Adjuvant Arthritis Research Articles

Pharmacodynamic mechanism of modified Ganlu Yaoyu San intreatment of rheumatoid arthritis based on MAPK signaling pathway

According to the findings, modified Ganlu Yaoyu San has a good anti-inflammatory activity, and can significantly alleviate the degree of arthritis. Its therapeutic effect for rheumatoid arthritis may be related to the regulation of MAPK pathway of synovial cells. In the study, the rat adjuvant arthritis(AA) model was established to further investigate the pharmacodynamic mechanism for regulating MAPK pathway of synovial cells. Enzyme-linked immune assay was used to determine the serum TNF-α level of AA rats administered with drug for two weeks, synovial tissue protein kinases ERK1/2 and p38 content were determined by immunohistochemistry, synovial tissue JNK1, ERK1, p38 gene(mRNA) expression were detected with fluorescence quantitative PCR(RT-PCR) method. According to the results, after administration for two weeks, the levels of serum TNF-α of AA rat was significantly decreased(P<0.05). After administration for four weeks, the protein expressions of p38 and ERK1/2 in synovial tissue were reduced(P<0.05 or P<0.01), the gene expressions of JNK1, p38 and ERK1 in knee joint synovial tissue were reduced(P<0.05 or P<0.01). In conclusion, modified Ganlu Yaoyu San can effectively treat rheumatoid arthritis. Its mechanism might be related to the reduction of TNF-α levels in serum, protein expression of p38 and ERK1/2 in synovial tissue, and JNK1, p38 and ERK1 gene expressions, and regulation of MAPK pathway.

Anti-inflammatory activity of two varieties of pumpkin seed oil in an adjuvant arthritis model in rats

The aim of the present research was to evaluate the anti-inflammatory activity of pumpkin seed oils (PSOs) of an Egyptian and European variety, in a rat model of adjuvant arthritis. Edema thickness, plasma tumor necrosis factor-α (TNF-α) and erythrocyte sedimentation rate (ESR) were determined as inflammatory biomarkers while malondialdehyde (MDA) and total antioxidant capacity (TAC) were assessed as indicative of oxidative stress. Chromosomal aberration, sperm shape abnormalities, and DNA fragmentations are cytogenetic parameters which aid in tracing inflammatory and oxidative activity. Phenolic contents and β-carotene were determined in PSOs. The results showed elevated ESR, plasma TNF-α, plasma MDA, liver cellular DNA fragmentation, bone marrow chromosomal aberration, sperm shape abnormalities with a reduction in plasma TAC and body weight gain in an adjuvant arthritis control compared to a healthy control. Administration of low and high doses of either Egyptian or European PSO improved all the aforementioned parameters with variable degrees.

Effects of Tibetan medicine Manu-Xitang granules on the expression of prostaglandin E2 and TNF-α on adjuvant arthritis rats

Objective To study the effect of Tibetan Medicine Manu-Xitang granules on the expression of prostaglandin E2 and TNF-α on adjuvant arthritis (AA) rats. Methods A total of 60 rats were randomly divided into the control group, the model group, the Aspirin group, the low-, medium- and high- dose Manu-Xitang granules groups (each group with 10). Complete Freund's adjuvant method was used for the adjuvant arthritis model except the control group. All the groups started treatment at 8th day, and the treatment last 20 days. The low-, middle- high-dosage groups were treated with 2.0, 1.0, 0.5g/(kg body weight) Manu-Xitang granules, while the the Aspirin group with ASP (0.27 g/kg), control and medol groups with the equal volumn saline. The body weight, the swelling of primary side of arthritis index were observed. The levels of PGE2 and TNF-α were measured by ELISA. Results At 6th, 12th, 18th day, compared with the model group, the body weights in Aspirin group, the low-, middle-, high-dosage groups significantly increased (P<0.05 or P<0.01); at the 6th day, the foot swelling (0.40% ± 0.18%, 0.50% ± 0.25%, 0.55% ± 0.35% vs. 0.85% ± 0.15%) in the Aspirin group, middle-, and high-dosage groups significantly decreased (P<0.05 or P<0.01). And the PGE2 (0.66 ± 0.31 vs. 0.39 ± 0.11) and TNF-α (0.72 ± 0.24 vs. 0.50 ± 0.15) in the high- dose group were significantly lower than the model group (P<0.05). Conclusions The Tibetan medicine Manu-Xitang granules could treat AA through the changes of PGE2 and TNF-α. Key words: Tibetan drugs; Manu-Xitang granules; Adjuvant arthritis; Prostaglandin E2; Rats

Mechanism studies of Xinfeng capsule on improving cardiovascular function though toll-like receptor 4/nuclear factor kappa B pathway in a rat model of adjuvant arthritis

OBJECTIVETo observe the impact of Xinfeng capsule (XFC) on cardiovascular function in adjuvant arthritis (AA) model rats and investigate the mechanism though toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κβ) signaling pathway. METHODSSeventy rats were randomly divided into seven groups: normal control (NC), model control (MC), tripterygium glycosides tablet (TPT), methotrexate (MTX), high, moderate and low dose XFC group. The administration began from day 19 after modeling for 30 day. Paw swelling, arthritic index (AI), cardiac function indexes and myocardial pathological pattern were detected. The expression of TLR4, myeloid differentiation factor (MyD) 88, interleukin-1 receptor-associated kinase (IRAK) 1, tumor necrosis factor receptor associated factor (TRAF) 6, NF-κβ, tumor necrosis factor-alpha (TNF-α) proteins in myocardial tissue were determined by western blot method. RESULTSPaw swelling and AI in MC group increased in MC group (P < 0.01), and decreased in high and moderate dose XFC groups (P < 0.01 or P > 0.05). Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR) were elevated in MC group (P < 0.01), and ± dp/dtmax and CI were reduced (P < 0.01); while LVSP, LVEDP and HR declined and ±dp/dtmax, CI improved in high dose XFC group (P < 0.05 or P < 0.01). LVSP in high dose XFC group were reduced more than other treatment groups (P < 0.05 or P < 0.01). The improvements on LVEDP, dp/dt-max were superior to MTX and low dose XFC group, and the improvement on CI was better than low dose XFC group (P < 0.05 or P < 0.01). Myocardial fibers arranged irregular in MC group with intracellular edema and mitochondria damage. The modifications on myocardial structural were shown in each treatment group, but more prominent in TPT, high and moderate dose XFC group. The proteins of TLR4, MyD88, IRAK1, TRAF6, NF-κβ, TNF-α were highly expressed in MC group, and those proteins declined in high and moderate dose XFC group (P < 0.05 or P < 0.01). High dose XFC group was superior to MTX and low dose XFC group on reducing TLR4, NF-κβ, TNF-α (P < 0.05). CONCLUSIONXFC can not only inhibit the excessive activation of TLR4/NF-κB signaling pathway and the increased inflammatory mediators, but also reduce the damage of myocardial tissue and cells.

Effects of Adjuvant-Induced Inflammation on Disposition of Diclofenac and Its Metabolites in Perfused Rat Liver

The reactive metabolites of diclofenac (DF) such as 1-O-acyl glucuronide (DF-Glu) are hypothesized to result in idiosyncratic hepatotoxicity. However, it is unclear whether inflammation affects the hepatic disposition of DF and its metabolites. To clarify the alterations in the disposition of DF and its metabolites in inflammatory conditions, we performed in situ perfused rat liver experiments. Using adjuvant arthritis rats as a model of inflammation, the elimination of DF, 4’-hydroxydiclofenac, and DF-Glu from the perfusate was observed to be delayed in comparison with the control. Parameter sensitivity analysis for hepatic DF disposition revealed that the area under the plasma concentration-time curve (AUC) and the maximum concentration (Cmax) of DF-Glu in the liver markedly increased along with a decrease in intrinsic excretion clearance of DF-Glu (CLint,bile,Glu) and an increase in intrinsic glucuronidation clearance (CLint,Glu) of DF-Glu. It is possible that the elimination of DF-Glu from the perfusate in adjuvant arthritis rats was delayed via reduction of biliary excretion of DF-Glu.

Boi-ogi-to (TJ-20), a Kampo Formula, Suppresses the Inflammatory Bone Destruction and the Expression of Cytokines in the Synovia of Ankle Joints of Adjuvant Arthritic Rats.

TJ-20 is a formula consisting of 6 herbs that has been used in the clinical treatment of rheumatoid arthritis (RA) in China and Japan for centuries. However, scientific evidence of the effects of TJ-20 has not been established. In the present study, we focused on the therapeutic effects and investigated the main function of TJ-20 on adjuvant arthritis (AA), an animal model of RA, which was induced with complete Freund's adjuvant (CFA). TJ-20 was administered orally at 600 mg/kg once a day from 0, 7, and 10 days to 8 weeks after the CFA treatment. TJ-20 significantly ameliorated inflammatory progression and bone destruction in AA in a time-dependent manner. Furthermore, TJ-20 significantly reduced the increased changes in a number of macrophages and helper T cells. Moreover, TJ-20 suppressed the expression of TNF-α whereas it augmented the expression of IL-10 and attenuated Th1 cells responses in the synovia of the ankle joint. Therefore, TJ-20 regulated the expression of proinflammatory and anti-inflammatory cytokines in macrophages and Th1/Th2 balance in the synovia of ankle joints in AA rats. These results suggest the positive anti-inflammatory effect of TJ-20 and provide a scientific basis for the clinical use of TJ-20 for RA.

PATHOMORPHOLOGICAL AND COMPUTERIZED TOMOGRAPHY STUDY OF IBUPROFEN AND COMBINATION WITH 2-PHENYL-3-CARBETHOXY-4-DIMETHYLAMINOMETHYL-5-OKSIBENZOFURAN HYDROCHLORIDE (VINBORON) INFLUENCE ON THE JOINTS OF LOWER EXTREMITIES OF RATS WITH EXPERIMENTAL RHEUMATOID ARTHRITIS

The most frequent adverse reactions to nonsteroidal anti-inflammatory drugs are upper and lower gastrointestinal tract disorders, hepatotoxicity, nephrotoxicity, a decrease in the synthesis of proteoglycans of the cartilaginous tissue and allergic reactions. In our previous studies we have found out that Ukrainian vinboron, the antispasmodic with polytrophic pharmacological effects, can improve the safety profile of the popular non-steroidal antiphlogistics ibuprofen and can also potentiate its anti-inflammatory effect. That fact has become the background to a detailed study of the therapeutic action mechanisms of these drugs when they are used together. Aim of the study. To characterize the influence of vinboron (11 mg/kg, i.g.) on the anti-inflammatory activity of ibuprofen (218 mg/kg, i.g.) when combined, in a model of experimental rheumatoid arthritis according to pathomorphological, morphometric and cone-ray computerized tomography of rats’ hind limbs. Materials and methods . To reproduce the conditions for the administration of ibuprofen corresponding to its clinical use, we selected a model of experimental rheumatoid arthritis-adjuvant arthritis (AA) in rats. On the 28 th day the animals were excluded from the experiment. It was followed by the amputation of the hind limbs at the level of the hocks (art. coxae) and fixation of the material in 10.0% aqueous solution of neutral formalin. The ankle joints were decalcified. The drugs were prepared according to the standard procedure. Microscopy and photographing of histological preparations were carried out with the light microscope OLIMPUS BX 41 at magnifications of 40, 100, 200 and 400 times. The obtained images were processed and morphometry was carried out with the program “Quick PHOTO MICRO 2.3”. The contents of the cellular elements was determined on the basis of the equivalent unit area (1 mm2). Cone-ray computerized tomography was carried out with a digital panoramic cone-ray tomographic scanner “Point 3D Combi 500” (PointNix, South Korea), using a planar sensor with a scanning site of 9 × 12 mm and a radial load of 30-100 μSv. Scanning was carried out with a voltage of 52.0 kV and the electric current of 6.0 mA. For the integral characteristics of the linear dimensions of rats’ hind limbs joints we proposed a formula for calculating the joint volume (Patent of Ukraine No. UA 117538). For the integral characteristics of the dimensions of rats’ hind limbs linear joints we proposed a formula for calculating the joint volume (Patent of Ukraine No. UA 117538). Results and discussion. The use of ibuprofen in combination with vinboron was accompanied by restoration of the structure and function of the cartilage and bone tissues, evidenced by the appearance of acidic and sulfated glycosaminoglycans. The restoration of the bone tissue was realized by the proliferation of the connective tissue and its transformation into cartilage. The synovial membrane was hyperplastic with proliferating synoviocytes of both macrophage and secretory types. In rare cases, the phenomenon of moderate edema and minor focal lymphohistiocytic infiltration persisted. Thus, the use of ibuprofen and ibuprofen in combination with vinboron in rats with adjuvant arthritis resulted in a reduction or absence of inflammation in both the joint and periarticular tissues. The analysis of the results of cone-ray computerized tomography and digital radiography showed that against the background of simultaneous administration of ibuprofen and vinboron there was a tendency to increase the anti-inflammatory effect of the studied antiphlogistics on the rats’ joints affected by adjuvant arthritis. On the 28-th day of the experiment there was the indicatation of decrease of the volume of the hind limb joints by an average of 33.8% compared to the control animals and a near-complete restoration of the width of the joint gaps of the joints examined. It was also determined that vinboron can harmonize the anti-inflammatory effect of ibuprofen when they are used together. Сonclusion. The use of vinboron in combination with ibuprofen leads to a more pronounced pathogenetic anti-inflammatory structural-modifying effect of this antiphlogistics.

The Anti-Inflammatory Activity of Toonaciliatin K against Adjuvant Arthritis.

Toonaciliatin K is a natural limonoid purified from the Toona ciliata Roem. var. ciliata (Meliaceae). This study is to reveal the inflammatory suppression effect of toonaciliatin K and further the intrinsic mechanism. Firstly, anti-inflammatory effect of toonaciliatin K was evaluated in lipopolysaccharide- (LPS-) induced RAW264.7 cells. RT-PCR results indicated that the mRNA expressions of TNF-α, IL-6, and IL-1β were downregulated by toonaciliatin K. The toonaciliatin K inhibited TNF-α, IL-6, and IL-1β levels stimulated by LPS. Furthermore, LPS elicited the excess iNOS and COX-2 mRNA and protein production and toonaciliatin K attenuated the excess production. Western blot assay demonstrated that MAPK and NF-κB signaling pathways play critical roles in the toonaciliatin K's anti-inflammatory activity. Secondly, toonaciliatin K inhibited carrageenan-induced paw edema in rats. Thirdly, toonaciliatin K alleviated the paw swelling and improved arthritis clinical scores in the adjuvant arthritis rats. Toonaciliatin K decreased the proinflammatory cytokines levels and Mankin scores in adjuvant arthritis rats. The HE staining, safranin O-fast green, and toluidine blue staining results demonstrated that toonaciliatin K alleviated the histological changes of paw, for example, pannus formation, focal loss of cartilage, bone erosion, and presence of extra-articular inflammation. Hence, toonaciliatin K is a promising agent for treatment of arthritis.

Evaluation of liposomal carnosine in adjuvant arthritis.

Liposomal carnosine could overcome the problems associated with direct application of this peptide. Anti-inflammatory and antioxidant effects of liposomal and non-liposomal carnosine in adjuvant arthritis were compared. The experiments were done on healthy animals, untreated arthritic animals, arthritic animals with oral administration of carnosine, and with subcutaneous administration of liposomal carnosine, both administered in the same daily dose of 150 mg/kg b.w. during 28 days. Carnosine reduced hind paw volume on day 28. Both forms markedly decreased interleukin-1β, matrix metalloproteinase-9 and monocyte chemoattractant protein-1 (MCP-1) in plasma on day 14. Only liposomal carnosine reduced significantly MCP-1. Malondialdehyde, 4-hydroxynonenal, resistance to Fe2+-induced oxidation and protein carbonyls were significantly corrected after administration of any form of carnosine. Liposomal carnosine corrected more effectively the oxidative stress in plasma than did carnosine. In brain tissue, our results showed protective ability of both forms of carnosine against oxidation of proteins and lipids. They also corrected the resistance to Fe2+-induced oxidation in arthritic animals. We found that only liposomal carnosine decreased the mRNA expression of inducible nitric oxide synthase in cartilage tissue. It can be concluded that the liposomal drug-delivery system is improving the pharmacological properties of carnosine administered in arthritis.

Antiinflammatory effect of herbal preparations on adjuvant arthritis in rats

The present study aimed to investigate the antiinflammatory effects of 2 herbal extracts (CDS and CDSM) in rats with experimental adjuvant arthritis (AA). Joint swelling, development of polyarthritis, blood indices, pro/antioxidant activity, and proinflammatory cytokine IL-17 in blood serum, as well as the liver and paw histopathology and histochemistry, were assessed. Oral injections of extracts showed an antiinflammatory effect analogous to that of diclofenac (DF), improved blood indices and suppressed level of IL-17, significantly diminished joint swelling, and histological changes in the joints. Increase in CAT activity and AOA and reduction in lipid peroxidation was noted. The preparations did not show toxic effects on the liver. The obtained results suggest that the prophylactic use of CDS and CDSM have a significant effect on slowing AA progression, similar to the effect of DF. Both preparations could be potential preventive or therapeutic candidates for the treatment of autoimmune processes in combination with other drugs.

Effect of Baihu Guizhi decoction on characteristic methylation genes expression of pyretic arthralgia rat model

To investigate the characteristic methylation genes of pyretic arthralgia model in hot and dampness environment and the regulation effect of Baihu Guizhi decoction on this characteristic methylation genes. Plantar injection of CFA was used in hot and dampness environment to induce the pyretic arthralgia rat models. From 15th day after modeling, Baihu Guizhi decoction was given for 30 days. Foot volume was detected every 4 days after modeling, and HE staining was used to detect the histopathology of all rats' ankle joint at day 45.MeDIP-Seq sequencing method was used to detect the methylation level of knee joint synovial, and the method of difference sets was used to screen the characteristic methylation genesinpyretic arthralgia models.The contents of IL-1β, IL-17, TNF-α, EGF, IL-12p70, IL-4, IL-6 and IFN-γ in serum were measured by using suspension chips. The mRNA expression level of characteristic methylation genes was measured by qRT-PCR. The results suggested that as compared with adjuvant arthritis rat models(AA), the foot swelling and histopathology inpyretic arthralgia models (PA) were only slightly increased. As compared with normal group (NG), the wholegenome CpG island in both AA and PA groups was kept in a lower methylation state, furthermore, the methylation level was lowest in PA group; with 705 difference methylation genes in AA group and 2 418 difference methylation genes in PA group. As compared with AA, there were 1 287 difference methylation genes, including 974 down-regulated methylation genesand 313 up-regulated methylation genes. This difference methylation genes were mostly enriched in 32 KEGG pathways. Moreover, there were 52 characteristic methylation genes of PA models in promoter region, including 36 down-regulatedmethylation genes and 16 up-regulatedmethylation genes. After drug intervention, Baihu Guizhi decoction improved the foot swelling and pathological injury in PA models, significantly decreased the levels of IL-1β, TNF-α, EGF, VEGF, IL-17, IL-12p70, inhibited the mRNA expression levels of down-regulated methylation genes AHCY and RPL3, and promoted the mRNA expression levels of up-regulated methylation gene Agxt. In conclusion, unique methylation changes of synovial genes were present in PA models, and Baihu Guizhi decoction may adjust the methylation level of PA's characteristic methylation genes to achieve the therapeutic effect of pyretic arthralgia.

Anti-Arthritic Activity of Schistosoma mansoni and Trichinella spiralis Derived-Antigens in Adjuvant Arthritis in Rats: Role of FOXP3+ Treg Cells.

A growing body of evidence supports the concept of helminths therapy in a variety of autoimmune diseases. Here, we aimed to investigate the protective effects of autoclaved Schistosoma mansoni antigen (ASMA) and Trichinella spiralis antigen (ATSA) on the clinical and immunopathological features of rheumatoid arthritis (RA). Adjuvant arthritis was induced by subcutaneous and intradermal injections of complete Freund’s adjuvant into the plantar surface of the right hind paw and the root of the tail, respectively. Rats were randomly assigned to serve as normal control, untreated arthritis, ASMA or ATSA-treated arthritis groups. Antigens were given by intradermal injection in two doses, two weeks apart. The development, progression of arthritic features, and the impact on animals’ gait and body weight were followed up for 4 weeks. The associated changes in serum cytokines (IL-17, IFN-γ and IL-10), joints’ histopathology and immunohistochemistry of Foxp3+ T regulatory cells (Tregs) were evaluated at the end of the study. Treatment with either ASMA or ATSA attenuated the progression of clinical features of polyarthritis, improved gait and body weight gain, reduced the elevated serum IL-17 and further increased both IFN-γ and IL-10. Histopathologically, this was associated with a remarkable regression of paws’ inflammation that was limited only to the subcutaneous tissue, and a significant increase in the number of Foxp 3+ cells versus the untreated arthritis group. In conclusion, both Schistosoma mansoni and Trichinella spiralis derived antigens exerted protective effect against adjuvant arthritis with better effect achieved by ASMA treatment. This anti-arthritic activity is attributed to upregulation of the Foxp3+ Tregs, with subsequent favorable modulation of both pro- and anti-inflammatory cytokines. The use of autoclaved parasitic antigens excludes the deleterious effects of imposing helminthic infection by using live parasites, which may pave the way to a new therapeutic modality in treating RA.

Evaluation of Anti-inflammatory and Anti-arthritic Activity of Ajmodadi Churna- A Polyherbal Formulation

Anti-inflammatory and anti- arthritic activity of aqueous extract of Ajmodadi Churna (AJM) were evaluated by three methods namely, Carrageenan paw edema, Carrageenan induced Air Pouch Model in Rats and Freunds’ complete adjuvant Arthritis. The Carrageenan paw edema was carried out to test the effect of the extract on acute phase of inflammation. Carrageenan induced Air Pouch Model was used for local inflammation and Freunds’ complete adjuvant Arthritis was used for evaluation of chronic inflammation. Results showed that AJM have significant anti-inflammatory activity and Anti-arthritic Activity in both the doses (200 mg/kg and 400 mg/kg) when compared to the Diclofenac but higher dose was found more effective.Key Words: Anti-inflammatory, Anti-arthritic Activity, Freunds’ complete adjuvant Arthritis, Ajmodadi churna.

Recombinant human endostatin inhibits TNF-alpha-induced receptor activator of NF-κB ligand expression in fibroblast-like synoviocytes in mice with adjuvant arthritis.

Bone loss is a critical pathology responsible for the functional disability in patients with rheumatoid arthritis (RA). It is well known that receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) plays a crucial role in bone loss in RA. The purpose of this study was to determine whether recombinant human endostatin (rh-endostatin) mediates bone erosion in RA by regulation of RANKL expression in an experimental model of RA, consisting of mice with adjuvant-induced arthritis (AA). Cultured AA fibroblast-like synoviocytes (FLSs) obtained from these mice were induced by tumor necrosis factor-α (TNF-α) combined with or without rh-endostatin. The levels of RANKL and osteoprotegerin (OPG) mRNA, soluble and membrane-bound proteins were assessed by real-time PCR, ELISA, and Western blotting. Western blotting and the luciferase reporter assay were used to study related signaling pathways. Rh-endostatin inhibited RANKL mRNA expression, soluble and membrane-bound protein expression in AA FLSs but not in CD4+ T cells. However, OPG expression and secretion was not affected by rh-endostatin in AA FLSs. Molecular analysis demonstrated that rh-endostatin significantly inhibited TNF-α-induced MAPK and AP-1 signaling pathways. Moreover, rh-endostatin attenuated TNF-α-induced NF-κB signaling by suppressing the phosphorylation level of inhibitor kappaBα (IκBα) and nuclear translocation of NF-κB p65 in FLSs from mice with AA. These results provide the first evidence that rh-endostatin inhibits TNF-α-induced RANKL expression in AA FLSs.

Identification of Tricyclic Agonists of Sphingosine-1-phosphate Receptor 1 (S1P1) Employing Ligand-Based Drug Design.

Fingolimod (1) is the first approved oral therapy for the treatment of relapsing remitting multiple sclerosis. While the phosphorylated metabolite of fingolimod was found to be a nonselective S1P receptor agonist, agonism specifically of S1P1 is responsible for the peripheral blood lymphopenia believed to be key to its efficacy. Identification of modulators that maintain activity on S1P1 while sparing activity on other S1P receptors could offer equivalent efficacy with reduced liabilities. We disclose in this paper a ligand-based drug design approach that led to the discovery of a series of potent tricyclic agonists of S1P1 with selectivity over S1P3 and were efficacious in a pharmacodynamic model of suppression of circulating lymphocytes. Compound 10 had the desired pharmacokinetic (PK) and pharmacodynamic (PD) profile and demonstrated maximal efficacy when administered orally in a rat adjuvant arthritis model.

Amelioration of oxidative and inflammatory changes by Swertia chirayita leaves in experimental arthritis.

The present study was aimed to determine the therapeutic effects of Swertia chirayita leaves against oxidative and inflammatory injuries in Freund's complete adjuvant (FCA) induced arthritic rats. The extract was evaluated for its phytoconstituents and various invitro antioxidant properties followed by its in vivo effects. The hydroethanolic extract of S. chirayita leaves (SCE) was orally administered (200mg/kg body weight, per day, p.o.) and the effect on the liver lipid peroxidation (LPO), antioxidant status, protein carbonyl formation along with the histopathology of liver were evaluated after induction of adjuvant arthritis. The markers of inflammation and arthritis, such as tumor necrosis factor-α (TNF-α), interleukin 1α (IL-1α), inhibition of paw edema, along with the histological and radiographic changes in the arthritic ankle joint were studied with and without SCE administration. The result showed the presence of major phytoconstituents, such as phenolic, flavonoid and terpenoid content in SCE. HPLC analysis revealed the presence of swertiamarin and amarogentin in high concentration. The extract also showed in vitro antioxidant potential which has positive correlation with the phytoconstituents. The result of in vivo study showed elevated malondialdehyde (MDA) and carbonyl content indicative of LPO and protein oxidation, respectively, with compromised intracellular antioxidant defense system in arthritic rats, which were significantly normalized after SCE treatment. The increase in serum proinflammatory cytokines (TNF- α and IL-1α) and paw edema of arthritic rats was significantly suppressed by SCE. Histology and radiographic analysis of arthritic ankle joints indicated abnormal changes. Marked reduction in inflammation and arthritic changes were observed after treatment with SCE. The present investigation suggests that hydroethanolic extract of S. chirayita leaves exhibit potential immunomodulatory effects, which may possibly be due to boosting the intracellular antioxidant defense.

Hesperidin derivative-11 inhibits fibroblast-like synoviocytes proliferation by activating Secreted frizzled-related protein 2 in adjuvant arthritis rats

Hesperidin (HDN), a flavanone glycoside derived from the citrus cultivation, has a multitude of pharmacological properties, which include antioxidant, anti-inflammatory, hypolipidaemic and anti-carcinogenic actions, but the underlying mechanisms by which treatment of HDN attenuates Rheumatoid Arthritis (RA) remain elusive. Here we engaged to determine whether Hesperidin derivative-11(HDND-11), a HDN derivative with enhanced water-solubility and bioavailability, is effective on treating arthritis in rats. In this study, results of 3-(4, 5-dimethylthiazol-2-yl)−2, 5-diphenyltetra-zolium bromide (MTT) assay and Flow cytometry indicated that administration of HDND-11 inhibited proliferation of fibroblast-like synoviocytes (FLS). Results of Western blot, Real-time quantitative PCR (RT-qPCR) analysis and Immunofluorescence staining demonstrated that HDND-11 was able to up-regulate the expression of Secreted frizzled-related proteins 2 (SFRP2) and diminish DNA methyltransferase 1(DNMT1) expression. We also identified that the effect of DNMT1 inhibition was completely similar to the effects of HDND-11 on SFRP2 gene expression. Furthermore, our results indicated that treatment with HDND-11 could suppress activation of Wnt pathway. Taken together, we found that the HDND-11diminished inhibitory effect of DNMT1 on SFRP2, thereby down-regulated β-catenin expression and inhibited the activation of Wnt signaling pathways to inhibit FLS growth.

Inhibitory effect of recombinant human endostatin on the proliferation of hypertrophic scar fibroblasts in a rabbit ear model

Hypertrophic scar (HS) is a pathological scar that particularly occurs after traumatic injuries, surgical procedures and burning. Abnormal activation of hypertrophic scar fibroblasts (HSFs) intensifies fibrosis during wound healing. Our previous studies demonstrated that recombinant human endostatin (rhEndostatin) prevented synovial thickening in adjuvant arthritis (AA) rats via inhibition of proliferation and enhancement of apoptosis in synovial fibroblasts. However, the effect of this protein on HSF proliferation is not known. This study investigated the inhibitory effect of rhEndostatin on the proliferation of cultured HSFs in a rabbit ear model. MTT assay and flow cytometric detection were performed to investigate HSF proliferation and cell cycle progression, respectively. The expression levels of p53, p21, cyclinD1, cyclin-dependent kinase 4 (CDK4) and proliferating cell nuclear antigen (PCNA) in HSFs were detected using real-time PCR and Western blotting. Our data revealed that HSFs treated with rhEndostatin were significantly inhibited in a concentration-dependent manner with an IC50 value of 100mg/L. Also, rhEndostatin (100mg/L) primarily induced G0/G1 and partially G2/M cell cycle arrest of HSFs. There were significant decreases in the expression levels of p53, p27, CDK4, cyclinD1 and PCNA in HSFs treated with rhEndostatin. In conclusion, rhEndostatin inhibited HSF proliferation via G0/G1 and/or G2/M phase arrest of the cell cycle, which was partially due to the down-regulation of cyclinD1, CDK4 and PCNA. These findings suggest that rhEndostatin may reduce scar hypertrophy in vivo via inhibition of HSF proliferation and may be a novel agent for HS treatment.

Experimental substantiation of effectively administration of vinboron for analgesic activity increase of ibuprofen

Background. The increase of NSAIDs safety is current direction of modern pharmacology, because of so-called class-specific adverse reactions, which are common to this class, and the leading place among them is occupied by gastro-intestinal toxicity. In previous studies we have proved the ability of vinboron to neutralize ulcerogenic effect of ibuprofen (Hladkykh F.V. and al., 2014). The presence of the proven analgesic activity in the domestic antispasmodics (Stepaniuk H.I. and al., 2007) serves as the basis for research of vinboron action on analgesic aspect of ibuprofen pharmacotherapeutic effec.Aim is to conduct research in silico of the relation «molecular structure – anelgetic activity» of vinboron and to prove experimentally in vivo the practicability of vinboron using with the aim to increase the analgesic activity of ibuprofen on the model of adjuvant arthritis in rats.Materials and methods. The study of the relation «molecular structure – activity anelgetic» of vinboron was conducted in silico by PASS- analysis of biological activity spectrum. The analysis was set online with direct insertion of structural formula of vinboron in browser using Marvin JS web-resource «PASS Online» (http://www.way2drug.com/passonline). Analgesic activity in vivo was studied on the model of acute thermal pain, which was simulated in the conventional behavioral test of nociception «Hot plate». The lag of pain reaction was determined at the beginning («0» day), on 7, 14, 21 and 28 days of experiment.Results and discussion. According to the PASS-forecast the mechanisms of vinboron analgesic activity is caused by agonism towards the vanilloid (TRPV1) receptors (Pa=0,490; Pi=0,008), agonism to the μ (mu) – receptor (Pa=0,323; Pi=0,036), inhibition of GABA (Pa=0,329; Pi=0,089) and others. Experimental studies have shown that the combined administration of ibuprofen and vinboron analgesic activity was higher than the results by ibuprofen monotherapy at a dose of 73 mg/kg up by 15.3%, and at a dose of 218 mg/kg up by 20.4%. Besides, it is worthwhile to note that analgesic activity during the integrated administration of ibuprofen at a dose of 218 mg/kg and vinboron (+39.8%) was higher than the total analgesic activity of ibuprofen monotherapy at the same dose (+19.4%) and monotherapy of vinboron (+10.9%), that points at potentiation of analgesic effect of both drugs during their integrated administration.Conclusions. According to the PASS-analysis, the leading mechanism in analgesic component implementation of vinboron is its agonism to TRPV1. The results of our in vivo studies point at the ability of vinboron to intensify the antinociceptive activity of ibuprofen.

Autophagy and mitochondrial dysfunction in adjuvant-arthritis rats treatment with resveratrol.

Resveratrol is a polyphenol derivatives which exhibits a pro-apoptotic effect in a variety of human cancers by triggering mitochondria apoptosis pathway and autophagy. However, there are scarcely reports on its apoptosis-promoting effect in abnormal proliferation fibroblast-like synoviocytes (FLSs). In this study, we investigated the underlying mechanism and apoptosis-inducing effects of resveratrol on the abnormal proliferation of FLSs in adjuvant-arthritis (AA) rats. Since using resveratrol for 12 days resulted in a significant decreasing the swelling degree of the paw, reducing malondialdehyde (MDA) content and enhancing superoxide dismutase (SOD) activity, antioxidant capacity, glutathione peroxidase and glutathione reductase ratio in AA rats. Moreover, we found that 5 μMH2O2 could increase cells viability, Beclin1, LC3A/B, MnSOD, SIRT3 protein expression in FLSs. But, resveratrol could reverse these effects by changing mitochondrial membrane potential (Δψm) to promote mitochondrial reactive oxygen species (mtROS) generation in 5 μMH2O2-treatment FLSs. These results suggest that oxidative stress existed in AA rats. Resveratrol could suppress oxidative stress in AA rats and increase mtROS production by reducing autophagy protein Beclin1, LC3A/B and oxidative stress protein MnSOD to promoted the apoptosis of FLSs. Thus, targeting of mtROS may be a crucial mechanism of resveratrol confers patients with rheumatoid arthritis.