Complete Freund's Adjuvant Group Research Articles

TRPA1 Ion Channel Mediates the Analgesic Effects of Acupuncture at the ST36 Acupoint in Mice Suffering from Arthritis.

Acupuncture (ACU) has been demonstrated to alleviate inflammatory pain. Mechanoreceptors are present in acupuncture points. When acupuncture exerts mechanical force, these ion channels open and convert the mechanical signals into biochemical signals. TRPA1 (T ransient receptor potential ankyrin 1) is capable of sensing various physical and chemical stimuli and serves as a sensor for inflammation and pain. This protein is expressed in immune cells and contributes to local defense mechanisms during early tissue damage and inflammation. In this study, we investigated the role of TRPA1 in acupuncture analgesia. We injected complete Freund's adjuvant (CFA) into the mouse plantars to establish a hyperalgesia model. Immunohistochemistry and immunofluorescence analyses were performed to determine the effect of acupuncture on the TRPA1 expression in the Zusanli (ST36). We used TRPA1-/- mouse and pharmacological methods to antagonize TRPA1 to observe the effect on acupuncture analgesia. On this basis, collagenase was used to destroy collagen fibers at ST36 to observe the effect on TRPA1. We found that the ACU group vs the CFA group, the number of TRPA1-positive mast cells, macrophages, and fibroblasts at the ST36 increased significantly. In CFA- inflammatory pain models, the TRPA1-/- ACU vs TRPA1+/+ ACU groups, the paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) downregulated significantly. In the ACU + high-, ACU + medium-, ACU + low-dose HC-030031 vs ACU groups, the PWL and PWT were downregulated, and in carrageenan-induced inflammatory pain models were consistent with these results. We further found the ACU + collagenase vs ACU groups, the numbers of TRPA1-positive mast cells, macrophages, and fibroblasts at the ST36 were downregulated. These findings together imply that TRPA1 plays a significant role in the analgesic effects produced via acupuncture at the ST36. This provides new evidence for acupuncture treatment of painful diseases.

Effects of inflammation on the properties of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative afferent mechanoreceptors in the hindpaw glabrous skin of mice.

We recently used Nav1.8-ChR2 mice in which Nav1.8-expressing afferents were optogenetically tagged to classify mechanosensitive afferents into Nav1.8-ChR2-positive and Nav1.8-ChR2-negative mechanoreceptors. We found that the former were mainly high threshold mechanoreceptors (HTMRs), while the latter were low threshold mechanoreceptors (LTMRs). In the present study, we further investigated whether the properties of these mechanoreceptors were altered following tissue inflammation. Nav1.8-ChR2 mice received a subcutaneous injection of saline or Complete Freund's Adjuvant (CFA) in the hindpaws. Using the hind paw glabrous skin-tibial nerve preparation and the pressure-clamped single-fiber recordings, we found that CFA-induced hind paw inflammation lowered the mechanical threshold of many Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors but heightened the mechanical threshold of many Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors. Spontaneous action potential impulses were not observed in Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors but occurred in Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors with a lower mechanical threshold in the saline goup, and a higher mechanical threshold in the CFA group. No significant change was observed in the mechanical sensitivity of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors and Nav1.8-ChR2-positive C-fiber mechanoreceptors following hind paw inflammation. Collectively, inflammation significantly altered the functional properties of both Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors, which may contribute to mechanical allodynia during inflammation.

Obacunone Alleviates Inflammatory Pain by Promoting M2 Microglial Polarization and by Activating Nrf2/HO-1 Signaling Pathway.

Treating inflammatory pain (IP) continues to pose clinical challenge, because of the lack of effective pharmacological interventions. Microglial polarization serves as pivotal determinant in IP progress. Obacunone (OB), a low-molecular-weight compound with a diverse array of biological functions, having reported as an activator of nuclear factor E2-related factor 2 (Nrf2), exhibits anti-inflammatory property. However, it remains uncertain whether OB can alleviate IP by facilitating the transition of microglial polarization from the M1 to M2 state through modulating Nrf2/ heme oxygenase-1 (HO-1) pathway. We induced an mice IP model by subcutaneously administering Complete Freund's Adjuvant (CFA) into the hind paw. Paw withdrawal latency (PWL) in seconds (s) and paw withdrawal frequency (PWF) were employed to evaluate the establishment of the IP model, while a caliper was used to measure the maximal dorsoventral thickness of the mice paw. Nerve injury was assessed by Hematoxylin-Eosin (HE) Staining. Western blot and got conducted for detection of M1/M2 microglial polarization markers, Nrf2 and HO-1 in spinal cord tissues respectively. In comparison to the control cohort, PWF, M1 phenotype marker iNOS, CD86, paw thickness increased significantly within CFA cohort, while PWL, M2 phenotype marker Arg-1, interleukin-10 (IL-10) decreased in the CFA group. In comparison to model cohort, OB treatment decreased PWF, paw thickness, M1 phenotype marker iNOS, CD86 significantly, while PWL, M2 phenotype marker Arg-1, IL-10, Nrf2, HO-1 increased significantly. The morphological injuries of sciatic nerve in CFA mice were obviously improved by OB treatment. OB inhibited the release of M1-related IL-1β, CXCL1 but promoted M2-related TGF-β, IL-10 in serum in CFA mice. The intervention of the Nrf2 inhibitor ML385 mitigated analgesic effect of OB. We demonstrate that OB is able to attenuate inflammatory pain via promoting microglia polarization from M1 to M2 and enhancing Nrf2/HO-1 signal. OB treatment may be a potential alternative agent in the treatment of IP.

The Effect of Evogliptin Tartrate on Controlling Inflammatory Pain.

Background: Evogliptin tartrate inhibits dipeptidyl peptidase-4 (DPP-4), boosting glucagon-like peptide 1 (GLP-1) secretion and improving insulin release and glucose tolerance, while also exerting anti-inflammatory effects. We investigated its anti-inflammatory and analgesic effects. Methods: Forty male Sprague Dawley rats were divided into (N = 10 in each): (1) naïve, (2) complete Freund's adjuvant (CFA) inflammation + evogliptin tartrate (once for 10 mg/kg) (CFAE), (3) CFA + vehicle (same volume with normal saline with evogliptin tartrate/once) (CFAV), and (4) CFA + indomethacin (5 mg/mL/kg/1 time) (CFAI) groups. CFA was injected subcutaneously into rat plantar regions, and medications (evogliptin tartrate, vehicle, and indomethacin) were administered orally for 5 days. Post treatment, blood from the heart and plantar inflammatory tissue were collected to assess inflammatory cytokines. Evogliptin tartrate effects on controlling inflammation and pain were evaluated by measuring rat plantar paw thickness, paw withdrawal threshold, dorsal root ganglion (DRG) resting membrane potential, DRG action potential firing, and cytokine (TNF-α and IL-1β) levels. Results: Compared with the naïve group, plantar paw thickness, cytokine (TNF-α and IL-1β) levels, DRG resting membrane potential, and DRG action potential firing increased, whereas the paw withdrawal threshold decreased in all CFA groups. However, CFAE and CFAI rats showed recovery. The degree of CFAE recovery resembled that observed in the CFAI group. Conclusions: Evogliptin tartrate mirrored the anti-inflammatory pain relief of indomethacin. We aim to broaden its use as an anti-inflammatory drug or pain relief drug.

Identification and validation of Rab11a in Rat orofacial inflammatory pain model induced by CFA

Orofacial pain (OFP) is a clinically very common and the most troubling condition; however, there is few effective way to relieve OFP. Rab11a, a small molecule guanosine triphosphate enzyme, is one of the Rab member family playing a vital role in intracellular endocytosis and the pain process. Therefore, we investigated the hub genes of rat OFP model induced by Complete Freund's Adjuvant (CFA) via re-analyzing microarray data (GSE111160). We found that Rab11a acted as a key hub gene in the process of OFP. During the validation of Rab11a, the OFP model was established by peripheral injection of CFA, which decreased the head withdrawal threshold (HWT) and head withdrawal lantency (HWL). Rab11a was observed in NeuN of Sp5C instead of GFAP/IBA-1, and double-IF of Rab11a and Fos positive cells were increased on the 7th day after CFA modeling statistically. Rab11a protein expression in TG and Sp5C of CFA group was also significantly increased. Interestingly, injection of Rab11a-targeted short hairpin RNA (Rab11a-shRNA) into Sp5C could reverse the decrease in HWT and HWL and reduce the expression level of Rab11a. Electrophysiological recording further demonstrated that the activity of Sp5C neuron was improved in CFA group, while Rab11a-shRNA considerably decreased the enhancement of Sp5C neuronal activity. Finally, we detected the expression level of p-PI3K, p-AKT, and p-mTOR in Sp5C of rats after injecting the Rab11a-shRNA virus. To our surprise, CFA upregulated the phosphorylation of PI3K, AKT and mTOR in Sp5C, and Rab11a-shRNA downregulated these molecules' expression. Our data suggest that CFA activates the PI3K/AKT signaling pathway through up-regulating Rab11a expression, which can induce OFP hyperalgesia development furtherly. Targeting Rab11a may be a novel treatment strategy for OFP.

Metabolic changes induced by complete Freund’s adjuvant resemble the features of trained immunity

Abstract Using the mouse model of viral myocarditis, we recently reported that animals immunized with complete Freund’s adjuvant (CFA) conferred complete protection against Coxsackievirus B3 infection. Because that Bacillus Calmette-Guérin (BCG) is a well-known inducer of trained immunity and both CFA and BCG possess similar mycobacterial components, we sought to test the hypothesis that the trained immunity might be the underlying mechanism of CFA-mediated protection against CVB3. By using incomplete Freund’s adjuvant (IFA) as a negative control, we investigated the metabolic profiles by mass spectrometry in bone marrow-derived monocytes and CD11b+ cells sorted from splenocytes using LPS as a secondary stimulus. The analysis revealed elevated levels of lactate, α-ketoglutarate, fumarate, malate, and oxaloacetate in the CFA group as compared with the control or IFA groups. In contrast, S-adenosyl methionine and homoserine levels were high in the IFA group. Overall, our data suggest that the metabolites produced in response to CFA were uniquely different from those of IFA. Whether these profiles correlate with the epigenetic changes are currently being investigated. Supported by grants from NIH (R21, A116673)

TRPV1 and GABAB1 in the Cerebrospinal Fluid-Contacting Nucleus are Jointly Involved in Chronic Inflammatory Pain in Rats.

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. 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. TRPV1 and GABAB1 were co-expressed in the CSF-contacting nucleus in rats with CIP, and their expression was upregulated. 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.

Early life nociceptive stimulus and fentanyl exposure increase hippocampal neurogenesis and anxiety but do not affect spatial learning and memory

This study aimed to determine whether preemptive fentanyl administration in neonatal rats reduces the impact of a nociceptive stimulus initiated during the first day of life (P1) on hippocampal neurogenesis, behavior, and learning. At P1, Wistar rat pups received either a subcutaneous injection of fentanyl (F) before intraplantar injection of complete Freund’s adjuvant (CFA) (CFA + F group), an isolated injection of CFA (CFA group), or subcutaneous injection of fentanyl without CFA injection (F). Control animals received saline injections using the same route and volume as the treatment groups. Hippocampal neurogenesis was evaluated by 5′ –bromo-2′-deoxyuridine (BrdU) staining on P10 and P39 to assess neuronal proliferation and survival, respectively. Anxiety behavior in adulthood was assessed using an open field test (OF) and an elevated plus maze test (EPM). Spatial memory was assessed on a Morris water maze test (MWM), where the animals were trained for seven days, beginning on P81, and the probe trial was performed to evaluate memory retention. Although the CFA + F group showed an increased number of proliferative cells on P10, this finding did not persist on P39. The CFA + F group spent more time in the closed arms in the EPM, revealing more anxious behavior, although the early noxious experience, both with and without fentanyl, did not alter neurogenesis in adolescence and learning in adulthood. This study highlights that the impact of pain in early life pain combined with fentanyl on hippocampal neurogenesis on P10 did not persist on P39. In addition, this combined intervention during the first week of life was associated with higher anxiety levels.

Evaluation of Antiarthritic and Antinociceptive Effects of Cedrol in a Rat Model of Arthritis.

Pharmacological studies revealed that cedrol, a natural sesquiterpene, has antioxidant, anti-inflammatory, and analgesic properties. This study is aimed at evaluating the potential antiarthritic activity of cedrol in a rat experimental model of arthritis induced by using complete Freund's adjuvant (CFA). Arthritis was induced in Wistar rats by CFA (0.1 ml) injection. Cedrol (10 and 20 mg/kg) and indomethacin (5 mg/kg) were orally administered from day one and continued for 21 days. The antiarthritic activity was assessed through mechanical allodynia and thermal hyperalgesia responses, paw edema assessment, and arthritis scores. Serum TNF-α and IL-1β levels were measured for the evaluation of inflammation. Furthermore, serum oxidative stress markers, including malondialdehyde (MDA) and thiol levels, as well as superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, were also assessed. Oral administration of cedrol and indomethacin significantly decreased paw edema and arthritis score. Besides, cedrol and indomethacin significantly decreased pain responses. In the serum of the CFA group, TNF-α, IL-1β, and MDA were higher, while thiol and SOD and GPx were lower than the control group. Treatment by cedrol and indomethacin corrected the biochemical parameters in the serum. In this study, cedrol offers potential antiarthritic properties through its anti-inflammatory and antioxidant effects.

Benefits of electroacupuncture and a swimming association when compared with isolated protocols in an osteoarthritis model

Background and aimOsteoarthritis (OA) is characterized by pain and inflammation. Electroacupuncture (EA) and swimming (SW) are non-pharmacological interventions recommended for treating OA. The study evaluated the benefits of electroacupuncture (EA) and swimming (SW) association when compared with isolated protocols in an OA rodent model. Experimental. ProceduresAn ankle monoarthritis model was induced in rats by applying Complete Freund's Adjuvant (CFA). After seven days of induced OA, the groups were submitted to EA (ST36 and the GB 30 Acupoint), SW, or the EA + SW protocol. The nociceptive behavior was measured by the Von Frey test, the Cold Stimulation test, and the Paw Flick Immersion test. Inflammatory activity was evaluated by measuring TNF levels, myeloperoxidase, NAGase, immunological parameters and the histology from the subcutaneous tissue. ResultsCompared to CFA group, EA decreased the nociceptive scores in the cold stimulation test (p < 0.05), and it also increased the latency time in thermal cold (p < 0.01) and heat hyperalgesia (p < 0.001). Also, EA reduced NAGase (p < 0.01). SW reduced the edema (p < 0.05) and did not increase the inflammatory infiltrates or congestion, neither in the histological measurements nor by analyzing the levels of TNF. The association of EA + SW decreased the neutrophils and the monocytes, MPO (p < 0.05), and the glutamate levels in the cerebrospinal fluid (CSF, p < 0.001). ConclusionThere were statistical differences between combination therapy and monotherapy as seen by the inflammatory parameters, which could be associate to the delay of the chronification osteoarthritis retardation. However, EA + SW did not show benefits when compared to isolated protocols in nociceptive behavior.

Anti-Inflammatory Effects of BoNT/A Against Complete Freund's Adjuvant-Induced Arthritis Pain in Rats: Transcriptome Analysis.

Arthritis is the most common cause to lead to chronic pain. Botulinum toxin type A (BoNT/A) has been widely used to treat chronic pain. In our previous study, we confirmed the anti-inflammatory and antinociceptive effects of BoNT/A in the Complete Freund’s Adjuvant (CFA)-induced arthritis model, but the underlying anti-inflammatory mechanism was not fully elucidated. The purpose of this study was to investigate the anti-inflammatory effects and mechanisms of BoNT/A on arthritis using transcriptomic analysis. The BoNT/A was injected into the rat ankle joint on day 21 after CFA injection. The von Frey and hot plate tests were applied to assess the pain-related behaviors at different time points. Five days after BoNT/A treatment, gene expression profiling in dorsal root ganglion (DRG) was performed using RNA sequencing (RNA-seq). The differentially expressed genes (DEGs) were analyzed by various tools. The mechanical allodynia and thermal hyperalgesia were significantly reversed after BoNT/A injection. RNA-seq revealed 97 DEGs between the CFA group and Sham group; these DEGs were enriched inflammatory response, IL-17 signaling pathway, etc. There are 71 DEGs between the CFA+BoNT/A group and the CFA group; these DEGs related to response to peptide, PI3K-Akt signaling pathway, ECM–receptor interactions, etc. Three key genes were significantly decreased after CFA-induced arthritis pain, while BoNT/A increased the expression of these genes. The identification of S100A9, S100A8, and MMP8 genes can provide new therapeutic targets for arthritis pain and affect the signaling pathway to play an anti-inflammatory role after the treatment of BoNT/A.

Se-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl] 4-Chlorobenzoselenolate Attenuates Inflammatory Response, Nociception, and Affective Disorders Related to Rheumatoid Arthritis in Mice.

Despite major advances, not all patients achieve rheumatoid arthritis (RA) remission, thus highlighting a pressing need for new therapeutic treatments. Given this scenario, this study sought to evaluate Se-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl] 4-chlorobenzoselenolate (Se-DMC) potential on a complete Freund's adjuvant (CFA)-induced unilateral arthritis model. The effects of Se-DMC (5 mg/kg; oral dose) and meloxicam (5 mg/kg; oral dose), both administered to animals daily for 14 days, on paw edema, mechanical sensitivity, neurobehavioral deficits (anxiogenic- and depressive-like behaviors), Na+/K+-ATPase activity, oxidative stress, and inflammation were evaluated in male Swiss mice exposed to CFA (intraplantar injection of 0.1 mL; 10 mg/mL). Se-DMC reduced the paw withdrawal threshold and CFA-induced paw edema. Histopathological results revealed the antiedematogenic potential of the compound, which was evidenced by lower quantities of dilated lymphatic vessels compared with the CFA group. Se-DMC reduced mRNA relative expression levels of tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) in the hippocampus and paw of CFA mice. The CFA-induced anxiogenic- and depressive-like behaviors were reversed by Se-DMC to the control levels in the elevated plus-maze and tail suspension tests. Se-DMC reduced the paw reactive species levels and restored the superoxide dismutase (hippocampus and paw) and Na+/K+-ATPase (hippocampus) activities previously increased by CFA. Moreover, CFA administration inhibited serum creatinine kinase activity, albeit the Se-DMC effects did not appear to involve the modulation of this enzyme and were equal to or greater than meloxicam. Se-DMC attenuates CFA-induced inflammatory response, nociception, and neurobehavioral deficits in mice.

Styphnolobium japonicum (L.) Schott Flower Extract Alleviates Oxidative Stress and Inflammatory Factors in the Adjuvant-Induced Arthritis Rat Model.

IntroductionThis research was to evaluate the beneficial effects of Styphnolobium japonicum (L.) Schott flower extract (SJF) on the adjuvant-induced arthritis rat model.MethodsArthritis was evoked by injection of complete Freund’s adjuvant (CFA) in the hind paw. SJF (150 or 300 mg/kg/day) or Celecoxib (5 mg/kg/day) were administered intragastrically from the 0th day to the 28th day. The arthritis symptoms (paw edema, arthritic scores, mechanical hyperalgesia, and thermal hyperalgesia), inflammation biomarkers (RT and CRP), related enzymes (MMP1 and MMP13), oxidative stress markers (CAT, SOD, GPx, and MDA), and inflammatory cytokines (IL-6, IFN-γ, TNF-α, and IL-1β) of SJF-treated CFA rats were evaluated.ResultsCFA rats exhibited severe arthritis symptoms, increased oxidative stress, and inflammatory cytokines. Interestingly, SJF treatment relieving arthritis symptoms and restored body weight gain compared with those in the CFA group. SJF treatment decreased the levels of CRP, RF, MMP1, and MMP13 in the CFA group. Besides, SJF treatment increased the activities of antioxidant enzymes and decreased the MDA content and inflammatory cytokines compared with those in the CFA group. Moreover, SJF could increase the mRNA expression of GPx-1 and CAT and inhibit the mRNA expression of IL-6 and TNF-α in the ankle tissue of CFA rats.

Polarization of macrophages in the trigeminal ganglion of rats with pulpitis

Dental pulp tissues are rich in pain-related afferent nerve fibers, which originate from primary sensory neurons in the trigeminal ganglion (TG). The mechanisms of central nervous system (CNS) underlying ectopic pain following peripheral inflammation have been reported that the macrophages as inflammatory and immunologic mediators in the TG play an important role in the process of pulpitis and hyperalgesia. To observe the polarization response and dynamic distribution of macrophages in the TG during the development of dental pulp inflammation. A rat model of pulpitis was established using complete Freund's adjuvant (CFA). Hematoxylin-eosin (HE), immunohistochemistry (IHC), immunofluorescence (IF), toluidine blue (TB) staining, and RT-qPCR were performed to observe the expression of macrophage-related factors in the TG. The results of IHC staining showed that M2 macrophages labeled with CD206 were observed in the TG of both the control and CFA groups. The statistical analysis indicated that the number of CD206-positive macrophages in the TG increased significantly at 24h after CFA-induced pulpitis, reached a peak at 2weeks, and then returned to the normal level after 6weeks. The ratio of M2/M1 in the CFA groups was significantly lower than that in the control group from 24 to 72h, and this pattern was reversed at 2weeks after CFA-induced pulpitis; then, the ratio increased significantly and was maintained at a high level for 4weeks. RT-qPCR results showed that the expression of IL-10 in the TG increased significantly from 1 to 4weeks after CFA-induced pulpitis. The trend of M2 macrophages was opposite to that of M1 macrophages in the TG during the process of pulpitis induced by CFA, which is consistent with the expression of macrophage-related cytokines. Macrophage polarization in the TG may participate in the neuroinflammation response induced by dental pulpitis.

Comparative evaluation of the protective effects of oral administration of auraptene and umbelliprenin against CFA-induced chronic inflammation with polyarthritis in rats

This study aimed to evaluate the anti-inflammatory effect of Auraptene (AUR) and Umbelliprenin (UMB) in a rat model of rheumatoid arthritis (RA) induced by using complete Freund's adjuvant (CFA).Paw swelling of adjuvant arthritis rats measured at various times after CFA injection. Over 15 days of RA induction, mediator/cytokine-mediated processes involved in managing the regulation and resolving RA's inflammation were also quantified with ELISA. Histopathological changes were also assessed under a microscope 15 days after the CFA injection.AUR at all doses and UMB administration only at a 16 mM /kg administration dose significantly reduced CFA-induced paw edema level compared to the control group. UMB (64 and 32 mM) and AUR (64, 32, and 16 mM) could reduce the PGE2 (p < .0001–.01) and NO (p < .0001–.05) levels in the treatment groups compared to the negative control group. However, these compounds showed no significant effect on the TNF-α, IFN-γ, TGF-β, IL-4, and IL-10 levels than the control group (p > .05). Unlike indomethacin and prednisolone, treatment of rats with AUR (16, 32, and 64 mM/kg) and UMB (16 and 32 mM/kg) reduced the level of IL-2 (p < .0001). In all treatment groups, the serum level of IL-17 was significantly reduced compared to the CFA group (p < .001–0.05).We suggested AUR and UMB could diminish inflammation by reducing the serum level of IL-17 and could be considered a proper alternative in the treatment of IL-17 related inflammatory diseases such as rheumatoid arthritis. Given that AUR and UMB apply their anti-inflammatory effects by changing distinct cytokine release/inhibition patterns, their potential application in diverse inflammatory diseases seems different.

Development and evaluation of a recombinant VP2 neutralizing epitope antigen vaccine candidate for infectious bursal disease virus.

Infectious bursal disease (IBD) is one of the most economically important infectious diseases. Currently, vaccination is the most effective method to prevent IBD. Medium-virulence vaccines can damage the bursa of Fabricius and result in immunosuppression. Therefore, it is essential to develop a safe and effective vaccine against infectious bursal disease virus (IBDV). In this study, the five neutralizing epitopes of the IBDV VP2 protein were confirmed by neutralizing single chain variable fragment antibodies. Then, the neutralizing epitopes antigen (NEA) protein was constructed with five neutralizing epitopes and expressed by pET-27b. Furthermore, the immune effect and protective immunity of the NEA protein with the following adjuvants were evaluated in specific-pathogen-free chickens: oil emulsion adjuvant (OEA), double emulsion adjuvant (DEA), granulocyte-macrophage colony-stimulating factor (GM-CSF) adjuvant and complete Freund's adjuvant (CFA). The experimental results demonstrated that chickens immunized with NEA vaccines elicited stronger humoral and/or cellular immune responses and inflammatory responses than those in the NEA protein group. Chickens were protected in OEA, CFA and GM-CSF adjuvant groups, which were challenged with virulent IBDV BC6/85. Furthermore, IBDV RNA was not measured, and there appeared to be little apoptosis in the bursa of Fabricius based on TUNEL histology and the expression of Bax and Bcl-2 in the OEA, CFA and GM-CSF adjuvant groups. Based on the experimental results, the advantages and disadvantages of adjuvants and industrial production methods, GM-CSF was found to be the optimal adjuvant. Therefore, NEA with GM-CSF adjuvant is a promising vaccine candidate against IBDV, and it provides a framework for developing other vaccines against infectious viral diseases.

The related mechanism of complete Freund's adjuvant-induced chronic inflammation pain based on metabolomics analysis.

Chronic inflammation pain is a debilitating disease, and its mechanism still remains poorly understood. This study attempted to illuminate the metabolic mechanism of chronic inflammation pain induced by complete Freund’s adjuvant (CFA) injection, especially at spinal level. The chronic inflammation pain model was established by CFA administration. Behavioral testing including mechanical allodynia and thermal hyperalgesia was performed. Meanwhile, a liquid chromatography–mass spectrometry‐based metabolomics approach was applied to analyze potential metabolic biomarkers. The orthogonal partial least squares discrimination analysis mode was employed for determining metabolic changes, and a western blot was performed to detect the protein expression change. The results showed that 27 metabolites showed obviously abnormal expression and seven metabolic pathways were significantly enriched, comprising aminoacyl‐tRNA biosynthesis, arginine and proline metabolism, histidine metabolism, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, glutathione metabolism, and phenylalanine metabolism. Meanwhile, the results showed that the expression of arginase I and nitric oxide levels were elevated in the CFA group compared with the control group, while the argininosuccinate synthetase and argininosuccinatelyase proteins were not significantly different between the groups. These findings demonstrate that metabolic changes of the spinal cord may be implicated in neurotransmitter release and pain conductivity following CFA administration.

Therapeutic potential of russelioside B as anti-arthritic agent in Freund's adjuvant-induced arthritis in rats

Ethnopharmacological relevanceCaralluma species are traditional edible herbs used in folkloric medicine as antidiabetic, antioxidant, antipyretic, antirheumatic, anti-inflammatory and anthelmintic agents. C. quadrangula was selected in this study to document the traditional use of the genus as anti-rheumatic treatment and the possible mechanisms of action. Aim of the studyThe higher mortality rates and shorter survival among the patients suffering from rheumatoid arthritis (RA) led to the increased interest on searching for new treatments for RA. Russelioside B (RB), a major pregnane glycoside found in C. quadrangula, was evaluated as a new anti-rheumatic agent. Materials and methodsThe n-butanol fraction of C. quadrangula was chromatographed on a silica gel column to isolate RB. The adjuvant-induced arthritis (AIA) model was established in rats by intradermal injection of complete Freund's adjuvant (CFA) to evaluate its anti-arthritic effect. Ibuprofen was used as a reference drug. Forty rats were randomly divided into 5 groups (n = 8): normal (NOR); CFA model (CFA); ibuprofen, 5 mg/kg; RB, 25 mg/kg and RB, 50 mg/kg. The treatments were initiated from day 16 when AIA model was established and continued up to day 40. Serum diagnostic rheumatoid markers, inflammatory cytokines, oxidative stress biomarkers, cartilage and bone degeneration enzymes were assessed. ResultsRB at 50 mg/kg b. wt., showed significant decreases in the activities of hyaluronidase and β-glucouronidase enzymes as well significant decreases in the levels of proinflammatory cytokines as nuclear factor-kappa-B (NF-κB), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) compared to the CFA group; 11.04 ± 0.61 pg/mg protein, 4.35 ± 0.25 pg/mg protein, 3.32 ± 0.13 pg/mg protein & 2.75 ± 0.14 pg/mg protein for RB, 50 mg/kg b. wt. group vs. 25.33 ± 2.13 pg/mg protein, 25.65 ± 2.1 pg/mg protein, 22.20 ± 1.34 pg/mg protein & 13.27 ± 1.40 pg/mg protein for the arthritic group, respectively. The total antioxidant capacity (TAC) was significantly restored to normal values in RB, 50 mg/kg treated rats (4.01 ± 0.09 nmol/mL vs. 3.71 ± 0.27 nmol/mL) and the levels of myeloperoxidase (MPO) reduced by 10-folds of the CFA arthritic group. Bone histomorphometry revealed that RB treatment significantly attenuated the CFA-induced bone loss in a dose-dependent manner. ConclusionsThese findings suggested that the anti-arthritic effect of RB was mediated through the reduction of the rheumatoid markers, anti-inflammatory and antioxidant action, inhibition of cartilage and bone degenerative enzymes as well as attenuation of bone loss and osteoclastogenesis.

A standardized extract of Centella asiatica (ECa 233) prevents temporomandibular joint osteoarthritis by modulating the expression of local inflammatory mediators in mice.

Objectives To investigate the effect of a standardized extract of Centella asiatica (ECa 233), which has anti-inflammatory properties, on the local expression of the transient receptor potential vanilloid 1 (TRPV1), the acid-sensing ion channel subunit 3 (ASIC3), and the calcitonin gene-related peptide (CGRP) in the temporomandibular joint (TMJ) structure 21 days after injecting the TMJ with complete Freund’s adjuvant (CFA).Methodology A mouse model was induced by analyzing the CFA-injected TMJ on days 7, 14, and 21. We assessed TMJ histology by the osteoarthritis cartilage grade score. Then, we observed the effect of different ECa 233 concentrations (30, 100, and 300 mg/kg) and of 140 mg/kg ibuprofen doses on TRPV1, ASIC3, and CGRP local expression on day 21.Results Osteoarthritis cartilage scores were 1.17±0.37 and 3.83±0.68 on days 14 and 21, respectively, in the CFA group (n=5). On day 21, TRPV1, ASIC3, and CGRP expression significantly increased in the CFA group. In the ibuprofen-treated group, TRPV1 expression significantly decreased, but ASIC3 and CGRP showed no significant difference. All ECa 233 doses reduced TRPV1 expression, but the 100 mg/kg ECa 233 dose significantly decreased ASIC3 expression.Conclusions TRPV1, ASIC3, and CGRP expression increased in mice with TMJ-OA on day 21. All ECa 233 and ibuprofen doses inhibited pathogenesis by modulating the local expression of TRPV1 and ASIC3. Therefore, ECa 233 was more effective than ibuprofen.

Evolution of mirror-image pain in temporomandibular joint osteoarthritis mouse model.

Mirror-image pain is a kind of pain that occurs on the contralateral side, but its pathogenesis remains unclear.ObjectiveTo develop an osteoarthritis mouse model for investigating mirror-image pain through observing nocifensive behaviors, histological changes, and nociceptive activity at days 3, 7, 14, 21, and 28 after the chemical induction of unilateral temporomandibular joint (TMJ) osteoarthritis.MethodologyWe randomly divided 6-week-old mice into sham and complete Freund adjuvant groups. To induce nocifensive behaviors, we applied 0.04 g of von Frey filament, 10 psi of air puff, and cold acetone on both sides of whisker pads at different days. The histology of TMJ on both sides was observed by hematoxylin/eosin staining and microcomputed tomography scanning. Furthermore, the nociceptive activity was evaluated using the phosphorylated cyclic AMP response element binding protein (pCREB) and a microglia marker at different days in the trigeminal subnucleus caudalis.ResultsNocifensive behaviors against mechanical and temperature stimuli on the contralateral side became stronger than the baseline on day 28, in agreement with the elevation of the pCREB and the microglia marker in the trigeminal subnucleus caudalis. Thus, hypernociception on the contralateral side occurred at day 28.ConclusionsClearly, the TMJ model with unilateral osteoarthritis exhibited mirror-image pain. Therefore, this model is useful in investigating the pathogenesis of pain and in developing treatments.