Freund's Adjuvant Research Articles

S1P/S1PR1 signaling is involved in the development of nociceptive pain.

Pain is a complex perception involving unpleasant somatosensory and emotional experiences. However, the underlying mechanisms that mediate its different components remain unclear. Sphingosine-1-phosphate (S1P), a metabolite of sphingomyelin and a potent lipid mediator, initiates signaling via G protein-coupled receptors (S1PRs) on cell surfaces. It serves as a second messenger in cellular processes such as proliferation and apoptosis. Nevertheless, the neuropharmacology of sphingolipid signaling in pain conditions within the central nervous system remains largely unexplored and controversial. Chronic nociceptive pain models were induced in vivo by intraplantar injection of 20μL complete Freund's adjuvant (CFA) into the left hind paws. We assessed S1P and S1PR1 expression in the spinal cords of CFA model mice. Functional antagonists of S1PR1 or S1PR1-specific siRNA were administered daily following CFA model establishment. Paw withdrawal response frequency (PWF) and paw withdrawal latency (PWL) were measured to evaluate mechanical allodynia and thermal hyperalgesia, respectively. RT-PCR assessed interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α levels. Western blotting and immunofluorescence were used to analyze glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule (Iba1), STAT3, ERK, and p38 MAPK protein expression. In the chronic nociceptive pain model induced by CFA, S1P and S1PR1 expression levels were significantly elevated, leading to activation of spinal cord glial cells. S1PR1 activation also promoted MMP2-mediated cleavage of mature IL-1β. Additionally, S1PR1 activation upregulated phosphorylation of STAT3, ERK, and p38 MAPK in glial cells, profoundly impacting downstream signaling pathways and contributing to chronic nociceptive pain. The S1P/S1PR1 axis plays a pivotal role in the cellular and molecular mechanisms underlying nociceptive pain. This signaling pathway modulates glial cell activation and the expression of pain-related genes (STAT3, ERK, p38 MAPK) and inflammatory factors in the spinal dorsal horn. These findings underscore the potential of targeting the S1P system for developing novel analgesic therapies.

N-methyl-D-aspartate Receptor Subunits 2A and 2B Mediate Connexins and Pannexins in the Trigeminal Ganglion Involved in Orofacial Inflammatory Allodynia during Temporomandibular Joint Inflammation.

Temporomandibular joint osteoarthritis (TMJOA) is a severe form of temporomandibular joint disorders (TMD), and orofacial inflammatory allodynia is one of its common symptoms which lacks effective treatment. N-methyl-D-aspartate receptor (NMDAR), particularly its subtypes GluN2A and GluN2B, along with gap junctions (GJs), are key players in the mediation of inflammatory pain. However, the precise regulatory mechanisms of GluN2A, GluN2B, and GJs in orofacial inflammatory allodynia during TMJ inflammation still remain unclear. Here, we established the TMJ inflammation model by injecting Complete Freund's adjuvant (CFA) into the TMJ and used Cre/loxp site-specific recombination system to conditionally knock out (CKO) GluN2A and GluN2B in the trigeminal ganglion (TG). Von-frey test results indicated that CFA-induced mechanical allodynia in the TMJ region was relieved in GluN2A and GluN2B deficient mice. In vivo, CFA significantly up-regulated the expression of GluN2A and GluN2B, Gjb1, Gjb2, Gjc2 and Panx3 in the TG, and GluN2A and GluN2B CKO played different roles in mediating the expression of Gjb1, Gjb2, Gjc2 and Panx3. In vitro, NMDA up-regulated the expression of Gjb1, Gjb2, Gjc2 and Panx3 in satellite glial cells (SGCs) as well as promoted the intercellular communication between SGCs, and GluN2A and GluN2B knocking down (KD) altered the expression and function differently. NMDAR regulated Gjb1 and Panx3 through ERK1/2 pathway, and mediated Gjb2 and Gjc2 through MAPK, PKA, and PKC intracellular signaling pathways. These findings shed light on the distinct functions of GluN2A and GluN2B in mediating peripheral sensitization induced by TMJ inflammation in the TG, offering potential therapeutic targets for managing orofacial inflammatory allodynia.

Comparison of disease-modifying anti-rheumatic drugs and hyperbaric oxygen therapy in the experimental model of rheumatoid arthritis in rats.

In this study, we wanted to investigate the effectiveness of combining disease-modifying anti-rheumatic drugs (DMARD) with hyperbaric oxygen therapy (HBOT) in reducing inflammation in a rheumatoid arthritis (RA) model using rats. We divided 56 male Sprague-Dawley rats into seven groups and induced RA using complete Freund's adjuvant. Some groups received HBOT, whereas others were given etanercept or leflunomide. We started the treatment on the 10th day after inducing RA and continued it for 18 days. To evaluate the effectiveness of the treatments, we measured paw swelling and used X-rays to examine the joints before and after the treatment. We also analysed the levels of two inflammatory markers, tumour necrosis factor (TNF)-α and interleukin (IL)-1β, using an enzyme-linked immunosorbent assay. Additionally, we conducted histological analysis and assessed the expressions of anti-IL-1β and anti-TNF-α antibodies. All the treatment groups showed a significant decrease in arthritis scores, paw swelling and levels of TNF-α and IL-1β. The X-ray images revealed improvements in joint structure, and the histopathological analysis showed reduced inflammation and collagen abnormalities. Combining DMARD with HBOT had similar effects to individual therapies, suggesting a cost-effective and potentially safer approach for improving outcomes in rats with RA.

Hormone replacement therapy did not alleviate temporomandibular joint inflammatory pain in ovariectomized rats.

This study had the aim of examining the relationships between variations in estrogen levels resulting from ovariectomy, and estrogen hormone replacement therapy (HRT) in rats subjected to an orofacial inflammatory pain model. Eighty adult female Wistar rats were initially divided into 2 groups: Sham or ovariectomy (OVX-D1). Seven days later (D7), the rats were subjected to an unilateral infiltration of Freund's Complete Adjuvant (CFA) or saline solution into the right temporomandibular joint (TMJ). Then, rats received 17β-estradiol (28µg/kg/day) or placebo for 21days (D10-D31). Nociception was evaluated by the von Frey (VF) and the Hot Plate (HP) tests, and depressive-like behavior by the Forced Swimming (FS) test. On D32 all rats were euthanized and serum, hippocampus and brainstem were collected. The CFA groups presented a mechanical hyperalgesia until day 21 (p ≤ 0.05). No differences were observed among groups in the HP (p = 0.735), and in the immobility and swimming time of the FS (p = 0.800; p = 0.998, respectively). In the brainstem, there was a significant difference in the TNF-ɑ levels (p = 0.043), and a marginal significant difference in BDNF levels (p = 0.054), without differences among groups in the hippocampal BDNF and TNF-ɑ levels (p = 0.232; p = 0.081, respectively). In conclusion, the hormone replacement therapy did not alleviate orofacial pain in ovariectomized rats. However, there is a decrease in brainstem TNF-ɑ levels in the animals submitted to both models, which was partially reverted by HRT.

Polyphenol-rich Sorghum bicolor supplement exhibits anti-nociceptive activity and protective effects against pathological changes associated with complete Freund's adjuvant induced arthritis in rodents

IntroductionArthritis is a common inflammatory joint disease; causing severe pain, anxiety, and depression. Sorghum bicolor L. Moench (Pocaeae) is used for treating cough, asthma, and arthritic pain in Traditional Chinese Medicine (TCM). This study evaluates the anti-nociceptive and anti-arthritic effects of the polyphenol-rich Sorghum bicolor supplement (SBS) in rodents. MethodsThe anti-nociceptive activity of SBS was assessed using hot plate, acetic acid mouse writhing, and formalin-induced paw licking tests in mice. In the arthritis study, rats received SBS (50, 100, and 200 mg/kg p.o) for 28 days. Thirty minutes after treatment on days, 1 and 20, rats received intra-articular injection of Complete Freund's adjuvant (CFA) for arthritis induction. Arthritic scores and paw edema were determined before assessing motor function, anxiety, depression, hyperalgesia and allodynia on day 28. Lipid profiles were determined, and ankle and brain tissues were analyzed for oxidative stress and inflammatory biomarkers, alongside histopathological studies. ResultsSBS reduced nociceptive responses induced by noxious heat, acetic acid and formalin in mice. In CFA-injected rats, SBS decreased paw volume and arthritic scores, and increased pain thresholds to thermal, cold, and mechanical stimuli. The SBS reduced motor activity, anxiety and depression, and improved serum lipid profiles in arthritic rats. It further modulated oxidative stress, pro-inflammatory cytokines, nuclear factor kappa (NF-kB), apoptotic markers, nuclear factor erythroid 2-related factor 2 (NRF2), glutamic acid decarboxylase (GAD) and brain-derived neurotrophic factor (BDNF) in CFA-treated rats. SBS produced histopathological improvements in joint tissues of arthritic rats. DiscussionThis study revealed that SBS exhibited anti-nociceptive activity in mice and reduces inflammation, hyperalgesia, anxiety and depression in CFA-arthritic rats. The ability of SBS to modulate key biomarkers such as pro-inflammatory cytokines, NF-κB, caspases, Nrf2, BDNF, and GAD highlights its multifaceted approach in addressing both the physical and psychological aspects of arthritis, providing a comprehensive therapeutic strategy, justifying its use in TCM for this debilitating condition.

Immunostimulatory effects of Toll-like receptor ligands as adjuvants in establishing a novel mouse model for pemphigus vulgaris.

The meticulous selection of appropriate vaccine adjuvants is crucial for optimizing immune responses. Traditionally, pemphigus vulgaris (PV), an autoimmune disorder, has been modelled using complete Freund's adjuvant (CFA). In this study, we aimed to discern potential variations in immune responses elicited by Toll-like receptor (TLR) ligands as compared to CFA. A comprehensive investigation was conducted, comparing the effects of these adjuvants in conjunction with ovalbumin or desmoglein-3. Flow cytometry was employed to analyse distinct cell subsets, while enzyme-linked immunosorbent assay quantified antigen-specific antibodies and cytokine levels. Histological examination of harvested skin tissues and transcriptome analysis of skin lesions were performed to identify differentially expressed genes. TLR ligands demonstrated efficacy in inducing PV-like symptoms in wild-type mice, in contrast to CFA. This underscored the substantial impact of the adjuvant on self-antigen tolerance. Furthermore, we proposed an enhanced method for establishing a PV model through adoptive transfer, substituting CFA with TLR ligands. Our results revealed that in contrast to the perception that CFA being the most potent immunopotentiator reported, CFA promoted regulatory T cells (Treg), follicular regulatory T cells and IL-10-producing neutrophils, whereas TLR ligands downregulated CCL17 and IL-10. This suggested potential implications for the recruitment and activation of Treg subsets. While B cell and CD8+ T cell responses exhibited similarity, CFA induced less activation in dendritic cell subsets. A novel mouse model of PV and systemic comparison of immunostimulatory effects of adjuvants were provided by this study. The systematic comparison of CFA and TLR ligands shed light on the distinctive properties of these adjuvants, presenting innovative mouse models for the investigation of pemphigus. This study significantly contributes to adjuvant research and advances our understanding of PV pathogenesis. Immunization with desmoglein 3 and Toll-like receptor (TLR) ligands effectively induces pemphigus symptoms in wild-type mice, whereas complete Freund's adjuvant (CFA) fails. TLR ligands heightened the autoreactivity of donor cells in the adoptive transfer pemphigus model. CFA promoted regulatory T cells and IL-10-producing neutrophils, whereas TLR ligands downregulated CCL17 and IL-10, leading to more effective immune responses.

PV and SST neurons in the anterior cingulate cortex regulate social disorders in adulthood induced by sensory abnormalities in childhood.

Childhood sensory abnormalities experience has a crucial influence on the structure and function of the adult brain. The underlying mechanism of neurological function induced by childhood sensory abnormalities experience is still unclear. Our study was to investigate whether the GABAergic neurons in the anterior cingulate cortex (ACC) regulate social disorders caused by childhood sensory abnormalities experience. We used two mouse models, complete Freund's adjuvant (CFA) injection mice and bilateral whisker trimming (BWT) mice in childhood. We applied immunofluorescence, chemogenetic and optogenetic to study the mechanism of parvalbumin (PV) neurons and somatostatin (SST) neurons in ACC in regulating social disorders induced by sensory abnormalities in childhood. Inflammatory pain in childhood leads to social preference disorders, while BWT in childhood leads to social novelty disorders in adult mice. Inflammatory pain and BWT in childhood caused an increase in the number of PV and SST neurons, respectively, in adult mice ACC. Inhibiting PV neurons in ACC improved social preference disorders in adult mice that experienced inflammatory pain during childhood. Inhibiting SST neurons in ACC improved social novelty disorders in adult mice that experienced BWT in childhood. Our study reveals that PV and SST neurons of the ACC may play a critical role in regulating social disorders induced by sensory abnormalities in childhood.

Evaluation of the possible effect of Ursodeoxycholic Acid to treat rheumatoid arthritis induced in the Rat model in comparison with Diclofenac.

Background: It is well known that ursodeoxycholic acid (UDCA) has notable impacts on the liver, However, tiny amount of knowledge available on how it affects autoimmune illness. This research was done to see an advantage that might exist of using UDCA in Rheumatoid arthritis (RA). Methods: In order to accomplish this objective, adult either sex rats were divided into 4 groups: Normal control (sodium bicarbonate 5% p.o. for 21 days),CFA (0.1 ml CFA footpad intradermal injection),FCA+ Diclofenac (5 mg/kg p.o for 21 days),FCA+ UDCA (100 mg/kg p.o. for 21 days). Results: UDCA (100 mg/kg p.o.) show significantly a rise in Body Mass also significantly reduce in Arthritic Oedema score in comparison with induction group . RBC, WBC, and ESR were Hematological UDCA therapy of animals Evaluation findings RBC increased noticeably and WBC ,ESR levels drop noticeably. a considerable drop in TNFα and RF biochemical parameters evaluated in UDCA treatment group. Keywords: rheumatoid arthritis (RA); ursodeoxycholic acid (UDCA) and Complete Freund adjuvant (CFA)

Development of dexibuprofen loaded nano transfersomal gel with enhanced biopharmaceutical performance in complete Freund's adjuvant induced arthritis model

The purpose of this study was to enhance the biopharmaceutical performance of Dexibuprofen (DXI) using transfersomal gel (DXI-TG) as drug delivery system. DXI loaded transfersomes (DXI-T) were prepared via thin film technique, characterized for particle size, polydispersity index, zeta potential, particle morphology and entrapment efficiency. The DXI-T was further incorporated into 2 % (w/v) chitosan gel to get the final dosage form DXI-TG, which was characterized for homogeneity, pH, spread-ability and rheological properties. In vitro release, ex vivo permeation and in vivo studies were also conducted along with stability study of the optimized formulation. DXI-T showed excellent vesicle properties including, size distribution (235.1 ± 4.66 nm), poly dispersity index (0.184 ± 0.001), zeta potential (−10.5 ± 0.23 mV) and entrapment efficiency (82.9 ± 1.07 %). TEM analysis exhibited spherical morphology and uniformly distributed nanoparticles, whereas FTIR showed no bond anomalies. DXI-TG showed suitable gel properties with a viscosity of 11310 ± 20 cP, pH 5.2 ± 0.3, good homogeneity, non-Newtonian flow behavior, good spread-ability (298 ± 7.63 %) and high drug content (97.01 ± 3.1 %). in vitro release studies demonstrated that DXI-TG has significantly controlled the release of DXI when compared with DXI-T, DXI-G and DXI-Suspension at pH 5.5 and 7.4. Similarly, ex vivo permeation analysis showed meaningfully increased permeation of DXI-T followed by DXI-TG when compared with test formulations. Like normal control group, skin irritation studies showed no erythema and edema in DXI-TG treated group. Additionally, DXI-TG demonstrated a significantly enhanced bioavailability (5-fold) when compared with DXI-Suspension and DXI-G. Furthermore, DXI-TG showed significantly enhanced therapeutic effect against acute arthritis model when compared with DXI-Suspension and DXI-G. Besides, no signs of evident toxicity on major body organs was observed in DXI-TG treated animal group. Also, the DXI-TG was found stable for a period of 6 months. It can be concluded that transfersomal gel may be a suitable carrier for DXI with enhanced bioavailability, improved therapeutic efficacy and no evident toxicity as exhibited in this study.

Bioinformatics design of a peptide vaccine containing sarcoma antigen NY-SAR-35 epitopes against breast cancer and evaluation of its immunological function in BALB/c mouse model.

The development of a cancer vaccine has become an essential focus in the field of medical biotechnology and immunology. In our study, the NY-SAR-35 cancer/testis antigen was targeted to design a novel peptide vaccine using bioinformatics tools, and BALB/c mice were used to evaluate the vaccine's immunological function. This evaluation involved assessing peptide-specific IgG levels in the serum via ELISA and measuring the levels of IFN-γ, IL-4, and granzyme B in the supernatant of cultured splenocytes. The final vaccine construct consisted of two T lymphocyte epitopes linked by the AAY linker. This construct displayed high antigenicity, non-allergenicity, non-toxicity, stability, and ability to induce IFN-γ and IL-4. It showed stable dynamics with both human MHC-I and II molecules, as well as mouse MHC-II molecules, and revealed strong Van der Waals and electrostatic energies. Emulsifying our peptide vaccine in incomplete Freund's adjuvant resulted in a remarkable increase in the levels of IgG. The splenocytes of mice that received the combination of peptide and adjuvant displayed a noteworthy increase in IFN-γ, IL-4, and granzyme B secretion. Additionally, their lymphocytes exhibited higher proliferation rates compared to the control group. Our data demonstrated that our vaccine could stimulate a robust immune response, making it a promising candidate for cancer prevention. However, clinical trials are necessary to assess its efficacy in humans.

Cellular Therapy in Experimental Autoimmune Encephalomyelitis as an Adjuvant Treatment to Translate for Multiple Sclerosis.

This study aims to evaluate and compare cellular therapy with human Wharton's jelly (WJ) mesenchymal stem cells (MSCs) and neural precursors (NPs) in experimental autoimmune encephalomyelitis (EAE), a preclinical model of Multiple Sclerosis. MSCs were isolated from WJ by an explant technique, differentiated to NPs, and characterized by cytometry and immunocytochemistry analysis after ethical approval. Forty-eight rats were EAE-induced by myelin basic protein and Freund's complete adjuvant. Forty-eight hours later, the animals received intraperitoneal injections of 250 ng/dose of Bordetella pertussis toxin. Fourteen days later, the animals were divided into the following groups: a. non-induced, induced: b. Sham, c. WJ-MSCs, d. NPs, and e. WJ-MSCs plus NPs. 1 × 105. Moreover, the cells were placed in a 10 µL solution and injected via a stereotaxic intracerebral ventricular injection. After ten days, the histopathological analysis for H&E, Luxol, interleukins, and CD4/CD8 was carried out. Statistical analyses demonstrated a higher frequency of clinical manifestation in the Sham group (15.66%) than in the other groups; less demyelination was seen in the treated groups than the Sham group (WJ-MSCs, p = 0.016; NPs, p = 0.010; WJ-MSCs + NPs, p = 0.000), and a lower cellular death rate was seen in the treated groups compared with the Sham group. A CD4/CD8 ratio of <1 showed no association with microglial activation (p = 0.366), astrocytes (p = 0.247), and cell death (p = 0.577) in WJ-MSCs. WJ-MSCs and NPs were immunomodulatory and neuroprotective in cellular therapy, which would be translated as an adjunct in demyelinating diseases.

Novel antiarthritic mechanisms of Azelaic acid against CFA-induced arthritis in rats by modulating pro- and anti-inflammatory cytokines network.

An immunologic system attacking the body's own tissues is a hallmark of autoimmune disorders, which encompass a wide range of unique conditions. Numerous essential biologic functions, including the regulation of the immune system, inflammation, cell division, and tissue repair, are carried out by cytokines. Natural compounds are an effective treatment for autoimmune illnesses by modulation of inflammatory cytokines and infiltration of leukocytes into the inflamed tissue. Here, anti-arthritic study was carried out using oral administration of Azelaic acid (AzA) for 28days with doses (20, 40, and 80mg/kg) in Complete Freund's Adjuvant (CFA) induced arthritis model. AzA ameliorated the adjuvant-induced arthritis by decreasing arthritic score, paw volume, improved body-weight alterations and serum levels of PGE2, 5-LOX and anti-ccp. AzA showed significant down regulation of NF-κB, COX-II, TNF-α, IL-17, IL-1β, IL-6, and up regulation of IL4 and IL10. Hemoglobin and RBCs count remarkably increased and ESR, CRP, platelets, WBCs levels markedly reduced in post treatment. In addition, the weakened SOD (superoxide dismutase), Catalase (CAT), Glutathione (GSH) activity and the increased levels of malondialdehyde (MDA) were all reversed by AzA treatment. And showed improved radiographical and histologic alterations in the structure of the joints. Molecular docking studies targeting COX-II, iNOS, TNF-α, 5-LOX, IL4, IL10, IL-6, and IL-17 establish a correlation between theoretical and experimental results. Results showed that AzA inhibit pro-inflammatory cytokines (COX-II, TNF-α, 5-LOX, IL-17, NF-κB, IL-1β, and IL-6) and increase anti-inflammatory cytokines, which supported the anti-arthritic and immunomodulatory potential of AzA.

Effect of heat-reinforcing needling on hypoxia-inducible factor 1α and glycolysis activity in rabbits with cold syndrome of rheumatoid arthritis.

To observe the effect of heat-reinforcing needling (HRN) on synovial inflammation, hypoxia-inducible factor-1α (HIF-1α) and glycolytic activity in serum and synovial tissue in rabbits with cold syndrome of rheumatoid arthritis (RA), so as to explore its mechanisms underlying improvement of RA. A total of 32 rabbits were randomly divided into normal, model, inhibitor and HRN groups, with 8 rabbits in each group. The RA with cold syndrome model was induced by injecting ovalbumin dry powder and Freund's complete adjuvant combined with cold freezing. Rabbits in the inhibitor group were intraperitoneally injected with 2-methoxyestradiol (2.5 mg/kg), rabbits in the HRN group were received HRN at bilateral "Zusanli" (ST36) for 30 min. The treatments were conducted once daily for 14 consecutive days. After the interventions, the knee circumference and pain threshold were measured. The contents of nicotinamide adenine dinucleotide phosphoric (NADPH), Hexokinase II (HK2) and 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3) in serum of rabbits were detected by ELISA. The pathological morphology of synovial tissue of the knee joints were observed by HE staining. The positive expressions of tumor necrosis factor (TNF-α), interleukin (IL)-1β, IL-6 and IL-17 in synovial tissue of knee joint were detected by immunohistochemistry. The content of lactic acid in synovial tissue of rabbit knee joint was detected by spectrophotometry. The expression levels of HIF-1α, pyruvate kinase 2 (PKM2) and lactate dehydrogenase (LDHA) in synovial tissue of knee joint were detected by Western blot. After intervention, compared with the normal group, the knee circumference was significantly enlarged (P<0.05), the pain threshold was significantly decreased (P<0.05)；the synovial tissue of knee joints showed significant cell proliferation and inflammatory infiltration, the pathological score was significantly increased (P<0.05)；positive expressions of TNF-α, IL-1β, IL-6 and IL-17, the content of lactic acid in synovial tissue, the contents of NADPH, HK2 and PFKFB3 in serum, and the protein expression levels of HIF-1α, PKM2 and LDHA in synovial tissue were increased (all P<0.05) in the model group. Compared with model group, the circumference of knee joint was significantly decreased (P<0.05), the pain threshold was significantly increased (P<0.05)；in synovial tissue, the pathological score was decreased (P<0.05)；the positive expressions of TNF-α, IL-1β, IL-6 and IL-17 in synovial tissue were decreased (P<0.05), the lactic acid content in synovial tissue was decreased (P<0.05)；the contents of NADPH, HK2 and PFKFB3 in serum and the protein expression levels of HIF-1α, PKM2 and LDHA in synovial tissue were decreased (P<0.05) in inhibitor group and HRN group. Compared with the inhibitor group, the synovial pathological score was significantly increased (P<0.05), positive expressions of TNF-α, IL-1β, IL-6 and IL-17, the content of lactic acid in synovial tissue, the contents of NADPH, HK2 and PFKFB3 in serum, and the protein expression levels of HIF-1α, PKM2 and LDHA in synovial tissue were increased (all P<0.05) in HRN group. HRN can increase the pain threshold, reduce the knee circumference and inhibit the inflammatory response in rabbits with cold syndrome of RA. The possible mechanism is related to the down-regulation of HIF-1α and glycolysis activity.

BNT12, a novel hybrid peptide of opioid and neurotensin pharmacophores, produces potent central antinociception with limited side effects

The development of multitarget opioid drugs has emerged as an attractive approach for innovative pain management with reduced side effects. In the present study, a novel hybrid peptide BNT12 containing the opioid and neurotensin (NT)-like fragments was synthesized and pharmacologically characterized. In acute radiant heat paw withdrawal test, intracerebroventricular (i.c.v.) administration of BNT12 produced potent antinociception in mice. The central antinociceptive activity of BNT12 was mainly mediated by μ-, δ-opioid receptor, neurotensin receptor type 1 (NTSR1) and 2 (NTSR2), supporting a multifunctional agonism of BNT12 in the functional assays. BNT12 also exhibited significant antinociceptive effects in spared nerve injury (SNI)-neuropathic pain, complete Freund's adjuvant (CFA)-induced inflammatory pain, acetic acid-induced visceral and formalin-induced pain after i.c.v. administration. Furthermore, BNT12 exhibited substantial reduction of acute antinociceptive tolerance, shifted the dose-response curve to the right by only 1.3-fold. It is noteworthy that BNT12 showed insignificant chronic antinociceptive tolerance at the supraspinal level. In addition, BNT12 exhibited reduced or no opioid-like side effects on conditioned place preference (CPP) response, naloxone-precipitated withdrawal response, acute hyperlocomotion, motor coordination, gastrointestinal transit, and cardiovascular responses. The present investigation demonstrated that the novel hybrid peptide BNT12 might serve as a promising analgesic candidate with limited opioid-like side effects.

Kv3.1 Interaction with UBR5 Is Required for Chronic Inflammatory Pain.

Chronic inflammatory pain caused by neuronal hyperactivity is a common and refractory disease. Kv3.1, a member of the Kv3 family of voltage-dependent K+ channels, is a major determinant of the ability of neurons to generate high-frequency action potentials. However, little is known about its role in chronic inflammatory pain. Here, we show that although Kv3.1 mRNA expression was unchanged, Kv3.1 protein expression was decreased in the dorsal spinal horn of mice after plantar injection of complete Freund's adjuvant (CFA), a mouse model of inflammatory pain. Upregulating Kv3.1 expression alleviated CFA-induced mechanical allodynia and heat hyperalgesia, whereas downregulating Kv3.1 induced nociception-like behaviors. Additionally, we found that ubiquitin protein ligase E3 component n-recognin 5 (UBR5), a key factor in the initiation of chronic pain, binds directly to Kv3.1 to drive its ubiquitin degradation. Intrathecal injection of the peptide TP-CH-401, a Kv3.1 ubiquitination motif sequence, rescued the decrease in Kv3.1 expression and Kv currents through competitive binding to UBR5, and consequently attenuated mechanical and thermal hypersensitivity. These findings demonstrate a previously unrecognized pathway of Kv3.1 abrogation by UBR5 and indicate that Kv3.1 is critically involved in the regulation of nociceptive behavior. Kv3.1 is thus a promising new target for treating inflammatory pain.

Deciphering the therapeutic potential of trimetazidine in rheumatoid arthritis via targeting mi-RNA128a, TLR4 signaling pathway, and adenosine-induced FADD-microvesicular shedding: In vivo and in silico study.

Rheumatoid arthritis (RA) is a debilitating autoimmune condition characterized by chronic synovitis, joint damage, and inflammation, leading to impaired joint functionality. Existing RA treatments, although effective to some extent, are not without side effects, prompting a search for more potent therapies. Recent research has revealed the critical role of FAS-associated death domain protein (FADD) microvesicular shedding in RA pathogenesis, expanding its scope beyond apoptosis to include inflammatory and immune pathways. This study aimed to investigate the intricate relationship between mi-RNA 128a, autoimmune and inflammatory pathways, and adenosine levels in modulating FADD expression and microvesicular shedding in a Freund's complete adjuvant (FCA) induced RA rat model and further explore the antirheumatoid potency of trimetazidine (TMZ). The FCA treated model exhibited significantly elevated levels of serum fibrogenic, inflammatory, immunological and rheumatological diagnostic markers, confirming successful RA induction. Our results revealed that the FCA-induced RA model showed a significant reduction in the expression of FADD in paw tissue and increased microvesicular FADD shedding in synovial fluid, which was attributed to the significant increase in the expression of the epigenetic miRNA 128a gene in addition to the downregulation of adenosine levels. These findings were further supported by the significant activation of the TLR4/MYD88 pathway and its downstream inflammatory IkB/NFB markers. Interestingly, TMZ administration significantly improved, with a potency similar to methotrexate (MTX), the deterioration effect of FCA treatment, as evidenced by a significant attenuation of fibrogenic, inflammatory, immunological, and rheumatological markers. Our investigations indicated that TMZ uniquely acted by targeting epigenetic miRNA128a expression and elevating adenosine levels in paw tissue, leading to increased expression of FADD of paw tissue and mitigated FADD microvesicular shedding in synovial fluid. Furthermore, the group treated with TMZ showed significant downregulation of TLR4/MYD88 and their downstream TRAF6, IRAK and NF-kB. Together, our study unveils the significant potential of TMZ as an antirheumatoid candidate, offering anti-inflammatory effects through various mechanisms, including modulation of the FADD-epigenetic regulator mi-RNA 128a, adenosine levels, and the TLR4 signaling pathway in joint tissue, but also attenuation of FADD microvesicular shedding in synovial fluid. These findings further highlight the synergistic administration of TMZ and MTX as a potential approach to reduce adverse effects of MTX while improving therapeutic efficacy.

Anti-arthritic Activity and Anti-inflammatory Mediators Modulation Effect of Bioactive Traditional Chaturbeeja Churna on Arthritis Experimental Model

Objectives: The purpose of this research was to determine whether or not Chaturbeeja Churnam (CC) had anti-arthritic properties and the effect it has on Wistar rats that have been induced with arthritis using Complete Freund's Adjuvant (CFA). Methods: Injection of 0.2 milliliters of CFA into the sub-plantar surface of the left hind paw resulted in the development of arthritis. The test samples CC-1 and CC-2 were administered to the animals for a period of 21 days in a row. After the arthritis was induced, the rise in swelling that was noticed was observed. The blood samples that were taken were then used in additional testing with a hematology analyzer to determine the number of red blood cells (RBC), white blood cells (WBC), erythrocytes sedimentation rate (ESR), and hemoglobin (Hb). Using rat ELISA kits, the levels of IL-6 and TNF- that were found in the serum were also analyzed. Results: CC showed a significant inhibitory effect in the protein denaturation assay. Results showed that a significant reduction in paw edema was observed in CC-2 treated rats. The maximum anti-inflammatory activity (59.12%) of the CC was noticed at a dose level of 400 mg/kg. The paw edema was restored on day 21 was 4.52 mm for CC-2, which is near to the control group. The arthritis score in treated rats was found to be considerably lower than in the control group i.e. 0.92 for CC-2 and 1.61 for CC-1. A decrease in levels of RBC and hemoglobin was observed in arthritic rats. Inflammation was significantly reduced and serum levels of IL-6 and TNF-α were lowered after treatment with the test drug. Conclusion: It can be concluded from the study that CC possess significant anti-arthritic activity. Furthermore, this condition was linked to a reduction in abnormal humoral immune responses.

The Minor Phytocannabinoid Delta-8-Tetrahydrocannabinol Attenuates Collagen-Induced Arthritic Inflammation and Pain-Depressed Behaviors.

Patients with arthritis report using cannabis for pain management, and the major cannabinoid delta-9-tetrahydrocannabinol (Δ9-THC) has anti-inflammatory properties, yet the effects of minor cannabinoids on arthritis are largely unknown. The goal of the present study was to determine the antiarthritic potential of the minor cannabinoid delta-8-tetrahydrocannabinol (Δ8-THC) using the collagen-induced arthritis (CIA) mouse model. Adult male DBA/1J mice were immunized and boosted 21 days later with an emulsion of collagen and complete Freund's adjuvant. Beginning on the day of the booster, mice were administered twice-daily injections of Δ8-THC (3 or 30 mg/kg), the steroid dexamethasone (2 mg/kg), or vehicle for two weeks. Dorsal-ventral paw thickness and qualitative measures of arthritis were recorded daily, and latency to fall from an inverted grid was measured on alternating days, to determine arthritis severity and functional impairment. On the final day of testing, spontaneous wire-climbing behavior and temperature preference in a thermal gradient ring were measured to assess CIA-depressed behavior. The Δ8-THC treatment (30 mg/kg) reduced paw swelling and qualitative signs of arthritis. Δ8-THC also blocked CIA-depressed climbing and CIA-induced preference for a heated floor without producing locomotor effects but did not affect latency to fall from a wire grid. In alignment with the morphologic and behavioral assessments in vivo, histology revealed that Δ8-THC reduced synovial inflammation, proteoglycan loss and cartilage and bone erosion in the foot joints in a dose-dependent manner. Together, these findings suggest that Δ8-THC not only blocked morphologic changes but also prevented functional loss caused by collagen-induced arthritis. SIGNIFICANCE STATEMENT: Despite increasing use of cannabis products, the potential effects of minor cannabinoids are largely unknown. Here, the minor cannabinoid delta-8-tetrahydrocannabinol blocked the development of experimentally induced arthritis by preventing both pathophysiological as well as functional effects of the disease model. These data support the development of novel cannabinoid treatments for inflammatory arthritis.

A Novel Diabetic Arthritic Model in Rats Induced by Streptozotocin, High-Fat Diet, and Complete Freund's Adjuvant.

Ankle arthrodesis is one of the treatments of choice, particularly in late-stage and unstable diabetic Charcot arthropathy. Unfortunately, poor healing capacity might play a role in the high nonunion rate (10-40%). The advancement in regenerative medicine opens a new horizon for enhancing fusion after ankle arthrodesis in patients with poor healing capacity. However, a suitable small animal model is warranted to study the effectivity of these regenerative medicine approaches. Streptozotocin (STZ)-induced diabetes models and adjuvant-induced arthritis models with complete Freund's adjuvant are two established models. However, no study has combined those two models to make a diabetic arthritic model that more closely resembles the condition in Charcot arthropathy. Twenty male Sprague-Dawley rats were assigned into five groups, consisting of one control group, and four diabetic groups which were induced by STZ injection and a high-fat diet. Among these diabetic rats, two groups received complete Freund's adjuvant (CFA) injections to the left ankle of the hind limb. The control group, one of the diabetic-only groups, and one of the arthritic-diabetic-induced groups were euthanized at 4 weeks after STZ induction, and the remainder were euthanized 6 weeks after STZ induction. Clinical, radiological, and histological examinations were then compared in all five groups. Diabetic status was successfully achieved in the model, which was maintained until the completion of the study. The CFA-induced ankles were significantly larger than the contralateral ankles in all groups (p<0.05). Histopathological evaluation confirmed arthritic changes in the CFA-induced group with less variability after 4 weeks of arthritis induction. This rat model of arthritic diabetic mimics the progressive and chronic nature of Charcot arthropathy in humans. This model can be further use to study treatments that might enhance the fusion rate in ankle arthrodesis in healing-defective patients such as those with diabetes. 5.

Transient receptor potential vanilloid 1 involved in the analgesic effects of total flavonoids extracted from Longxuejie ().

To evaluate the analgesic effects of total flavonoids of Longxuejie (Resina Dracaenae Cochinchinensis) (TFDB) and explore the possible analgesic mechanism associated with transient receptor potential vanilloid 1 (TRPV1). Whole-cell patch clamp technique was used to observe the effects of TFDB on capsaicin-induced TRPV1 currents. Rat experiments in vivo were used to observe the analgesic effects of TFDB. Western blot and immunofluorescence experiments were used to test the change of TRPV1 expression in DRG neurons induced by TFDB. Results showed that TFDB inhibited capsaicin-induced TRPV1 receptor currents in acutely isolated dorsal root ganglion (DRG) neurons of rats and the half inhibitory concentration was (16.7 ± 1.6) mg/L. TFDB (2-20 mg/kg) showed analgesic activity in the phase Ⅱ of formalin test and (0.02-2 mg per paw) reduced capsaicin-induced licking times of rats. TFDB (20 mg/kg) was fully efficacious on complete Freund's adjuvant (CFA)-induced inflammatory thermal hyperalgesia and capsaicin could weaken the analgesic effects. The level of TRPV1 expressions of DRG neurons was also decreased in TFDB-treated CFA-inflammatory pain rats. All these results indicated that the analgesic effect of TFDB may contribute to their modulations on both function and expression of TRPV1 channels in DRG neurons.