White adipose tissue, a novel antirheumatic target: Clues from its secretory capability and adipectomy-based therapy.

BACKGROUND AND PURPOSE: White adipose tissue (WAT) is involved in rheumatoid arthritis (RA). This study explored its potential as an antirheumatic target. EXPERIMENTAL APPROACH: WAT status of healthy and adjuvant-induced arthritis (AIA) rats were compared. The contribution of WAT to RA pathology was evaluated by pre-adipocyte transplant experiments and by dissecting perirenal fat pads of AIA rats. The impact of RA on WAT was investigated by culturing pre-adipocytes. Proteins differentially expressed in WAT of healthy and AIA rats were identified by the UPLC/MS2 method. These together with PPARγ siRNA and agonist were used to treat pre-adipocytes in vitro. The medium was used for THP-1 monocyte culture. KEY RESULTS: Compared with healthy controls, AIA WAT was smaller but secreted more leptin, eNAMPT, MCP-1, TNF-α, and IL-6. AIA rat pre-adipocytes increased the levels of these adipokines in healthy recipients. RA patients' serum induced a similar secretion change and impaired differentiation of pre-adipocytes. Adipectomy eased AIA-related immune abnormalities and arthritic manifestations. Hepatokines PON1, IGFBP4, and GPIHBP1 were among the differential proteins in high levels in RA blood, and induced inflammatory secretions by pre-adipocytes. GPIHBP1 inhibited PPARγ expression and caused differentiation impairment and inflammatory secretion by pre-adipocytes, a similar outcome to PPARγ-silencing. This endowed the cells with an ability to activate monocytes, which can be abrogated by rosiglitazone. CONCLUSION AND IMPLICATIONS: Certain hepatokines potentiate inflammatory secretions by pre-adipocytes and expedite RA progression by inhibiting PPARγ. Targeting this signalling or abnormal WAT secretion by various approaches may reduce RA severity.

Acupuncture alleviates inflammatory pain by activating CXCL1/CXCR2 signaling in the primary somatosensory cortex in adjuvant induced arthritis rats. / S1脑区CXCL1/CXCR2å‚ä¸Žé’ˆåˆºæ”¹å–„ä½å‰‚æ€§å ³èŠ‚ç‚Žå¤§é¼ ç‚Žæ€§ç—›çš„ä½œç”¨æœºåˆ¶.

OBJECTIVES: To observe whether acupuncture up-regulates chemokine CXC ligand 1 (CXCL1) in the brain to play an analgesic role through CXCL1/chemokine CXC receptor 2 (CXCR2) signaling in adjuvant induced arthritis (AIA) rats, so as to reveal its neuro-immunological mechanism underlying improvement of AIA. METHODS: BALB/c mice with relatively stable thermal pain reaction were subjected to planta injection of complete Freund adjuvant (CFA) for establishing AIA model, followed by dividing the AIA mice into simple AF750 (fluorochrome) and AF750+CXCL1 groups (n=2 in each group). AF750 labeled CXCL1 recombinant protein was then injected into the mouse's tail vein to induce elevation of CXCL1 level in blood for simulating the effect of acupuncture stimulation which has been demonstrated by our past study. In vivo small animal imaging technology was used to observe the AF750 and AF750+CXCL1-labelled target regions. After thermal pain screening, the Wistar rats with stable pain reaction were subjected to AIA modeling by injecting CFA into the rat's right planta, then were randomized into model and manual acupuncture groups (n=12 in each group). Other 12 rats that received planta injection of saline were used as the control group. Manual acupuncture (uniform reinforcing and reducing manipulations) was applied to bilateral "Zusanli" (ST36) for 4×2 min, with an interval of 5 min between every 2 min, once daily for 7 days. The thermal pain threshold was assessed by detecting the paw withdrawal latency (PWL) using a thermal pain detector. The contents of CXCL1 in the primary somatosensory cortex (S1), medial prefrontal cortex, nucleus accumbens, amygdala, periaqueductal gray and rostroventromedial medulla regions were assayed by using ELISA, and the expression levels of CXCL1, CXCR2 and mu-opioid receptor (MOR) mRNA in the S1 region were detected using real time-quantitative polymerase chain reaction. The immune-fluorescence positive cellular rate of CXCL1 and CXCR2 in S1 region was observed after immunofluorescence stain. The immunofluorescence double-stain of CXCR2 and astrocyte marker glial fibrillary acidic protein (GFAP) or neuron marker NeuN or MOR was used to determine whether there is a co-expression between them. RESULTS: In AIA mice, results of in vivo experiments showed no obvious enrichment signal of AF750 or AF750+CXCL1 in any organ of the body, while in vitro experiments showed that there was a stronger fluorescence signal of CXCL1 recombinant protein in the brain. In rats, compared with the control group, the PWL from day 0 to day 7 was significantly decreased (P<0.01) and the expression of CXCR2 mRNA in the S1 region significantly increased in the model group (P<0.05), while in comparison with the model group, the PWL from day 2 to day 7, CXCL1 content, CXCR2 mRNA expression and CXCR2 content, and MOR mRNA expression in the S1 region were significantly increased in the manual acupuncture group (P<0.05, P<0.01). Immunofluorescence stain showed that CXCR2 co-stained with NeuN and MOR in the S1 region, indicating that CXCR2 exists in neurons and MOR-positive neurons but not in GFAP positive astrocytes. CONCLUSIONS: Acupuncture can increase the content of CXCL1 in S1 region, up-regulate CXCR2 on neurons in the S1 region and improve MOR expression in S1 region of AIA rats, which may contribute to its effect in alleviating inflammatory pain.

CPD-002, a novel VEGFR2 inhibitor, relieves rheumatoid arthritis by reducing angiogenesis through the suppression of the VEGFR2/PI3K/AKT signaling pathway.

Synovial angiogenesis is a key player in the development of rheumatoid arthritis (RA), and anti-angiogenic therapy is considered a promising approach for treating RA. CPD-002 has demonstrated efficacy in suppressing tumor angiogenesis as a VEGFR2 inhibitor, but its specific impacts on RA synovial angiogenesis and possible anti-RA effects need further study. We examined the influences of CPD-002 on the migration and invasion of human umbilical vein endothelial cells (HUVECs) and its impacts on HUVECs' tube formation and vessel sprouting ex vivo. The therapeutic potential of CPD-002 in adjuvant-induced arthritis (AIA) rats and its suppression of synovial angiogenesis were examined. The involvement of the VEGFR2/PI3K/AKT pathway was assessed both in HUVECs and AIA rat synovium. Here, CPD-002 inhibited the migration and invasion of VEGF-stimulated HUVECs, decreased their chemotactic response to RA fibroblast-like synoviocyte-released chemoattractants, and exhibited anti-angiogenic effects in vitro and ex vivo. CPD-002's targeting of VEGFR2 was confirmed with molecular docking and cellular thermal shift assays, supported by the abolishment of CPD-002's effects upon using VEGFR2 siRNA. CPD-002 relieved paw swelling, arthritis index, joint damage, and synovial angiogenesis, indicating its anti-arthritic and anti-angiogenic effects in AIA rats. Moreover, the anti-inflammatory effects in vivo and in vitro of CPD-002 contributed to its anti-angiogenic effects. Mechanistically, CPD-002 hindered the activation of VEGFR2/PI3K/AKT pathway in VEGF-induced HUVECs and AIA rat synovium, as evidenced by reduced p-VEGFR2, p-PI3K, and p-AKT protein levels alongside elevated PTEN protein levels. Totally, CPD-002 showed anti-rheumatoid effects via attenuating angiogenesis through the inhibition of the VEGFR2/PI3K/AKT pathway.

Protective effects of piperlongumine against adjuvant-induced arthritis in rats through modulating OPG/RANKL/NF-κB signaling pathway.

OBJECTIVES: We examined the antirheumatoid effects of piperlongumine (PLM) on rat adjuvant-induced arthritis (AIA) and explored the underlying mechanisms involved. METHODS: PLM (2.5, 5, and 10 mg/kg) was administered intraperitoneally to AIA rats to assess its effectiveness. Blood, thymus, spleen, ankle joint, and synovial tissue samples were gathered for subsequent analyses, like enzyme-linked immunosorbent assay, thymus/spleen index measurement, ankle joint pathological examination, immunohistochemistry assay, polymerase chain reaction, and western blot assay. Moreover, the involvement of osteoprotegerin (OPG)/receptor activators of nuclear factor κB ligand (RANKL)/nuclear factor-κB (NF-κB) signaling was investigated. KEY FINDINGS: PLM effectively relieved inflammation and joint destruction in AIA rats, as indicated by reductions in hind paw swelling, arthritis index, thymus/spleen index, ankle joint pathological damage, production of TNF-α, IL-1ß, and IL-6 in both serum and synovium, and osteoclast formation. Also, PLM treatment raised OPG production, reduced RANKL expression, and elevated the OPG/RANKL ratio in synovial tissues. Furthermore, PLM prevented IκBα degradation and phosphorylation, resulting in a reduced expression of the nuclear NF-κB p65 protein in AIA rat synovial tissues. CONCLUSIONS: PLM demonstrated strong antiarthritic effects in rats with AIA by influencing the OPG/RANKL/NF-κB signaling pathway, highlighting its potential clinical relevance in treating rheumatoid arthritis.

Flavipin from fungi as a potential inhibitor of rheumatoid arthritis signaling molecules.

Flavipin, a fungal lower molecular weight biomolecule (MW 196.16 g/mol), has not been yet extensively studied for beneficial preclinical and clinical applications. In recent years, various preclinical mouse models including adjuvant-induced arthritis (AIA) were employed to understand mechanisms associated with Rheumatoid arthritis (RA) and to develop new therapeutic drugs. In the current study, we studied the inhibitory effect of Flavipin on major signaling molecules involved in the inflammatory response during RA using both in-silico virtual interaction and in vivo mouse model of AIA. Our in-silico results clarified that Flavipin interacts with the tumor necrosis factor alpha (TNF-α) through conventional hydrogen binding (H-H) at one of TNF-α critical amino acids tyrosine residues, Tyr119, with binding energy (b.e.) -5.9. In addition, Flavipin binds to ATP-binging sites of the Jesus kinases, JAK1, JAK2 and JAK3, through H-H (b. e. between -5.8 and -6.1) and then it may inhibit JAKs, regulators of RA signaling molecules. Moreover, our molecular dynamics stimulation for the docked TNF-α/Flavipin complex confirmed the specificity and the stability of the interaction. In vitro, Flavipin is not toxic to normal cells at doses below 50 µM (its IC50 in normal fibroblast cell line was above 100 µM). However, in vivo, the arthritis score and hind paw oedema parameters were modulated in Flavipin treated mice. Consistent with the in-silico results the levels of the TNF-α, the nuclear transcription factor kappaB (NF-κB) and the signal transduction and activator of transcription (STAT3, downstream of JAKs) were modulated at joint tissues of the hind-paw of Flavipin/AIA treated mice. Our data suggest Flavipin as a potential therapeutic agent for arthritis can inhibit RA major signaling molecules.

Discovery of novel NSAID hybrids as cPLA2/COX-2 dual inhibitors alleviating rheumatoid arthritis via inhibiting p38 MAPK pathway.

A series of NSAIDs hybrid molecules were synthesized and characterized, and their ability to inhibit NO release in LPS-induced RAW264.7 macrophages was evaluated. Most of the compounds showed significant anti-inflammatory activity in vitro, of which (2E,6Z,9Z,12Z,15Z)-1,1,1-trifluorohenicosa-2,6,9,12,15-pentaen-2-yl 2-(4-benzoylphenyl) propanoate (VI-60) was the most optimal (IC50 = 3.85 ± 0.25 µΜ) and had no cytotoxicity. In addition, VI-60 notably reduced the production of PGE2 in LPS-stimulated RAW264.7 cells compared to ketoprofen. Futhur more, VI-60 significantly inhibited the expression of iNOS, cPLA2, and COX-2 and the phosphorylation of p38 MAPK in LPS-stimulated RAW264.7 cells. The binding of VI-60 to cPLA2 and COX-2 was directly verified by the CETSA technique. In vivo studies illustrated that VI-60 exerted an excellent therapeutic effect on adjuvant-induced arthritis in rats by regulating the balance between Th17 and Treg through inhibiting the p38 MAPK/cPLA2/COX-2/PGE2 pathway. Encouragingly, VI-60 showed a lower ulcerative potential in rats at a dose of 50 mg/kg compared to ketoprofen. In conclusion, the hybrid molecules of NSAIDs and trifluoromethyl enols are promising candidates worthy of further investigation for the treatment of inflammation, pain, and other symptoms in which cPLA2 and COX-2 play a role in their etiology.

Anti-arthritic efficacy of Bombax ceiba ethanolic extract in a murine model for rheumatoid arthritis using in vivo, in vitro and radiological analysis.

Rheumatoid arthritis (RA) is a multisystem autoimmune disease that causes discomfort, synovial membrane inflammation, peripheral joint inflammation, morning stiffness, articular tissue loss, and restricted joint movement. In the present study, we aim to explore the anti-arthritic efficacy of Bombax ceiba ethanolic extract in a Freund's Complete Adjuvant-induced arthritis, in murine model. The hot soxhlet method was used to extract dried aerial components of Bombax ceiba using an ethyl alcohol: water (70:30) ratio. Bombax ceiba ethanolic extract at two doses of 200 and 400 mg/kg was investigated in Wistar rats against Freund's full adjuvant-induced chronic immunological arthritis. Anti-arthritis efficacy was studied utilising morphological research (paw volume, paw diameter, and body weight). On the 28th day, the animals were sacrificed, and haematological parameters, pro-inflammatory cytokines (TNF-alpha, IL-6), cell culture, histological and radiological analysis were performed. BCEE inhibited paw oedema significantly (P 0.05) at a dose of 40mg/Kg, which was corroborated by paw volume and diameter, as well as haematological parameters, in Freund's complete adjuvant-induced arthritis model. The BCEE also significantly reduced pro-inflammatory cytokine levels and the histopathological changes caused by Freund's full adjuvant model. BCEE preserves synovial membranes by enhancing health and has shown a significant anti-arthritic activity. Thus, data confirms the traditional usage of Bombax ceiba for arthritis.

Therapeutic Effect of Colony Stimulating Factor 1 Receptor Kinase Inhibitor, JTE-952, on Methotrexate-Refractory Pathology in a Rat Model of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation and the destruction of bone and cartilage in affected joints. One of the unmet medical needs in the treatment of RA is to effectively prevent the structural destruction of joints, especially bone, which progresses because of resistance to conventional drugs that mainly have anti-inflammatory effects, and directly leads to a decline in the QOL of patients. We previously developed a novel and orally available type II kinase inhibitor of colony-stimulating factor-1 receptor (CSF1R), JTE-952. CSF1R is specifically expressed by monocytic-lineage cells, including bone-resorbing osteoclasts, and is important for promoting the differentiation and proliferation of osteoclasts. In the present study, we investigated the therapeutic effect of JTE-952 on methotrexate (MTX)-refractory joint destruction in a clinically established adjuvant-induced arthritis rat model. JTE-952 did not suppress paw swelling under inflammatory conditions, but it inhibited the destruction of joint structural components including bone and cartilage in the inflamed joints. In addition, decreased range of joint motion and mechanical hyperalgesia after disease onset were suppressed by JTE-952. These results suggest that JTE-952 is expected to prevent the progression of the structural destruction of joints and its associated effects on joint motion and pain by inhibiting CSF1/CSF1R signaling in RA pathology, which is resistant to conventional disease-modifying anti-rheumatic drugs such as MTX.

An untargeted serum and urine lipidomics research based on UPLC-MS revealed the lipid alterations on adjuvant-induced arthritis rats.

Rheumatoid arthritis (RA) is a systemic autoimmune disease dominated by chronic inflammatory lesions of peripheral synovial joints. Growing evidence suggests that abnormal lipid metabolism levels contribute to the progression of RA. Although several metabolomics studies have shown abnormality in the RA lipidome, the relationship between the overall lipid metabolites and RA has not been systematically evaluated. In this study, an untargeted lipidomics method based on ultra performance liquid chromatography-mass spectrometry (UPLC-MS) was used to analyze the serum and urine lipidomes of adjuvant-induced arthritis rats to study the characteristics of lipid metabolism changes in the rats and search lipid markers for diagnosing RA. By combining with orthogonal partial least squares discriminant analysis, a total of 52 potential lipid markers were identified, mainly involved in sphingolipid metabolism, glycerophospholipid metabolism, sterol lipid metabolism, glycerolipid metabolism and fatty acid metabolism, which provided crucial insight into lipid metabolism disturbances in RA. Further receiver operating characteristic analysis revealed that the areas under the curve of PC(22:4/16:0), PI(18:1/16:0) and LacCer(d18:1/12:0) from serum and 25-hydroxycholesterol from urine were 0.94, 1.00, 1.00 and 1.00, respectively, indicating the high predictive ability of this method for RA. In this study, our results indicated that a combination of serum and urine analysis can provide a more comprehensive and reliable assessment of RA, and a UPLC-MS-based lipidomics strategy is a powerful tool to search for potential lipid markers associated with RA and explore the pathogenesis of RA.

Effect of Leflunomide-Metal Complexes on ROS, TNF, and Brain Indolamines in Comparison with Anti-Depressants as Adjunct Therapy in Rheumatoid Arthritic Model.

Leflunomide is an isoxazole immunomodulating drug used to treat rheumatoid arthritis (RA). It is adopted as a metal-containing molecule to proceed with saturated salts of essential and detected metals; it amends the pharmacokinetic and pharmacodynamics activity of leflunomide to provide [M(Lef)4]X2-type complexes. Earlier it has been reported that after forming complexes with metals, leflunomide anti-arthritic activity was significantly altered in an acute arthritic model. In the present study, we evaluated the possible modification in anti-arthritic activities of leflunomide-metal complexes (Mg+2, Ca+2, Fe+2, Zn+2) with and without an anti-depressant drug, i.e., fluoxetine (10 mg/kg) in a chronic AIA model. Rats (n = 5) were administered with 0.1 mL of CFA into the right hind paw while treated groups received leflunomide and its metal complexes orally (3.2 mg/kg) for 24 days. On the final day of experiment, rats were sacrificed; a specific rat immunoassay ELISA kit was used to assess TNF-α in serum samples and read at 450 nm; a tissue sample of a paw was homogenized in a phosphate buffer using DCFH-DA dye for binding to assess ROS. A rat's brain sample was homogenized and evaluated for tryptophan, serotonin (5-HT), and HIAA by RP-HPLC with EC detector. The overall TNF production was altered in treated rats. In addition, a decreased ROS was observed in all categories, except lef+Mg+2 group. Moreover, depletion in the brain indolamine levels were found in treated groups; an upraised level of these indolamines was observed when fluoxetine was added. It is concluded that metals affect leflunomide activity on complexation and simultaneous administration of fluoxetine cope up with the depression in arthritic-induced rats.

Macrophage Polarization Towards M2 Phenotype by Curcuminoids Through NF-κB Pathway Inhibition in Adjuvant-Induced Arthritis.

Macrophage polarization is decisive for homeostasis maintenance and tissue repair. Anti-inflammatory properties of curcumin (CUR) have been demonstrated in several studies. It used in the treatment of bone disorders includingrheumatoid arthritis. The present study aims to explore the potential mechanisms of curcumin on macrophage polarization, expression, activation, and cytokine secretion in adjuvant-induced arthritis as well as its possible role in enhancing the therapeutic action of methotrexate (MTX) together with minimizing MTX initiated side-effects. Rats were divided into eight groups as follows; Control group, MTX group: was weekly injected with MTX, CUR group: was treated with a daily oral dose of curcumin, MTX + CUR group: was treated with both methotrexate and curcumin, Adjuvant arthritis group (AIA): was injected with complete Freund's adjuvant for arthritis induction, AIA/MTX group: arthritic rats treated with methotrexate, AIA/CUR group: arthritic rats treated with curcumin and AIA/MTX + CUR: arthritic rats treated with both methotrexate and curcumin. Paw swelling, haematological analysis, immunological studies, histological observations and quantitative immunohistochemical investigations were performed. The present results showed that treating arthritic rats with curcumin either alone or in combination with methotrexate resulted in amelioration in paws inflammation, growth rate, absolute and relative spleen weights, and haematological analyses. Antinuclear antibodies, IL-1ß, IL-8, IL-10, NF-kB levels, and CD68 + joint expression were also ameliorated. The microscopic examination of joint and spleen showed more improvement as apparently normal tissues in treated groups. It can be concluded that curcumin seems to be most promising in regulating macrophage expression, activation, cytokine secretion, and polarization, thus providing a novel insight in the application of curcumin-based treatments.

LncRNA XIST promotes adjuvant-induced arthritis by increasing the expression of YY1 via miR-34a-5p.

Objectives: This study aims to explore the mechanism by which long non-coding ribonucleic acids (lncRNA) X-inactive specific transcript (XIST) affects the progression of adjuvant-induced arthritis (AIA). Materials and methods: Freund's complete adjuvant was used to induce arthritis in rats. The polyarthritis, spleen and thymus indexes were calculated to evaluate AIA. Hematoxylin-eosin (H&E) staining was used to reveal the pathological changes in the synovium of AIA rats. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-8 in the synovial fluid of AIA rats. The cell continuing kit (CCK)-8, flow cytometry, and Transwell assays were used to assess the proliferation, apoptosis, migration and invasion of transfected fibroblast-like synoviocytes (FLS) isolated from AIA rats (AIA-FLS). Dual-luciferase reporter assay was performed to verify the binding sites between XIST and miR-34b-5p or between YY1 mRNA and miR-34b-5p. Results: The XIST and YY1 were highly expressed, and miR-34a-5p was lowly expressed in the synovium of AIA rats and in AIA-FLS. Silencing of XIST impaired the function of AIA-FLS in vitro and inhibited the progression of AIA in vivo. The XIST promoted the expression of YY1 by competitively binding to miR-34a-5p. Inhibition of miR-34a-5p strengthened the function of AIA-FLS by upregulating XIST and YY1. Conclusion: The XIST controls the function of AIA-FLS and may promote the progression of rheumatoid arthritis via the miR-34a-5p/YY1 axis.

Mesenchymal Stem Cells and Myeloid-Derived Suppressor Cells Interplay in Adjuvant-Induced Arthritis Rat Model.

There has not been much researchs on the biological relationship between myeloid-derived suppressor cells (MDSCs) and mesenchymal stem cells (MSCs). The goal of the current work is to examine how these cells cooperate with one another in a rat model of adjuvant-induced arthritis (AIA). Three groups of equal numbers of rats were created; the first group served as the control. Complete Freund's adjuvant (CFA) was injected into the second group to induce AIA. The third group underwent MSCstreatment. Three weeks later, ANA, IL-1ß, IL-4, IL-6, IL-10, TNF-α, IFN-γ, M-CSF, iNOS and Arg-1 were determined using ELISA. Flowcytometric studies for MDSCs using CD11bc + and His48 + antibodies were performed. Current results showed significantly higher levels of WBCs, ANA, IL-1, IL-4, IL-6, IL-10, TNF-α, M-CSF, iNOS and Arg-1 along with a significant rise in MDSCs % in the AIA group compared to the control group. As opposed to AIA animals, MSCs administration resulted in a considerable improvement in cytokine levels, supporting the immunomodulation function of MSCs. Histological examination of the joints in the AIA group revealed articular cartilage degradation as well as infiltration of inflammatory cells and fibroplasia. These several evidences suggested that MDSCs may perform various roles in autoimmunity. Understanding how MDSCs and MSCs contribute to arthritis may help their prospective application in immunotherapy. Therefore, the reciprocal collaboration of MSCs and MDSCs must therefore be the subject of new investigations, which can offer new platforms for the development of more effective and individualized therapies for the treatment of immunological illnesses.

A Non-targeted Metabolomics Reveals Therapeutical Effect and Mechanism of Sanmiao Pill on Adjuvant-induced Arthritis Rats.

BACKGROUND AND OBJECTIVE: Rheumatoid arthritis (RA) is an increasingly serious disease worldwide that can damage the joints and bones of sufferers. Sanmiao Pill (SMP), a classical traditional Chinese medicine (TCM) prescription, has been used for effective treatments for RA in the clinic. To comprehensively illuminate the therapeutic mechanism of SMP in the treatment of RA, the effects of SMP on biomarkers and metabolic pathways in rats with adjuvant-induced arthritis (AIA) were examined. > Methods: Sprague Dawley rats were randomly divided into two control (CC, Control) groups, two model (MM, Model) groups, a methotrexate group (MTX, 7.6 mg/kg body weight per week), and two SMP groups (San-L, 28.7 mg/kg body weight per day and San-H, 57.4 mg/kg body weight per day). Rats' body weight, paw swelling, arthritis scores, biochemical parameters, histopathology, and so on were used to evaluate the success of the model and the therapeutic effects of SMP. The metabolic techniques were used to characterize the metabolic profile and biomarkers of the serum and urine samples of rats to reveal the metabolic changes that occurred after SMP treatment. > Results: After 21 days of treatment, SMP improved weight gain, reduced the severity of paw swelling, lowered the levels of biochemical indicators (CCP-Ab, IL-6, TNF-α, RF), decreased destruction of articular cartilage and bone erosion, and protected the affected joints.Additionally, 17 and 19 potential biomarkers associated with RA were identified in the serum and urine, respectively. SMP significantly reversed 14 potential biomarkers, such as arachidonic acid, lysoPC(20:4(5Z,8Z,11Z,14Z)), L-tryptophan, 9-cis-Retinoic acid, hippuric acid, pyridoxine, and pantothenic acid. These metabolites are associated with arachidonic acid metabolism, glycerophospholipid catabolism, tryptophan metabolism, phenylalanine metabolism, vitamin B6 metabolism, etc. > Conclusion: These results indicated that RA-related biomarkers reflected the metabolic profile of AIA rats. Meanwhile, SMP could effectively treat RA mainly by reducing inflammation and regulating abnormal lipid metabolic pathways and amino acid metabolisms. It showed that metabolomics could be used to analyze the metabolic profiles involved in RA and reveal the mechanism of SMP treatment of RA.>.

Therapeutic effects of shikonin on adjuvant-induced arthritis in rats and cellular inflammation, migration and invasion of rheumatoid fibroblast-like synoviocytes via blocking the activation of Wnt/ß-catenin pathway.

BACKGROUND: Shikonin (SKN), the main bioactive component isolated from Lithospermum erythrorhizon Sieb et Zucc, has multiple activities including anti-rheumatic effect, but its specific roles and the precise mechanisms in regulating biological properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) are unclear and need further clarification. PURPOSE: This study explored the therapeutic roles of SKN on rat adjuvant-induced arthritis (AIA) and cellular inflammation, migration and invasion of TNF-α-induced RA FLS (MH7A cells), and further demonstrated the involved mechanisms. METHODS: SKN was intraperitoneally given to AIA rats and its therapeutic role was valued. The effects of SKN in vivo and in vitro on the production of pro-inflammatory factors were examined by ELISA and western blot. Wound-healing, transwell and phalloidin staining assay were carried out to evaluate the effects of SKN on TNF-α-induced migration and invasion in RA FLS. The involvement of Wnt/ß-catenin pathway was checked by immunohistochemistry or immunofluorescence assay for ß-catenin and western blot for pathway-related proteins. RESULTS: SKN treatment in AIA rats reduced paw swelling, arthritis index and pathological damage of ankle joints, indicating its anti-arthritic effect in vivo. SKN had anti-inflammatory roles in vivo and in vitro, evidenced by inhibiting the production of pro-inflammatory factors (like IL-1ß, IL-6, IL-8, TNF-α, MMP-2 and MMP-9) in sera and synovium of AIA rats, and in TNF-α-induced MH7A cells. Gelatin zymography result revealed the suppression of SKN on TNF-α-induced MMP-2 activity in vitro. Moreover, SKN inhibited TNF-α-induced migration, invasion and cytoskeletal reorganization in MH7A cells. Mechanistically, SKN suppressed the activation of Wnt/ß-catenin signaling in AIA rat synovium and in TNF-α-induced MH7A cells, indicated by the reduced protein levels of Wnt1, p-GSK-3ß (Ser9) and ß-catenin, the raised protein level of GSK-3ß and the decreased nuclear translocation of ß-catenin. Interestingly, the combination of LiCl (Wnt/ß-catenin agonist) canceled the therapeutic functions of SKN on cellular inflammation, migration and invasion in TNF-α-induced MH7A cells, whereas XAV939 (Wnt/ß-catenin inhibitor) enhanced the therapeutic roles of SKN. CONCLUSION: SKN showed therapeutic effects on rat AIA and cellular inflammation, migration and invasion of TNF-α-stimulated RA FLS via interrupting Wnt/ß-catenin pathway.

Inflammation and subsequent nociceptor sensitization in the bone marrow are involved in an animal model of osteoarthritis pain.

AIMS: This study aimed to determine whether pathological changes in the bone marrow cause Osteoarthritis (OA) pain based on magnetic resonance imaging (MRI), immunohistochemistry, and electrophysiology. MAIN METHODS: Adjuvant-induced arthritis (AIA) was achieved by injecting 150 µL of complete Freund's adjuvant into the right knee joints of male Sprague-Dawley rats. AIA rats were compared with saline-injected rats. KEY FINDINGS: AIA significantly induced mechanical hyperalgesia and spontaneous pain in the right hind paw 1-14 days after induction. Intratibial injection of 50 µL of 1 % lidocaine significantly suppressed AIA-induced mechanical hyperalgesia (p = 0.0001) and spontaneous pain (p = 0.0006) 3 days after induction. In T2-weighted MRI, AIA induced high-signal intensity within the proximal tibial metaphysis, and the mean T2 values in this area significantly increased on days 3 (p = 0.0043) and 14 (p = 0.0012) after induction. AIA induced intraosseous edema and significantly increased the number of intraosseous granulocytes on days 3 (p < 0.0001) and 14 (p < 0.0001) after induction. The electrophysiological study on days 3-7 after induction showed significantly increased spontaneous firing rates (p = 0.0166) and evoked responses to cutaneous stimuli (brush, p < 0.0001; pinching, p = 0.0359) in the right hind paw plantar surface and intratibial stimuli (p = 0.0002) in wide-dynamic-range neurons of the spinal dorsal horn. SIGNIFICANCE: Intraosseous changes caused by OA induce hypersensitivity in the sensory afferents innervating bone marrow may be involved in OA pain. Novel bone marrow-targeted therapies could be beneficial for treating OA pain.

Formulation of a dual drug-loaded nanoparticulate co-delivery hydrogel system and its validation in rheumatoid arthritis animal model.

Rheumatoid arthritis (RA), a systemic autoimmune disease that dramatically affects patients' quality of life. Given the intricacy of RA's pathophysiology, no single treatment can completely halt the disease progression. Here, we attempted to treat RA holistically and synergistically by co-delivering methotrexate (MTX), a standard slow-acting anti-rheumatic drug, and phenethyl isothiocyanate (PEITC), a bioactive phytochemical, using a sodium alginate (SA)-pluronic F127 (PF-127) in situ hydrogel formulation. Therefore, in the current study, the co-delivery of MTX and PEITC in the nanoparticulate form could help enhance stability and solubility and facilitate greater penetration in the target arthritic tissues. The fabricated MTX NP and PEITC NE were found to have a minimum particle size, PDI, and good zeta potential. Results from in vitro release studies showed that MTX and PEITC were simultaneously released from the DD NP HG matrix over 6-7 days through diffusion and erosion mechanisms. An intra-articular (IA) injection of DD NP HG dramatically reduced chronic inflammation in adjuvant-induced arthritis (AIA) rats, delayed the onset of bone erosion, significantly reduced synovitis, and down-regulated the inflammatory cytokine expression. Most notably, the co-delivery strategy almost entirely restored the morphological features of the ankle joints of RA rats. The hepatic and renal function tests indicated good biological safety for DD NP HG in RA conditions. Taken together, these findings indicated that DD NP HG could achieve good anti-inflammatory activity and reverse cartilage disruption through a synergistic effect between two nanoparticulate forms of MTX and PEITC, which can effectively improve the drawbacks of their free forms.

A nanostructured dual-drug loaded smart hydrogel (DD NP HG) was successfully constructed for the intra-articular delivery of MTX and PEITC to the affected joints of RA.The fabrication of the nanoformulation of both MTX (MTX NP) and PEITC (PEITC NE) aided in mitigating the drawbacks and drug-related side effects of the free form of drugs.DD NP HG markedly suppressed joint inflammation and protect against bone destruction in arthritic rats.This combination approach of PEITC and MTX (DD NP HG) synergistically improved anti-arthritic activity and reduced the adverse side effects in vivo.

Comparison of the effects of peficitinib and tofacitinib in the adjuvant-induced arthritis rat model.

We investigated and compared the pharmacologic properties of two Janus kinase (JAK) inhibitors, peficitinib and tofacitinib, in an adjuvant-induced arthritis rat model. Repeated administration of peficitinib (3 - 30 mg/kg) or tofacitinib (1 - 10 mg/kg) exhibited a dose-related and significant attenuation of arthritis score, paw swelling, pain threshold, grip strength and histopathologic injuries in the model; peficitinib 10 mg/kg and tofacitinib 3 mg/kg demonstrated comparable efficacy. Equivalent Cmax and AUC0-12h values were observed with peficitinib 10 mg/kg and tofacitinib 3 mg/kg, suggesting that the two drugs may demonstrate comparable efficacy on arthritis-associated symptoms at comparable plasma concentration levels. However, peficitinib 10 mg/kg had greater efficacy than tofacitinib 3 mg/kg on some inflammation- and bone destruction-associated parameters in the paw fluid, including the production of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), receptor activator of nuclear factor kappa-B ligand, and matrix metalloproteinase-3, which are associated with arthritis exacerbation. Peficitinib 10 mg/kg also showed significantly greater inhibitory effects than tofacitinib 3 mg/kg on loss of bone mineral density and synovial thickening score, which might be a result of the VEGF and PDGF receptor kinase inhibitory effects of peficitinib, in addition to JAK inhibition. In conclusion, both tofacitinib and peficitinib potently improved arthritis and associated symptoms in adjuvant-induced arthritis rats; moreover, owing to possible differences in the mechanism of action of the two drugs, peficitinib may have exerted its effects through JAK inhibition and additional unique off-target properties.

Therapeutic effects of kefir peptides on adjuvant-induced arthritis in rats through anti-inflammation and downregulation of matrix metalloproteinases.

AIMS: Rheumatoid arthritis (RA) is a chronic autoimmune disease. Its pathological features are synovial inflammation, bone erosion, and joint structural damages. Our previous studies have shown that kefir peptides (KPs) can reduce cardiovascular disease, osteoporosis and renal inflammation. In this study, we further evaluate the efficacy of KPs on adjuvant-induced arthritis (AIA) in a rat model. MAIN METHODS: After the 14th day of adjuvant induction, rats were subsequently orally administered KPs (83 or 166 mg/day/kg) or tofacitinib (6.2 mg/day/kg) for 14 days. On the 28th day, the rats were anesthetized with isoflurane for ultrasonic, in vivo imaging system (IVIS), and radiographic imaging and then sacrificed for ankle tissues collection and analysis. In vitro, IL-1ß-treated human synovial cells (SW982) were subjected to anti-arthritis mechanism study in the presence of KPs. KEY FINDINGS: The results of ultrasonography, radiograph, histology, the expression of matrix metalloproteinases (MMPs), inflammatory cytokines and RANKL/OPG ratio demonstrated decreasing severity of synovitis and bone erosion in the ankle joints after KPs treatment. Activation of the NF-κB and MAPK pathways was significantly reduced in KPs treated AIA group. Furthermore, KPs attenuated IL-1ß-induced inflammatory cytokine production and the expression of MMPs in a human synovial cell line SW982. These results demonstrated that KPs alleviated adjuvant-induced arthritis in rats by inhibiting IL-1ß-related inflammation and MMPs production. SIGNIFICANCE: We concluded that KPs can exhibit anti-inflammatory effects by reducing the levels of macrophage-related inflammatory cytokines and MMPs, thus alleviating bone erosion in the ankle joint and constituting a potential therapeutic strategy for rheumatoid arthritis.

Effects of Multimodal Therapy, Blinding, and Multi-laboratory Protocol Conduct on the Robustness of the Rat Model of Adjuvant Induced Arthritis.

BACKGROUND/AIM: The aim of this study was to investigate the effects of multimodal therapy comprising buprenorphine (BUP) and indomethacin (IND) on key translational parameters in the rat adjuvant induced arthritis (AIA) model. Furthermore, we investigated the difference between visual assessment scores and histology scores generated by blinded and non-blinded assessors and the robustness and generalizability of results by conducting a multi-laboratory study. MATERIALS AND METHODS: The experiment was terminated on day 26 after 11 days (days 15-25) of voluntarily ingested buprenorphine and 7 days of gavage delivered indomethacin treatment (days 19-25). The treatment effects were assessed on the last day of the study, relying on body weight assessment, serum concentrations of α1- acid glycoprotein, and assessment of affected hind paws swelling, in-life and post mortem. RESULTS: Across two laboratories, the combined analgesic treatments had minimal effects on the measured model parameters indicating that multimodal treatment did not affect the translatability of the model. We found an improvement in clinical scores (a negative change in scores) in nearly all medicated animals when scored informed, whereas it was essentially 50:50 for the blinded scorings and no difference between the blinded and informed histological scoring. CONCLUSION: The present results support the use of more effective analgesic treatment regimens and the good practice recommendations advocating blinding as a mandatory practice in conduct of preclinical in vivo efficacy studies. In spite of minor differences between results obtained at the two sites, there was good agreement between them indicating robustness of the AIA model.