Freund's Adjuvant Research Articles

An iota-carrageenan isolated from marine alga Agardhiella ramosissima negatively modulates the inflammatory response in arthritis conditions

The present study involves the structural characterization of an iota-carrageenan from Agardhiella ramosissima (IC-Ar) and its ability to suppress the inflammatory response in experimental arthritis. The IC-Ar was structurally characterized by Size Exclusion Chromatography (SEC) and Nuclear Magnetic Resonance (NMR) techniques. Experimentally, the mice were treated with IC-Ar (3, 10, or 30 mg/kg) e submitted to arthritis with zymosan and Complete Freund's Adjuvant (CFA). IC-Ar has a molecular mass of 730 KDa and contains 3,6 anhydro-α-d-galactose-2 sulfate (DA2S), β-d-galactose-4 sulfate (G4S) and glucose, structural characteristic typical of an iota carrageenan. IC-Ar treatment reduced zymosan-induced hypernociception, in a dose-dependent manner, accompanied by reduced edema, vascular permeability, and histopathological scores. Likewise, IC-Ar also significantly decreases MPO activity, leukocytes, neutrophils, IL-1β, and nitrite levels in the synovial fluid. Therapeutically, IC-Ar inhibited the hypernociception, edema, and arthritic index in both acute, sub-chronic, and chronic phases of CFA-induced arthritis. In summary, we demonstrated the protective efficacy of an iota-carrageenan extracted from A. ramosissima in arthritis models by negatively modulating the inflammatory response and by reducing polymorphonuclear cell migration, IL-1β, and nitrite levels, culminating in the inhibition of hypernociception and edema. Thus, IC-Ar could be a promising molecule to treat arthritis conditions.

Can Chilodonella uncinata induce cross-protection in koi carp (Cyprinus carpio) against Ichthyophthirius multifiliis? Evidence from immune response and challenge experiments

The intraperitoneal injection of koi carp with inactivated Ichthyophthirius multifiliis or Chilodonella uncinata combined with Freund's adjuvant was evaluated for its capacity to protect the fish from I. multifiliis infection. Our results showed that the level of Ig M in serum significantly increased after vaccination, reaching up to 4.09 μg/mL on day 14 in fish vaccinated with C. uncinata, while C3 content also generally increased. In addition, genes related to immunity, including Ig M, IL-1β, TNF-α, TLR 9, TLR 20 and TLR 21, exhibited differential expression, which suggests that the koi carp did indeed produce immune responses after vaccination. In challenge experiments, each fish specimen was challenged with 30,000 theronts on day 14 post vaccination. The relative survival rate of fish vaccinated with 10,000 I. multifiliis theronts was 93.3%, while the survival rate of fish vaccinated with 10,000C. uncinata or with phosphate buffer saline (the latter being the control) was 0% in both cases. However, as the dose of C. uncinata was increased, the survival rate of the fish also increased, with fish vaccinated with 60,000C. uncinata cells achieving a higher survival rate than those vaccinated with 30,000 cells. Above all, we demonstrated that Ig M does not play a major role in protection against ichthyophthiriasis and the protection conferred by inactivated C. uncinata vaccination is related to the dose.

Evaluation of the Anti-Inflammatory Pain Effect of Ginsenoside-Conjugated O-Carboxymethyl Chitosan Particles.

Nanoparticle delivery of functional molecules or vaccines is an effective method for the treatment of many diseases. This study aims to design ginsenoside Rh2-conjugated O-carboxymethyl chitosan (O-CMC/Rh2) as a drug delivery system and explore its anti-nociceptive effects. O-CMC/Rh2 was synthesized with an esterification reaction, and its chemical composition and morphology were evaluated using proton nuclear magnetic resonance (1H NMR), the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, and scanning electron microscopy (SEM). In addition, the in vitro cumulative release of Rh2 from the O-CMC/Rh2 was also evaluated under different pH conditions. The results showed that the ginsenoside Rh2 was successfully conjugated to the O-CMC matrix and exhibited a highly porous structure after conjugation, facilitating the release of Rh2 from O-CMC. Complete Freund's adjuvant (CFA) and burn injury-induced pain models were used to evaluate the anti-nociceptive effects of O-CMC/Rh2 on inflammatory pain. O-CMC/Rh2 reduced CFA-induced pain hypersensitivity in a dose-dependent manner and had a longer analgesic effect than Rh2. In addition, O-CMC/Rh2 also relieved the chronic pain induced by bury injury. These results indicated that O-CMC/Rh2 could be useful in reducing inflammatory pain, thus possessing a potential medicinal application in pain therapy.

FRI374 Immunomodulatory Effects Of Progesterone And Glucocorticoids In Pregnancy Immunity

Abstract Disclosure: A. Yasuda: None. T. Seki: None. H. Kashiwagi: None. N. Kitajima: None. Y. Kametani: None. Immune tolerance to a semi-allogeneic fetus is crucial for a successful pregnancy. However, the mechanisms of tolerance to semi-allografts in the co-existence with normal defenses against cancer and pathogens is not clear. During pregnancy, levels of pregnancy-related proteins and hormones such as progesterone (P4), estrogen, testosterone, and glucocorticoids (GC) increase. We hypothesized that the most abundant pregnancy related hormone in the placenta, P4, might play an important role in the local tolerance in the placenta. Thus, we aimed to compare the effects of P4 and cortisol (COR) on human lymphocytes in vivo, to clarify their role in the systemic regulation of immunity during pregnancy. Healthy donor peripheral blood mononuclear cells (PBMCs) were treated with P4 or COR for 6 h. These PBMCs were washed and transplanted intravenously into 8- to 9-week-old NOG-hIL-4-Tg (formal name, NOD.Cg-PrkdcscidIl2rgtm1Sug/Tg(CMV-IL4)/Jic) mice. Human epidermal growth factor receptor-2 (HER2) peptide was emulsified in complete Freund's adjuvant and administered intraperitoneally to these PBMC-NOG-hIL-4-Tg mice at the same day. As a negative control, an equal volume of PBS was added to the culture and transplanted into NOG-hIL-4-Tg mice. Boosters were administered using incomplete Freund's adjuvant 2 weeks after the primary immunization. Peripheral blood was collected 4 weeks after transplantation and 2 weeks after booster administration. T and B cell profiles were analyzed using FCM 28 days later. The results revealed that significantly higher numbers of human CD45+ cells engrafted in the spleens of P4-treated mice compared to COR-treated mice 4 weeks post-transplantation. Transplanted human CD45+ cells included T and B cells, and CD8+ T cells were significantly higher in P4-treated mice compared to both control and COR-treated mice. Moreover, antigen-specific IgG production was maintained in the P4-treated mice, suggesting that the T cell/B cell function was maintained in the mice. Compared to control and P4-treated mice, COR-treated mice tended to engraft fewer human CD45+ cells. Moreover, B cell engraftment tended to be lower in COR-treated mice than in control mice. Antigen-specific IgG production was not maintained. There results suggest that lymphocytes which experienced a high P4 environment maintain engraftment and function of human lymphocytes in the NOG-hIL-4-Tg mice, while high COR environment rather dampens the engraftment and function of them. The high-P4 environment limited in the placenta may support pregnant immunity to protect the semi-allogeneic fetus from pathogen-induced infectious disease or cancer. Presentation: Friday, June 16, 2023

The Effects of Commercially-Relevant Disturbances on Sleep Behaviour in Laying Hens.

Ensuring the welfare of commercially kept animals is a legal and ethical responsibility. Sleep behaviour can be sensitive to environmental perturbations and may be useful in assessing welfare state. The objective of this study was to use behavioural and electrophysiological (EEG) measures to observe the effects of 24 h stressors followed by periods of no stressors on laying hen sleep behaviour, and to investigate the use of sleep behaviour as a means of welfare assessment in commercial poultry. Ten laying hens surgically implanted with EEG devices to record their brain activity over four batches were used. Hens were subjected to undisturbed, disturbed and recovery periods for 24 h. Disturbed periods consisted of either feed deprivation, increased ambient temperature (28 °C) or simulated footpad pain via injection of Freund's adjuvant into the footpad. Sleep state was scored using behaviour data from infrared cameras and EEG data. Over all periods, hens engaged in both SWS (average 60%) and REM sleep (average 12%) during the lights-off period. Feed deprivation and footpad pain had little to no effect on sleep states, while increased ambient temperature significantly reduced REM sleep (to near elimination, p < 0.001) and SWS (p = 0.017). During the lights-on period, footpad pain increased the proportion of time spent resting (p = 0.008) and in SWS (p < 0.001), with feed deprivation or increased ambient temperature (p > 0.05) having no effect. Increasing ambient temperatures are likely to affect sleep and welfare in commercially-kept laying hens in the face of global climate change.

Peripheral Activation of Formyl Peptide Receptor 2/ALX by Electroacupuncture Alleviates Inflammatory Pain by Increasing Interleukin-10 Levels and Catalase Activity in Mice.

In the context of the electroacupuncture (EA) neurobiological mechanisms, we have previously demonstrated the involvement of formyl peptide receptor 2 (FPR2/ALX) in the antihyperalgesic effect of EA. The present study investigated the involvement of peripheral FPR2/ALX in the antihyperalgesic effect of EA on inflammatory cytokines levels, oxidative stress markers and antioxidant enzymes in an animal model of persistent inflammatory pain. Male Swiss mice underwent intraplantar (i.pl.) injection with complete Freund's adjuvant (CFA). Mechanical hyperalgesia was assessed with von Frey monofilaments. Animals were treated with EA (2/10Hz, ST36-SP6, 20 minutes) for 4 consecutive days. From the first to the fourth day after CFA injection, animals received i.pl. WRW4 (FPR2/ALX antagonist) or saline before EA. Levels of inflammatory cytokines (TNF, IL-6, IL-4 and IL-10), antioxidant enzymes (catalase and superoxide dismutase), oxidative stress markers (TBARS, protein carbonyl, nitrite/nitrate ratio), and myeloperoxidase activity were measured in paw tissue samples. As previously demonstrated, i.pl. injection of the FPR2/ALX antagonist prevented the antihyperalgesic effect induced by EA. Furthermore, animals treated with EA showed higher levels of IL-10 and catalase activity in the inflamed paw, and these effects were prevented by the antagonist WRW4. EA did not change levels of TNF and IL-6, SOD and MPO activity, and oxidative stress markers. Our work demonstrates that the antihyperalgesic effect of EA on CFA-induced inflammatory pain could be partially associated with higher IL-10 levels and catalase activity, and that these effects may be dependent, at least in part, on the activation of peripheral FPR2/ALX.

Moxibustion enables protective effects on rheumatoid arthritis-induced myocardial injury transforming growth factor beta1 signaling and metabolic reprogramming.

To examine the effects of moxibustion on myocardial injury and myocardial metabolomics in rats with rheumatoid arthritis (RA) based on the transforming growth factor beta1 (TGF-β1)/Smads signaling pathway. One hundred rats were treated with saline [normal control (NC) group] or complete Freund's adjuvant (CFA) by right plantar injection for the RA model group, and the latter were randomly divided into 4 groups. Tripterygium wilfordii polyglycoside tablets (, TPT) have anti-inflammatory and are widely used in the clinical treatment of RA, therefore serving as a positive control group. Three days post injection rats were given TPT tablet (TPT group), acupuncture therapy (APT group), and moxibustion treatment (MOX group) for 15 consecutive days, while NC group and model group were equally grasped and fixed and received normal saline. Rat joint swelling scores and arthritis index (AI) were evaluated in each group before the CFA challenge, therapy and after receiving therapy. Myocardial ultrastructure was observed by electron microscope. Enzyme-linked immunosorbent assay was used to detect cardiac troponin I (cTnI) levels in rat myocardial tissue. Quantitative reverse transcription polymerase chain reaction and Western blotting analysis were used to measure the mRNA and protein levels of TGF-β signaling molecules including TGF-β1, Smad2, Smad3, Smad4, and Smad7. Myocardial metabolomics was analyzed using gas chromatography-mass spectrometer. Compared with model group, RA model rats receiving TPT, acupuncture, or moxibustion therapy all showed reduced joint swelling scores and AI (all P < 0.01) and improved myocardial damage, whereas rats treated with moxibustion were found to be more marked. Consistently, the expressions of cTnI, TGF-β1, Smad2, Smad3, and Smad4 were found to be elevated in model rat group in contrast to NC rats and were significantly downregulated in TPT, APT and MOX group when compared with model group, while the levels of Smad7 showed the opposite result (all P < 0.01). Moreover, the dissection of metabolomics suggested a novel metabolite biomarker panel including D-Xylulose 5-phosphate, dihydroxyacetone phosphate, arachidonic acid, etc was defined and implicated in amino acid, glucose, and fatty acid metabolic processes as revealed by principal component analysis and partial least squares discriminant analysis. Moxibustion prevents RA-induced inflammatory response and offers potent therapeutic effects on myocardial dysfunctions. The protective effects might be associated with its role in TGF-β1 inactivation and metabolic reprogramming.

Efficacy of electroacupuncture stimulating Zusanli (ST36) and Xuanzhong (GB39) on synovial angiogenesis in rats with adjuvant arthritis.

To investigate the efficacy of electroacupuncture (EA) stimulating Zusanli (ST36) and Xuanzhong (GB39) on synovial angiogenesis in rats with adjuvant arthritis (AA). AA models were established by bilateral injection of Freund's complete adjuvant (FCA) in male Sprague-Dawley rats. Three days after injection, rats were given EA at Zusanli (ST36) and Xuanzhong (GB39) acupoints, once every other day, for 16 d. The arthritis index score, paw volume, and hematoxylin-eosin (HE) staining was performed for each animal. Angiogenesis marker cluster of differentiation 34 (CD34) expression and synovial cell apoptosis in synovial tissue were observed. The levels of Notch1, hairy and enhancer of split homolog-1 (Hes1), transforming growth factor-beta (TGF-β) and basic fibroblast growth factor (bFGF) were subsequently detected. We found that EA significantly decreased arthritis index scores, paw volume, and HE staining scores. EA could significantly inhibit the expression of CD34, promoting apoptosis of synovial cells in the joint synovial tissue of AA rats. The expression of Notch1 signaling pathway proteins and mRNAs (Notch1, Hes1, TGF-β, and bFGF) were markedly downregulated by EA treatment. These results prove that EA attenuates synovial angiogenesis by inhibiting the Notch1 signaling pathway in AA rat models. Based on our findings, we propose that EA is a promising complementary and alternative therapy in rheumatoid arthritis.

Evaluating the effect of ursodeoxycholic acid (UDCA) in comparison with dexamethasone and diclofenac in a rat model of rheumatoid arthritis.

Ursodeoxycholic acid (UDCA) is known for its major effects on the liver, but its impact on autoimmune diseases is not well understood. This study aimed to assess the effectiveness of UDCA in controlling rheumatoid arthritis (RA) in an in vivo setting. Experimental RA was induced in rats using Freund's complete adjuvant, and the effects of UDCA (50,100 mg/kg) were compared to those of dexamethasone and diclofenac by measuring changes in paw size, IL-17, pro-inflammatory cytokines, oxidative stress (GSH, MDA), and radiological changes. The administration of UDCA resulted in decreased cartilage damage, reduced paw edema, and a decrease in the release of pro-inflammatory cytokines and oxidative stress. Additionally, X-ray joint alterations were observed in the UDCA-treated group compared to the dexamethasone and diclofenac groups. These results suggest that UDCA has anti-rheumatoid arthritis properties due to its ability to minimize oxidative stress and inflammation in arthritis-affected rats.

Antiarthritic Activity and Inflammatory Mediators Modulation Effect of Traditional Ajmodadi Churna on Arthritis Experimental Model.

The study was designed to evaluate anti-arthritic activity of Ajmodadi Churna (AC) and its effect on Complete freund's adjuvant (CFA)-induced arthritis in Wistar rats. Arthritis was induced by injecting 0.2 mL CFA into sub plantar surface of left hind paw. Test sample AC-1 and AC-2, 200 and 400 mg/kg, respectively was given to the animals for 21 consecutive days. The increase in swelling was observed after induction of arthritis. The paw edema was measured on 0, 3, 7, 14 and 21 day using Vernier calliper after the induction of arthritis. The collected blood samples further used for the estimation of red blood cells (RBC), white blood cells (WBC), erythrocytes sedimentation rate (ESR), and hemoglobin (Hb), using hematology analyzer. Serum concentration of IL-6 and TNF-α were also measured using rat ELISA kits. Results showed that a significant reduction in paw edema was observed in AC-2 treated rats. The paw edema was restored on day 21 was 4.48 mm for AC-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. 0.83 for AC-2 and 1.50 for AC-1. A decrease in levels of RBC and hemoglobin were 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. It can be concluded from the study that AC possess significant anti-arthritic activity. Furthermore, this condition was linked to a reduction in abnormal humoral immune responses.

Geraniol Suppresses Oxidative Stress, Inflammation, and Interstitial Collagenase to Protect against Inflammatory Arthritis.

Geraniol (GER) is a plant-derived acyclic isoprenoid monoterpene that has displayed anti-inflammatory effects in numerous in vivo and in vitro models. This study was therefore designed to evaluate the antiarthritic potential of GER in complete Freund's adjuvant (CFA)-induced inflammatory arthritis (IA) model in rats. IA was induced by intraplantar injection of CFA (0.1 mL), and a week after CFA administration, rats were treated with various doses of methotrexate (MTX; 1 mg/kg) or GER (25, 50, and 100 mg/kg). Treatments were given on every alternate day, and animals were sacrificed on the 35th day. Paw volume, histopathological, hematological, radiographic, and qPCR analyses were performed to analyze the severity of the disease. GER significantly reduced paw edema after 35 days of treatment, and these results were comparable to the MTX-treated group. GER-treated animals displayed a perfect joint structure with minimal inflammation and no signs of cartilage or bone damage. Moreover, GER restored red blood cell and hemoglobin levels, normalized erythrocyte sedimentation rate, platelet, and c-reactive protein values, and also attenuated the levels of rheumatoid factor. RT-qPCR analysis demonstrated that GER decreased mRNA expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta. GER also down-regulated the transcript levels of cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1, prostaglandin D2 synthase, and interstitial collagenase (MMP-1). Molecular docking of GER with COX-2, TNF-α, and MMP-1 also revealed that the antiarthritic effects of GER could be due to its direct interactions with these mediators. Based on our findings, it is conceivable that the antiarthritic effects of GER could be attributed to downregulation of pro-inflammatory mediators and protease like MMP-1.

Xanthohumol relieves arthritis pain in mice by suppressing mitochondrial-mediated inflammation.

Chronic pain is the most common symptom for people who suffer from rheumatoid arthritis and it affects approximately 1% of the global population. Neuroinflammation in the spinal cord induces chronic arthritis pain. In this study, a collagen-induced arthritis (CIA) mice model was established through intradermally injection of type II collagen in complete Freund's adjuvant solution. Following CIA inducement, the paws and ankles of mice were found to swell, mechanical pain and spontaneous pain were induced, and their motor coordination was impaired. The spinal inflammatory reaction was triggered, which presented as severe infiltration of inflammatory cells, and the expression levels of GFAP, IL-1β, NLRP3, and cleaved caspase-1 increased. Oxidative stress in the spinal cord of CIA mice was manifested as reduced Nrf2 and NDUFB11 expression and SOD activity, and increased levels of DHODH and Cyto-C. At the same time, spinal AMPK activity was decreased. In order to explore the potential therapeutic options for arthritic pain, Xanthohumol (Xn) was intraperitoneally injected into mice for three consecutive days. Xn treatment was found to reduce the number of spontaneous flinches, in addition to elevating mechanical pain thresholds and increasing latency time. At the same time, Xn treatment in the spinal cord reduced NLRP3 inflammasome-mediated inflammation, increased the Nrf2-mediated antioxidant response, and decreased mitochondrial ROS level. In addition, Xn was found to bind with AMPK via two electrovalent bonds and increased AMPK phosphorylation at Thr174. In summary, the findings indicate that Xn treatment activates AMPK, increases Nrf2-mediated antioxidant response, reduces Drp1-mediated mitochondrial dysfunction, suppresses neuroinflammation, and can serve to relieve arthritis pain.

Investigation of the Mechanism of Action of Periploca forrestii Schltr. Extract on Adjuvant Collagen Rats Based on UPLC-Q-Orbitrap-HRMS Non-Targeted Lipidomics.

Periploca forrestii Schltr. (P. forrestii) is a classical medicinal plant and is commonly used in traditional medicine for the treatment of rheumatoid arthritis, soft tissue injuries, and traumatic injuries. The aim of this study was to evaluate the anti-arthritic effects of three fractions of P. forrestii alcoholic extracts (PAE), P. forrestii water extracts (PWE), and total flavonoids from P. forrestii (PTF) on Freund's complete adjuvant (FCA)-induced arthritis in rats, and to use a non-targeted lipidomic method to investigate the mechanism of action of the three fractions of P. forrestii in the treatment of rheumatoid arthritis. To assess the effectiveness of anti-rheumatoid arthritis, various indicators were measured, including joint swelling, histopathological changes in the joints, serum cytokines (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)), and the joint inflammatory substance prostaglandin E2 (PGE2). Finally, ultra-performance liquid chromatography-quadrupole-orbitrap-high-resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS) was used to determine the non-targeted lipid histology of the collected rat serum and urine samples to investigate the possible mechanism of action. PWE, PAE, and PTF were all effective in treating FCA-induced rheumatoid arthritis. The administered groups all reduced joint swelling and lowered serum inflammatory factor levels in rats. In the screening of lipid metabolite differences between serum and urine of the rat model group and the normal group, a total of 52 different metabolites were screened, and the levels of lipid metabolites in PWE, PAE, and PTF were significantly higher than those in the normal group after administration. In addition, PWE, PAE, and PTF may have significant therapeutic effects on FCA-induced arthritis by modulating nicotinic acid, nicotinamide, and histidine metabolic pathways.

Soft Tissue Manipulation Alters RANTES/CCL5 and IL-4 Cytokine Levels in a Rat Model of Chronic Low Back Pain.

Low back pain (LBP) is a common musculoskeletal complaint that can impede physical function and mobility. Current management often involves pain medication, but there is a need for non-pharmacological and non-invasive interventions. Soft tissue manipulation (STM), such as massage, has been shown to be effective in human subjects, but the molecular mechanisms underlying these findings are not well understood. In this paper, we evaluated potential changes in the soft tissue levels of more than thirty pro- or anti-inflammatory cytokines following instrument-assisted STM (IASTM) in rats with chronic, induced LBP using Complete Freund's Adjuvant. Our results indicate that IASTM is associated with reduced soft tissue levels of Regulated on Activation, Normal T cell Expressed and Secreted (RANTES)/Chemokine (C-C motif) ligand 5 (CCL5) and increased soft tissue levels of Interleukin (IL)-4, which are pro-inflammatory and anti-inflammatory factors, respectively, by 120 min post-treatment. IASTM was not associated with tissue-level changes in C-X-C Motif Chemokine Ligand (CXCL)-5/Lipopolysaccharide-Induced CXC Chemokine (LIX)-which is the murine homologue of IL-8, CXCL-7, Granulocyte-Macrophage-Colony Simulating Factor (GM-CSF), Intercellular Adhesion Molecule (ICAM)-1, IL1-Receptor Antagonist (IL-1ra), IL-6, Interferon-Inducible Protein (IP)-10/CXCL-10, L-selectin, Tumor Necrosis Factor (TNF)-α, or Vascular Endothelial Growth Factor (VEGF) at either 30 or 120 min post-treatment. Combined, our findings raise the possibility that IASTM may exert tissue-level effects associated with improved clinical outcomes and potentially beneficial changes in pro-/anti-inflammatory cytokines in circulation and at the tissue level.

The role of the gut–microbiota–brain axis via the subdiaphragmatic vagus nerve in chronic inflammatory pain and comorbid spatial working memory impairment in complete Freund's adjuvant mice

Chronic inflammatory pain (CIP) is a common public medical problem, often accompanied by memory impairment. However, the mechanisms underlying CIP and comorbid memory impairment remain elusive. This study aimed to examine the role of the gut–microbiota–brain axis in CIP and comorbid memory impairment in mice treated with complete Freund's adjuvant (CFA). 16S rRNA analysis showed the altered diversity of gut microbiota from day 1 to day 14 after CFA injection. Interestingly, fecal microbiota transplantation (FMT) from healthy naive mice ameliorated comorbidities, such as mechanical allodynia, thermal hyperalgesia, spatial working memory impairment, neuroinflammation, and abnormal composition of gut microbiota in the CFA mice. Additionally, subdiaphragmatic vagotomy (SDV) blocked the onset of these comorbidities. Interestingly, the relative abundance of the bacterial genus or species was also correlated with these comorbidities after FMT or SDV. Therefore, our results suggest that the gut–microbiota–brain axis via the subdiaphragmatic vagus nerve is crucial for the development of CIP and comorbid spatial working memory impairment in CFA mice.

Modeling of rheumatoid arthritis is accompanied by a change in the cortico-papillary ratio of hyaluronidase activity of the kidneys

Background. Rheumatoid arthritis is a disease that is accompanied by impaired kidney function in most patients, which attracts the attention of researchers to this problem. Both in the joints and in the kidneys, hyaluronic acid plays an important role, the degree of polymerization of which depends on the functioning of these organs. The degradation of hyaluronic acid by hyaluronidases may be a factor involved in the development of the inflammatory process in rheumatoid arthritis as well as in the realization of osmoregulatory functions of the body. In this regard, the purpose of this work was to find out whether the development of this pathology can affect on the hyaluronidase activity in the kidneys. THE AIM: to clarify is in the experiment formation of inflammatory changes in the joints accompanied by a change in the cortico-papillary ratio of hyaluronidase activity in the kidneys.Materials and Methods. A model of rheumatoid arthritis was created in Wistar rats by a single administration of a complete Freund adjuvant. After 7 weeks after injection, the degree of pathological changes in the joints was assessed by radiographic method and the hyaluronidase activity in various kidney zones was determined using zymography.Results. It was revealed that in the cortex and papillary zone of the kidneys, hyaluronidase activity was characteristic of proteins with a molecular weight of 63 and 73 kDa. It has been shown that the development of the inflammatory process is accompanied by a change in the corticopapillary ratio of hyaluronidase activity in the kidneys. In the papillary zone, a decrease in hyaluronidase activity was observed, on average, by 1.6 times, while in the renal cortex, hyaluronidase activity increased by 1.5 times.Conclusion. Such a redistribution of hyaluronidase activity in the cortical and papillary zones of the kidneys in rheumatoid arthritis can lead to a decrease in the effectiveness of the mechanism of osmotic concentration of urine and impaired kidney function.

Toxicity Test on the Combination of Caesalpinia sappan and Zingiber officinale in Rattus norvegicus Induced by Complete Freund's Adjuvant

Highlights:1. This study determined the toxic effects of combining Caesalpinia sappan and Zingiber officinale extracts, aspeople need to be aware of the potential side effects of these common herbal remedies for the treatment ofrheumatoid arthritis.2. The combination of Caesalpinia sappan and Zingiber officinale extracts is efficacious and non-toxic as an antiarthritis treatment, hence, the ingredients can be upgraded to standardized herbal medicines andphytopharmaceuticals. Abstract Sappanwood (Caesalpinia sappan) and red ginger (Zingiber officinale) are plant species that have been studied for theirefficacy in treating inflammation related to rheumatoid arthritis. This study aimed to examine the effects of combiningsappanwood and red ginger in order to determine the potential toxicity of the herbal extracts in medicine. The toxicitytesting was carried out in vivo using 32 Wistar strain male white rats (Rattus norvegicus) grouped into eight groups of four.The rats were injected with complete Freund's adjuvant to induce a chronic inflammatory effect. The eight groups consistedof the negative control group, the positive control group, the normal group, and five treatment groups. This study wasconducted by observing the animals for toxic symptoms and death to determine the safety of the extracts and drugs. Theobservation results were analyzed using a one-way analysis of variance (p&lt;0.05). The analysis results showed that weightgain and relative organ weight among the groups had no significant differences (p&gt;0.05). Microscopic examination of theorgan preparations observed under a light microscope revealed no significant changes or adverse effects in rats treated withthe extracts or drugs. In conclusion, a combination of sappanwood and red ginger ethanol extracts administered orally hasno toxic effect in rats injected with complete Freund's adjuvant.

Mechanisms involved in the antinociceptive and anti-inflammatory effects of xanthotoxin.

Xanthotoxin (XAT) is a natural furanocoumarin clinically used in the treatment of skin diseases such as vitiligo and psoriasis. Recent studies have also investigated its effects on anti-inflammatory, anti-cognitive dysfunction, and anti-amnesia as a guideline for clinic application. However, little is known about its effects on pain relief. Here, we tested the analgesic effects of XAT in serious acute pain and chronic pain models. For acute pain, we used hot-, capsaicin- and formalin-induced paw licking. Nociceptive threshold was measured by mechanical stimuli with von Frey filaments. For chronic pain, we injected complete Freund's adjuvant (CFA) into the mice's plantar surface of the hind paw to induce inflammatory pain. Heat and mechanical hyperalgesia were evaluated by radiant heat and von Frey filament tests, respectively. To investigate the mechanisms underlying the analgesic effect of XAT, we used calcium imaging and western blot to assess transient receptor potential vanilloid 1 (TRPV1) activity and expression in isolated L4-L6 dorsal root ganglion (DRG) neurons. Haematoxylin and eosin (HE) staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine immune cell recruitment and proinflammatory factor release from skin tissue from paw injection sites. Our results demonstrated that XAT not only reduced acute pain behaviors generated by hot, capsaicin, and formalin but also attenuated CFA-induced heat and mechanical hyperalgesia. The analgesic activity of XAT may be achieved by controlling peripheral inflammation, lowering immune cell infiltration at the site of inflammatory tissue, reducing inflammatory factor production, and therefore inhibiting TRPV1 channel sensitization and expression.

Eichhornia crassipes Ameliorated Rheumatoid Arthritis by Modulating Inflammatory Cytokines and Metalloproteinase Enzymes in a Rat Model.

Background and Objectives: This study was planned to investigate the anti-arthritic property of flowers of E. crassipes in a Sprague-Dawley rat model by administering Freund's Complete Adjuvant (FCA). Materials and Methods: Arthritis was induced at day 0 in all rats except negative controls, while arthritic progress and paw edema were analyzed on specific days (8th, 13th, 18th, and 23rd) via the macroscopic arthritic scale and a digital Vernier caliper, respectively. Histopathological parameters were examined using a Hematoxylin and Eosin (H&E) staining method. Blood samples were withdrawn from rats to investigate the effects of the E. crassipes flower on the mRNA expression values of inflammatory markers, via a reverse transcription PCR technique. Serum samples were used to determine prostaglandin E2 (PGE2) levels using enzyme-linked immunosorbent assay (ELISA). Values of alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine, and urea, besides hematological parameters, i.e., the hemoglobin (Hb) content and complete blood count (CBC), were investigated. Results: The data showed that E. crassipes inhibited the arthritic progress and ameliorated the paw edema. The amelioration of parameters assessed via the histopathological analysis of ankle joints, as well as via hematological analysis, confirmed the diminution of rheumatoid arthritis (RA) in the plant-treated groups. Treatment with E. crassipes inhibited the expression levels of tumor necrosis factor-α (TNF-α), interleukins (IL-1β and IL-6), nuclear factor KappaB (NF-κB), matrix metalloproteinase (MMP-2 and MMP-3), and vascular endothelial growth factor (VEGF). Serum PGE2 levels were also found to be reduced in treatment groups. A biochemical investigation revealed the improvements in hepatic markers in plant-treated groups. The data indicated that the plant has no hepatotoxic or nephrotoxic effects at the studied dose. GC-MS (Gas Chromatography-Mass Spectrometry) analysis displayed the presence of phytochemicals having known anti-inflammatory and antioxidant properties. Conclusions: Therefore, it may be concluded that E. crassipes possesses anti-arthritic characteristics that could be attributed to the modulation of pro-inflammatory cytokines, MMPs, and PGE2 levels.

Anti-arthritic and Antioxidant Effects of Trehalose in an Experimental Model of Arthritis.

The purpose of the present study was to study the potential anti-arthritic and antioxidant effects of trehalose in an experimental model of complete Freund's adjuvant (CFA)-induced arthritis. Arthritis was induced via subcutaneous injection of CFA (0.1) into the right footpad of each rat. Trehalose (10 mg/kg per day) and indomethacin (5 mg/kg) as a reference drug were intraperitoneally injected into CFA-induced arthritic rats from days 0 to 21. Changes in paw volume, pain responses, arthritic score, and oxidative/antioxidative parameters were determined. Trehalose administration could significantly decrease arthritis scores (p <0.01) and paw edema (p <0.001), and significantly increase the nociceptive threshold (p <0.05) in CFA-induced arthritic rats. Trehalose also significantly reduced the pro-oxidant-antioxidant balance values when compared to CFA treatment alone. In addition, no significant difference was found between the trehalose group and indomethacin as a positive control group. The current study suggests that trehalose has a protective effect against arthritis, which may be mediated by antioxidative effects of this disaccharide.