mechanisms are not well understood. In this study, we aimed to understand the molecular mechanisms in MIP using quantitative proteomics and whole-cell patch clamp recordings. Behavioral test results showed that complete Freund's adjuvant could induce MIP in the mice. The results of isobaric tags for relative and absolute quantification (iTRAQ) quantitative proteomics showed that 108 proteins were dysregulated, and these proteins may represent potential targets. Furthermore, bioinformatics analysis was applied to explore the potential molecular mechanisms during MIP after complete Freund's adjuvant (CFA) treatment. Parallel reaction monitoring (PRM) results showed that PKCδ and seven other dysregulated proteins were related to MIP after CFA treatment. Patch clamp recording results showed that CFA treatment could increase intrinsic excitability and spontaneous firing in spinal cord neurons during MIP. In summary, we found that CFA could induce MIP. The results of proteomic research on the spinal cord after CFA treatment could provide new insight into the molecular mechanisms of MIP. Moreover, the neuronal activity of spinal cord neurons was upregulated during MIP after CFA treatment. In summary, the results of the spinal cord proteomic profile provide a potential molecular mechanism for understanding MIP.

Carbamylated erythropoietin improves recognition memory by modulating microglia in a rat model of pain.

Patients with chronic pain often complain about memory impairments. Experimental studies have shown neuroprotective effects of Carbamylated erythropoietin (Cepo-Fc) in the treatment of cognitive dysfunctions. However, little is currently known about its precise molecular mechanisms in a model of inflammatory pain. Therefore, this study aimed to investigate neuroprotective effects of Cepo-Fc against cognitive impairment induced by the inflammatory model of Complete Freund's Adjuvant (CFA). Carbamylated erythropoietin was administrated Intraperitoneally (i.p) on the day CFA injection, continued for a 21-days period. After conducting the behavioral tests (thermal hyperalgesia and novel object recognition test), western blot and ELISA were further preformed on days 0, 7, and 21. The results of this study indicate that Cepo-Fc can effectively reverse the CFA induced thermal hyperalgesia and recognition memory impairment. Additionally, Cepo-Fc noticeably decreased the hippocampal microglial expression, production of hippocampal IL-1ß, and hippocampal apoptosis and necroptosis induced by the inflammatory pain. Therefore, our findings suggest that neuroprotective effects of Cepo-Fc in the treatment of pain related recognition memory impairment may be mediated through reducing hippocampal microglial expression as well as IL-1ß production.

Appraisal of Anti-Arthritic and Anti-Inflammatory Potential of Folkloric Medicinal Plant Peganum harmala.

BACKGROUND & OBJECTIVE: Peganum harmala has been traditionally used to manage rheumatoid arthritis (RA) and other inflammatory conditions. However, its use against RA has not been scientifically evaluated. The current study was designed to assess the anti-arthritic and anti-inflammatory activities of the methanolic extract of P. harmala leaves by in vitro and in vivo methods. METHODS: The in vitro assays were carried out to determine the effect of plant extract on inhibition of egg albumin denaturation and human red blood cell membrane (HRBC) stabilization. Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity was performed to determine the antioxidant potential. In vivo anti-arthritic activity was performed by determining the curative effect against Complete Freund's adjuvant (0.1 ml). The plant extract was administered to rats orally at 200, 400 and 600 mg/kg/day for 21 days. RESULTS: The values of IC50 of plant extract in protein denaturation, stabilization of HRBC and DPPH assays were 77.54 mg/ml, 23.90 mg/ml and 58.09 µg/ml, respectively. Moreover, the plant extract significantly attenuated the poly-arthritis and weight loss, anemia and paw edema. The plant extract restored the level of C-reactive protein, rheumatoid factor, alanine transaminase, aspartate transaminase and alkaline phosphatase in poly-arthritic rats. Moreover, the plant extract restored the immune organs' weight in treated rats. Treatment with P. harmala also significantly subdued the oxidative stress by reinstating superoxide dismutase, reduced glutathione, catalase and malondialdehyde in poly-arthritic rats. The plant extract notably restored the prostaglandin-E2 and tumor necrosis factor (TNF)-α in the serum of poly-arthritic rats. CONCLUSION: It was concluded that P. harmala extract had potential antioxidant, anti-inflammatory and antiarthritic activities, which primarily might be attributed to alkaloids, flavonoids and phenols.

Referral and adjuvant treatment patterns after nephrectomy in high-risk locoregional renal cell carcinoma.

BACKGROUND: It is unclear whether patients with renal cell carcinoma (RCC) are routinely assessed for recurrence risk post-nephrectomy and whether patients at high recurrence risk are seen by providers who can evaluate candidacy for adjuvant systemic therapy (AST) and clinical trials. MATERIALS AND METHODS: We identified all patients with locoregional RCC who underwent nephrectomy via an institutional database within Duke University Health System between 1 April 2015 and 31 December 2019. Medical records were reviewed to identify patient characteristics, post-nephrectomy referrals, treatment, and follow-up. Patients with tumor stage ≥3 and grade ≥2, regional lymph node metastasis, or both, were classified as high recurrence risk. RESULTS: Of 618 patients with locoregional RCC who underwent nephrectomy, 136 (22%) had high recurrence risk. Of those, 25 patients with high-risk disease (18%) were referred to medical oncology for discussion of AST; 23 (92%) of these referrals took place in 2018-2019. One patient received adjuvant sunitinib and two patients participated in adjuvant immunotherapy trials. The decision not to receive AST was primarily made by the oncologist in 10 (46%), the patient in 8 (36%), and unrecorded in 4 (18%) of 22 cases, for multiple reasons. Individual surgeons referred high-risk patients for discussion of AST with varying frequency, ranging from 0% to 100% in 2019. CONCLUSIONS: Despite increasing number of patients with locoregional RCC at high recurrence risk referred to medical oncologists after nephrectomy, few patients received AST, including participation in clinical trials. With increasing AST options and ongoing clinical trials in this space, these findings highlight the need for continued efforts at identifying effective AST and referring patients most likely to benefit to medical oncologists. ClinicalTrials.gov, NCT04309617.

NLRP2 Is Overexpressed in Spinal Astrocytes at the Peak of Mechanical Pain Sensitivity during Complete Freund Adjuvant-Induced Persistent Pain.

Our earlier findings revealed that interleukin-1 receptor type-1 (IL-1R1) was overexpressed in spinal neurons, and IL-1R1-deficient mice showed significant attenuation of thermal and mechanical allodynia during the course of the Complete Freund adjuvant (CFA)-induced persistent pain model. In the present study, we found that a ligand of IL-1R1, termed interleukin-1ß (IL-1ß), is also significantly overexpressed at the peak of mechanical pain sensitivity in the CFA-evoked pain model. Analysis of cellular distribution and modeling using IMARIS software showed that in the lumbar spinal dorsal horn, IL-1ß is significantly elevated by astrocytic expression. Maturation of IL-1ß to its active form is facilitated by the formation of the multiprotein complex called inflammasome; thus, we tested the expression of NOD-like receptor proteins (NLRPs) in astrocytes. At the peak of mechanical allodynia, we found expression of the NLRP2 inflammasome sensor and its significantly elevated co-localization with the GFAP astrocytic marker, while NLRP3 was moderately present and NLRP1 showed total segregation from the astrocytic profiles. Our results indicate that peripheral CFA injection induces NLRP2 inflammasome and IL-1ß expression in spinal astrocytes. The release of mature IL-1ß can contribute to the maintenance of persistent pain by acting on its neuronally expressed receptor, which can lead to altered neuronal excitability.

Neuromedin B receptor stimulation of Cav3.2 T-type Ca2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity.

Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.

Evaluation of acute and chronic nociception in subchronically administered MK-801-induced rat model of schizophrenia.

Patients diagnosed with schizophrenia have been reported to exhibit atypically low pain sensitivity and to vary in their experience of chronic pain. To the best of our knowledge, there has yet to be an animal study that provides information concerning the relationship between models of schizophrenia and pain. In the present study, we investigated several distinct nociceptive behaviors in a translational rat model of schizophrenia (0. 5 mg/kg MK-801, twice a day for 7 days followed by a 7-day washout period). The presence of the expected cognitive deficit was confirmed with novel object recognition (NOR) paradigm prior to nociception testing. MK-801-treated rats with lack of novelty interest in NOR testing showed: hyposensitivity to thermal and mechanical stimuli; short-term hypoalgesia followed by augmented hyperalgesia in response to formalin-induced spontaneous nociception and increased thermal and mechanical hyperalgesia in the complete Freund's adjuvant (CFA) induced chronic pain model. In conclusion, MK-801 induced antinociception effects for thermal stimuli in rats that were consistent with the decreased pain sensitivity observed in schizophrenia patients. Additionally, the amplified biphasic response exhibited by the MK-801 group in the formalin-induced spontaneous nociception test affirms the suitability of the test as a model of acute to delayed pain transition.