[Effect and mechanism of aqueous extract of Strychni Semen on bone destruction in rats with type â ¡ collagen-induced rheumatoid arthritis].

To investigate the mechanism of action of aqueous extract of Strychni Semen(SA) on bone destruction in rats with type Ⅱ collagen-induced arthritis(CIA), the SD rats were randomly divided into normal group, model group, low, medium, and high dose(2.85, 5.70, and 11.40 mg·kg~(-1)) groups of SA, and methotrexate group. Except for the normal group, the CIA model was prepared for the other groups. After the second immunization, different doses of SA were given to the low, medium, and high dose groups of SA once a day, and the methotrexate group was given once every three days. 0.3% sodium hydroxymethylcellulose(CMC-Na) was given once a day to the normal and model groups for 28 d. The clinical score of arthritis was evaluated every three days. Micro computed tomography(Micro-CT) method was used to evaluate the degree of bone destruction. Histopathological changes in the joint tissue and the number of osteoclasts in CIA rats were evaluated by hematoxylin-eosin(HE) staining and tartrate-resistant acid phosphatase(TRAP) staining. The expression of interleukin-1ß(IL-1ß) in the joint tissue of rats was detected by immunohistochemistry. Western blot was used to detect key protein expression in mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathways in the joint tissue of rats. The results showed that different doses of SA were able to improve the red and swollen inflammatory joint and joint deformity in CIA rats to varying degrees, reduce the clinical score, inhibit synovial inflammation, vascular opacification, cartilage erosion, and bone destruction, and reduce the number of TRAP-positive cells in bone tissue. Micro-CT results showed that the SA was able to increase bone mineral density, bone volume fraction, trabecular reduce, and trabecular number and reduce bone surface/bone volume and trabecular separation/spacing. Different doses of SA could down-regulate the protein expression of IL-1ß, p-JNK, p-ERK, p-p38, PI3K, and p-Akt to varying degrees. In conclusion, SA can improve disease severity, attenuate histopathological and imaging changes in joints, and have osteoprotective effects in CIA rats, and its mechanism of action may be related to the inhibition of the overactivation of MAPK and PI3K/Akt signaling pathways.

Triterpenes G-A and G-E from Galphimia glauca with anti-inflammatory and anti-arthritic activity in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Galphimia glauca is a medicinal plant that treats inflammatory and anti-rheumatic problems. Its anti-inflammatory capacity has been reported pharmacologically, attributed to the triterpenes G-A and G-E. AIM: The objective of the present work was to measure the anti-inflammatory and immunomodulatory effect of the methanolic extract (GgMeOH) of Galphimia glauca and the isolated galphimines G-A and G-E, first in an acute test of plantar edema with carrageenan, and later in the model of experimental-induced arthritis with CFA. The effect was measured by quantifying joint inflammation, the concentration of pro- (TNF-α, IL-6, IL-17) and anti-inflammatory (IL-10, and IL-4) cytokines, and the ADA enzyme in joints, kidneys, and spleen from mice with experimental arthritis. METHOD: The extract and the active triterpenes were obtained according to established methods using different chromatographic techniques. Female ICR strain mice were subjected to intraplantar administration with carrageenan and treated with different doses of GgMeOH, G-A, and G-E; edema was monitored at different times. Subsequently, the concentration of TNF-a and IL-10 in the spleen and swollen paw was quantified. Meloxicam (MEL) was used as an anti-inflammatory control drug. The most effective doses of each treatment were analyzed using a complete Freunds adjuvant (CFA)-induced experimental arthritis model. Joint inflammation was followed throughout the experiment. Ultimately, the concentration of inflammation markers, oxidant stress, and ADA activity was quantified. In this experimental stage, methotrexate (MTX) was used as an antiarthritic drug. RESULTS: Treatments derived from G. glauca, GgMeOH (DE50 = 158 mg/kg), G-A (DE50 = 2 mg/kg), and G-E (DE50 = 1.5 mg/kg) caused an anti-inflammatory effect in the plantar edema test with carrageenan. In the CFA model, joint inflammation decreased with all natural treatments; GgMeOH and G-A inhibited the ADA enzyme in all organs analyzed (joints, serum, spleen, left and right kidneys), while G-E inhibited the enzyme in joints, serum, and left kidney. CFA caused an increase in the weight index of the organs, an effect that was counteracted by the administration of G. glauca treatments, which also modulate the response to the cytokines analyzed in the different organs (IL-4, IL-10, IL-17, IL-6, and TNF- α). CONCLUSION: It is shown, for the first time, that the GgMeOH extract and the triterpenes G-A and G-E of Galphimia glauca have an anti-arthritic effect (anti-inflammatory, immunomodulatory, antioxidant, and ADA inhibitor), using an experimental arthritis model with CFA. Therefore, knowledge of the plant as a possible therapeutic agent for this rheumatic condition is expanding.

Chemometric profiling and anti-arthritic activity of aerial parts of Glinus oppositifolius (L.) Aug. DC.

ETHNOPHARMACOLOGICAL RELEVANCE: Glinus oppositifolius (L.) Aug. DC. belongs to the family Molluginaceae, an annual prostrate herb traditionally used to treat inflammations, arthritis, malarial, wounds, fevers, diarrhoea, cancer, stomach discomfort, jaundice, and intestinal parasites. However, the anti-arthritic activity of the aerial part has still not been reported. AIM OF THE STUDY: To investigate the antioxidant and anti-arthritic activity of G. oppositifolius in Complete Freund's Adjuvant (CFA) induced rats. MATERIALS AND METHODS: The dried aerial parts of this plant material were defatted with n-hexane and extracted by methanol using a soxhlet apparatus. The in vitro anti-arthritic activity of methanolic extract of G. oppositifolius (MEGO) was evaluated in protein denaturation, membrane stabilization, and inhibition of proteinase assay at 25, 50, 100, 200, and 400 µg/ml concentrations. Female Wistar rats were immunized sub-dermally into the right hind paw with 0.1 ml of CFA. Rats were administered with MEGO at doses of 200 and 400 mg/kg once daily for fourteen days after arthritis induction. Assessment of arthritis was performed by measuring paw diameter, arthritic index, arthritic score, body weight, organ weight, and hematological and biochemical parameters, followed by the analysis of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), interleukin-1-beta (IL-1ß), cyclooxygenase-2 (COX-2), interleukin 13 (IL-13) and interleukin 10 (IL-10) and histopathological study. In vivo antioxidant effect was investigated in enzymatic assays. The presence of phytoconstituents was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS), respectively. In silico molecular docking study of the compounds was carried out against COX-2, IL-1ß, IL-6, and TNF-α using AutoDock 4.2 and BIOVIA-Discovery Studio Visualizer software. RESULTS: MEGO's in vitro anti-arthritic activity showed dose-dependent inhibition of protein denaturation, membrane stabilization, and proteinase inhibition, followed by significant in vivo anti-arthritic activity. The rats treated with MEGO showed tremendous potential in managing arthritis-like symptoms by restoring hematological, biochemical, and histological changes in CFA-induced rats. MEGO (200 and 400 mg/kg) showed a significant alleviation in the levels of hyper expressed inflammatory mediators (TNF-α, IL-1ß, and IL-6) and oxidative stress (SOD, CAT, GSH, and LPO) in CFA-induced rats. Spergulagenin-A as identified by LC-MS analysis, exhibited the highest binding affinity against COX-2 (-8.6), IL-1ß (7.2 kcal/mol), IL-6 (-7.4 kcal/mol), and TNF-α (-6.5 kcal/mol). CONCLUSIONS: Provided with the comprehensive investigation, methanolic extract of G. oppositifolius against arthritic-like condition is a proof of concept that revalidates its ethnic claim. The presence of Spergulagenin-A might be responsible for the anti-arthritic activity.

Estragole Ameliorates CFA Induced Rheumatoid Arthritis Symptoms in Wistar Rats by Inhibiting JAK-2/STAT-3 Pathway.

The present study was conducted to scrutinize the pharmacological effect of Estragole (ESG) against CFA-induced arthritis in rats. The rats underwent induction of arthritis using the administration of CFA and after that, the rats were randomly divided into five different groups, where three groups correspond to diverse dosages of ESG, and the other two were control and CFA-arthritic control. Results of the study suggested that ESG in a dose-dependent manner, improves body weight and arthritis score of rats as evidenced by reduction of hind-paw volume. ESG also improved the antioxidant status of rats by reducing MDA levels and enhancing the concentration of endogenous antioxidants SOD and GPx. The level of pro-inflammatory cytokines was also found to be reduced in the case of ESG treated group as compared to CFA-group. In a western blot analysis, ESH showed downregulation of p-JAK-2/STAT-3. The study provided concrete evidence for the protective effect of ESG against rheumatoid arthritis in rats.

Effects of Viscum coloratum (Kom.) Nakai on collagen-induced rheumatoid arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Viscum coloratum (Kom.) Nakai has been traditionally used in China for nearly a thousand years to treat rheumatic diseases. However, its efficacy and mechanisms in treating rheumatoid arthritis (RA) have not been demonstrated. AIM OF THE STUDY: To investigate the anti-arthritic effects and molecular mechanisms of Viscum coloratum (Kom.) Nakai on collagen-induced arthritic mice through network pharmacology technology and experimental validation. MATERIALS AND METHODS: First, the main ingredients of the extract of Viscum coloratum (Kom.) Nakai (EVC) were identified through chemical composition characterization using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS). Then, the collagen-induced arthritis (CIA) model was established in DBA/1 J mice and the ameliorative effects of EVC on the progression of CIA mice were evaluated by oral treatment with different doses of the EVC for 28 days. After that, cytokine antibody microarray assay was used to detect the levels of multiple inflammation-related cytokines and chemokines in each group, and performed Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis. Subsequently, the potential target for the effective chemical components of EVC in treating RA was identified using various databases. Additionally, a drug-disease target protein-protein interaction network (PPI) was conducted using Cytoscape for visualization and clustering, while GO and KEGG enrichment analyses were performed with the Metascape database. Finally, identified phenotypes and targets by network pharmacology analysis were experimentally validated in vivo. RESULTS: Treatment with EVC significantly suppressed the severity of CIA with a dramatic reduction of paw swelling, arthritis index, levels of IgGs (IgG, IgG1, IgG2a, and IgG2b), multi-inflammation-related cytokines and chemokines on the progression of CIA. Histopathological examinations showed EVC could markedly inhibit inflammatory cell infiltration, tartrate-resistant acid phosphatase (TRAP) activity of osteoclast, and bone destruction. Furthermore, GO and KEGG enrichment analyses revealed that EVC could ameliorate RA by inhibiting osteoclast differentiation and regulating multiple signaling pathways including Osteoclast differentiation, IL-17, and TNF. PPI network analysis demonstrated that AKT1, MMP9, MAPK3, and other genes were highly related to EVC in treating RA. Finally, we proved that EVC could inhibit the expression of NFTAc1, MMP9, Cathepsin K, and AKT which were closely related to osteoclast activity. CONCLUSIONS: EVC could treat RA through multiple components, multiple targets, and multiple pathways. The present study demonstrated the therapeutic efficacy of EVC and its molecular mechanisms in treating RA, indicating that it would be a potent candidate as a novel botanical drug for further investigation.

Opuntia monacantha: Validation of the anti-inflammatory and anti-arthritic activity of its polyphenolic rich extract in silico and in vivo via assessment of pro- and anti-inflammatory cytokines.

ETHNOPHARMACOLOGICAL RELEVANCE: Opuntia monacantha belongs to the cactus family Cactaceae and is also known by cochineal prickly pear, Barbary fig or drooping prickly pear. It was traditionally used to treat pain and inflammation. O. monacantha cladodes showed pharmacological effects such as antioxidant potential owing to the presence of certain polysaccharides, flavonoids, and phenols. AIM OF THE STUDY: This research aimed to evaluate the anti-inflammatory as well as the anti-arthritic potential of ethanol extract of Opuntia monacantha (E-OM). MATERIALS AND METHODS: In vivo edema in rat paw was triggered by carrageenan and used to evaluate anti-inflammatory activity, while induction of arthritis by Complete Freund's Adjuvant (CFA) rat model was done to measure anti-arthritic potential. In silico studies of the previously High performance liquid chromatography (HPLC) characterized metabolites of ethanol extract was performed by using Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA) within active pocket of glutaminase 1 (GLS1) (PDB code: 3VP1; 2.30 Å). RESULTS: EOM, particularly at 750 mg/kg, caused a reduction in the paw edema significantly and decreased arthritic score by 80.58% compared to the diseased group. It revealed significant results when histopathology of ankle joint was examined at 28th day as it reduced inflammation by 18.06%, bone erosion by 15.50%, and pannus formation by 24.65% with respect to the diseased group. It restored the altered blood parameters by 7.56%, 18.47%, and 3.37% for hemoglobin (Hb), white blood count (WBC), and platelets, respectively. It also reduced rheumatoid factor RF by 13.70% with concomitant amelioration in catalase (CAT) and superoxide dismutase (SOD) levels by 19%, and 34.16%, respectively, in comparison to the diseased group. It notably decreased mRNA expression levels of COX-2, IL-6, TNF-α, IL-1, NF-κß and augmented the levels of IL-4 and IL-10 in real time PCR with respect to the diseased group and piroxicam. HPLC analysis previously performed showed that phenolic acids and flavonoids are present in E-OM. Molecular docking studies displayed pronounced inhibitory potential of these compounds towards glutaminase 1 (GLS1), approaching and even exceeding piroxicam. CONCLUSIONS: Thus, Opuntia monacantha could be a promising agent to manage inflammation and arthritis and could be incorporated into pharmaceuticals.

Dietary oleacein, a secoiridoid from extra virgin olive oil, prevents collagen-induced arthritis in mice.

Olacein (OLA), one of the main secoiridoids derived from extra virgin olive oil (EVOO), has been shown to modulate oxidative and inflammatory responses in various pathological conditions; however, its potential benefit in joint disorders such as rheumatoid arthritis (RA) is unknown. Therefore, this study was designed to evaluate the preventive role of the effects of an OLA-supplemented diet in the murine model of collagen-induced arthritis (CIA), delving into the possible mechanisms and signaling pathways involved. Animals were fed an OLA-enriched preventive diet for 6 weeks prior to CIA induction and until the end of the experimental time course. On day 43 after the first immunization, mice were sacrificed: blood was collected, and paws were histologically and biochemically processed. Dietary OLA prevented collagen-induced rheumatic bone, joint and cartilage conditions. Circulating matrix metalloproteinase (MMP)-3 and proinflammatory cytokine (IL-6, IL-1ß, TNF-α, IL-17) levels were significantly decreased in the joint, as well as MMP-9 and cathepsin-K (CatK) expression in secoiridoid-fed animals. In addition, dietary OLA was able to decrease COX-2, mPGES-1 and iNOS protein expressions and, also, PGE2 levels. The mechanisms possibly involved in these protective effects could be related to the activation of the Nrf-2/HO-1 axis and the inhibition of proinflammatory signaling pathways, including JAK-STAT, MAPKs and NF-κB, involved in the production of inflammatory and oxidative mediators. These results support the interest of OLA, as a nutraceutical intervention, in the management of RA.

The therapeutic effect of Loranthus parasiticus lignan derivatives on collagen-induced arthritis in rats through the SHBG/NFκB pathway.

Lignan-rich beans, nuts, and various seeds are the main foods with antioxidative and hormone-modulating activities. Although the role of lignans in mediating hormone-dependent cancers and cardiovascular diseases is well characterized, the function of lignans in anti-arthritic activity and its underlying mechanisms remain unknown. Three new lignan derivatives, (-)-nortrachelogenin, trachelogenin, and matairesinol, were extracted from Loranthus parasiticus. After establishing the collagen-induced arthritis (CIA) model by intradermal injection of collagen, rats were treated with three new lignan derivatives ((-)-nortrachelogenin: 37%; trachelogenin: 27%; matairesinol: 25.7%) at a concentration of 50 mg/kg and 100 mg/kg, or methotrexate at 0.3 mg/kg. Mixed lignan derivatives significantly attenuated the immune responses in the joints of CIA rats, leading to lower levels of proinflammatory cytokines (IL-6 and TNF-α) and higher levels of free androgen in the serum compared to the CIA model. The results of molecular docking using AutoDock Vina showed that the lignan derivative (-)-nortrachelogenin was the most effective compound for binding to sex hormone-binding globulin (SHBG), thus inhibiting the activity of NFκB in LPS-stimulated macrophages. In this study, (-)-nortrachelogenin was identified as a novel natural lignan derivative with previously unrecognized anti-inflammatory activity. Its molecular mechanism appears related to the regulation of the NFκB/SHBG pathway. Our findings suggest that further application of sex hormone-like compounds in the treatment of rheumatoid arthritis and the potential clinical applications of (-)-nortrachelogenin are promising.

The effects of the cholinergic system on carrageenan-induced arthritis.

The cholinergic system has been found to make an anti-inflammatory effect through the cholinergic anti-inflammatory pathway (CAIP), which suppresses the production of pro-inflammatory cytokines by secreting acetylcholine, a major neurotransmitter. However, no studies have been conducted on the effects of CAIP on joint pain and inflammation. In this study, we investigated the effects of muscarinic acetylcholine receptors (mAChRs) in knee arthritis. To examine pain behavioral changes, atropine (or saline for sham control) was pretreated in the joint cavity of rats at 1 % carrageenan + 5, 10, and 30 µL and the dynamic weight-bearing evaluation was performed. Synovial membranes were collected and cyclooxygenase-2 (COX-2) and interleukin-1ß (IL-1ß) were measured using a western blot. Hematoxylin and eosin staining was performed. Compared to that of the sham group, the weight-bearing of the affected knee joint significantly increased in the 1 % carrageenan + 10 µL atropine group (p < 0.05). However, no significant changes were observed in the 1 % carrageenan + 5 and 30 µL atropine groups. COX-2 and IL-1ß and the number of inflammatory cells in synovial membrane significantly increased with 1 % carrageenan + 10 µL of atropine (p < 0.05). These results suggest that cholinergic system is involved in knee joint pain and inflammation and that mAChRs are potential therapeutic targets for knee joint arthritis.

Study of the mechanism underlying the anti-inflammatory effect of Miao medicine comprising raw and processed Radix Wikstroemia indica using the "sweat soaking method".

ETHNOPHARMACOLOGICAL RELEVANCE: To explore the differences in the anti-inflammatory efficacy and mechanisms of the Miao medicine, both raw and after processing, using the "sweat soaking method" of Radix Wikstroemia indica (RWI). AIM OF THE STUDY: The purpose of this study was to explore the differences in the anti-inflammatory efficacy and mechanism of action before and after the processing of the Miao medicine (RWI) using the "sweat soaking method." MATERIALS AND METHODS: Network pharmacology technology was used to construct the "drug-component target-pathway-disease" network, and the main anti-inflammatory pathways of RWI were identified. Rat models of collagen-induced arthritis were established. The changes in body weight, swelling rate of the foot pad and ankle joint, arthritis index, thymus index, spleen index, pathological changes of the ankle joint, and the content of inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-10, TNF-α, and NO) were used as indices to evaluate the effect of RWI on rats with collagen-induced arthritis before and after its processing. Plasma and urine samples were collected from the rats, and the potential biomarkers of, and metabolic pathways underlying the anti-inflammatory effects of RWI before and after processing were identified using 1H-Nuclear magnetic resonance metabolomics combined with a multivariate statistical analysis. RESULTS: Eleven key anti-inflammatory targets of IL6, IL-1ß, TNF, ALB, AKT1, IFNG, INS, STAT3, EGFR, TP53, and SRC were identified by network pharmacology. The PI3K-Akt signaling pathway, steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, tryptophan metabolism, and other pathways were mainly involved in these effects. Pharmacodynamic studies found that both raw and processed RWI products downregulated inflammatory factors in rats with collagen-induced arthritis and alleviated the pathological changes. A total of 41 potential pathways for the anti-inflammatory effects of raw RWI products and 36 potential pathways for the anti-inflammatory effects of processed RWI products were identified by plasma and urine metabolomics. The common pathways of network pharmacology and metabolomics were steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, and tryptophan metabolism. CONCLUSIONS: The anti-inflammatory effect of RWI was mainly related to the regulation of steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, and tryptophan metabolism. Finally, the "sweat soaking method" enhanced the anti-inflammatory effect of RWI.

Sinomenine ameliorates fibroblast-like synoviocytes dysfunction by promoting phosphorylation and nuclear translocation of CRMP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and arthritic pain. Sinomenine (SIN), derived from the rhizome of Chinese medical herb Qing Teng (scientific name: Sinomenium acutum (Thunb.) Rehd. Et Wils), has a longstanding use in Chinese traditional medicine for treating rheumatoid arthritis. It has been shown to possess anti-inflammatory, analgesic, and immunosuppressive effects with minimal side-effects clinically. However, the mechanisms governing its effects in treatment of joint pathology, especially on fibroblast-like synoviocytes (FLSs) dysfunction, and arthritic pain remains unclear. AIM: This study aimed to investigate the effect and underlying mechanism of SIN on arthritic joint inflammation and joint FLSs dysfunctions. MATERIALS AND METHODS: Collagen-induced arthritis (CIA) was induced in rats and the therapeutic effects of SIN on joint pathology were evaluated histopathologically. Next, we conducted a series of experiments using LPS-induced FLSs, which were divided into five groups (Naïve, LPS, SIN 10, 20, 50 µg/ml). The expression of inflammatory factors was measured by qPCR and ELISA. The invasive ability of cells was detected by modified Transwell assay and qPCR. Transwell migration and cell scratch assays were used to assess the migration ability of cells. The distribution and content of relevant proteins were observed by immunofluorescence and laser confocal microscopy, as well as Western Blot and qPCR. FLSs were transfected with plasmids (CRMP2 T514A/D) to directly modulate the post-translational modification of CRMP2 protein and downstream effects on FLSs function was monitored. RESULTS: SIN alleviated joint inflammation in rats with CIA, as evidenced by improvement of synovial hyperplasia, inflammatory cell infiltration and cartilage damage, as well as inhibition of pro-inflammatory cytokines release from FLSs induced by LPS. In vitro studies revealed a concentration-dependent suppression of SIN on the invasion and migration of FLSs induced by LPS. In addition, SIN downregulated the expression of cellular CRMP2 that was induced by LPS in FLSs, but increased its phosphorylation at residue T514. Moreover, regulation of pCRMP2 T514 by plasmids transfection (CRMP2 T514A/D) significantly influenced the migration and invasion of FLSs. Finally, SIN promoted nuclear translocation of pCRMP2 T514 in FLSs. CONCLUSIONS: SIN may exert its anti-inflammatory and analgesic effects by modulating CRMP2 T514 phosphorylation and its nuclear translocation of FLSs, inhibiting pro-inflammatory cytokine release, and suppressing abnormal invasion and migration. Phosphorylation of CRMP2 at the T514 site in FLSs may present a new therapeutic target for treating inflammatory joint's destruction and arthritic pain in RA.

Collagen-Induced Arthritis Models.

Due to the limitations of using patient-derived samples for systemic kinetic studies in rheumatoid arthritis (RA) research, animal models are helpful for further understanding the pathophysiology of RA and seeking potential therapeutic targets or strategies. The collagen-induced arthritis (CIA) model is one of the standard RA models used in preclinical research. The CIA model shares several pathological features with RA, such as breach of tolerance and generation of autoantibodies targeting collagen, synovial inflammatory cell infiltration, synovial hyperplasia, cartilage destruction, and bone erosion. In this chapter, a protocol for the successful induction of CIA in mice is described. In this protocol, CIA is induced by active immunization by inoculation with type II heterologous collagen in Freund's adjuvant in susceptible DBA/1 mice.

Histological Analyses of Arthritic Joints in Collagen-Induced Arthritis Model Mice.

Histological analysis is a morphological technique and an effective method for understanding the pathology of rheumatoid arthritis (RA). RA is an inflammatory disease characterized by increased synovial tissue and osteoclasts, angiogenesis, infiltration of inflammatory cells, and pannus formation. These pathologies can be observed in a collagen-induced arthritis model mouse using formaldehyde-fixated paraffin-embedded (FFPE) samples. For the preparation of FFPE samples, the conditions of the fixation and decalcification process significantly affect tissue staining results. Since the lesion sites include bone tissue, a decalcification process is necessary when preparing an FFPE sample. Therefore, selecting an optimal condition for the fixating and decalcifying solution is important. In this chapter, we describe the procedures of preparing paraffin samples, including fixation, decalcification, embedding, and sectioning from the RA model mouse, as well as different staining methods (hematoxylin and eosin, tartrate-resistant acid phosphatase).

Jolkinolide B ameliorates rheumatoid arthritis by regulating the JAK2/STAT3 signaling pathway.

BACKGROUND: Jolkinolide B (JB), an ent­abietane-type diterpenoid in Euphorbia plants, has various pharmacological activities, including anticancer, anti-inflammatory, and anti-tuberculosis activities. However, no previous studies have proven whether JB can be regarded as a targeted drug for the treatment of rheumatoid arthritis (RA). PURPOSE: This study aimed to evaluate the anti-RA effects of JB and explore the potential mechanisms. METHODS: Components and targets of JB and RA were identified in different databases, and potential targets and pathways were predicted by protein-protein interaction (PPI) network analysis and pathway enrichment analysis. Then, molecular docking and surface-plasmon resonance (SPR) were used to confirm the predict. The anti-arthritic effects of JB were studied in vivo with collagen-induced arthritis (CIA) rat model and in vitro with lipopolysaccharide (LPS) and interleukin-6 (IL-6)-induced RAW264.7 macrophage. Potential mechanisms were further verified by in vivo and in vitro experiments. RESULTS: The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that Th17 cell differentiation, prolactin signaling pathway, and JAK/STAT signaling pathway might be associated with anti-RA effects of JB. Molecular docking and SPR results showed that JB bound effectively to JAK2. JB significantly decreased body weight loss, arthritis index, paw thickness, and synovial thickness in CIA rats. Histomorphological results suggested the protective effects of JB on CIA rats with ankle joint injury. Molecular biology analysis indicated that JB suppressed the mRNA expression of inflammatory factors in ankle joints for CIA rats and reduced the concentration of these factors in LPS- induced RAW264.7 macrophage. The protein expression level of the JAK2/STAT3 pathway was also significantly decreased by JB. CONCLUSION: JB had a novel inhibitory effect on inflammation and bone destruction in CIA rats, and the mechanism might be related to the JAK2/STAT3 signaling pathway.

Leonurine alleviates rheumatoid arthritis by regulating the Hippo signaling pathway.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause joint inflammation and damage. Leonurine (LE) is an alkaloid found in Leonurus heterophyllus. It has anti-inflammatory effects. HYPOTHESIS/PURPOSE: The molecular mechanisms by which LE acts in RA are unclear and further investigation is required. METHODS: Mice with collagen-induced arthritis (CIA), and RA-fibroblast-like synoviocytes (FLSs) isolated from them were used as in vivo and in vitro models of RA, respectively. The therapeutic effects of LE on CIA-induced joint injury were investigated by micro-computed tomography, and staining with hematoxylin and eosin and Safranin-O/Fast Green. Cell Counting Kit-8, a Transwell® chamber, enzyme-linked immunosorbent assays, RT-qPCR, and western blotting were used to investigate the effects of LE on RA-FLS viability, migratory capacity, inflammation, microRNA-21 (miR-21) levels, the Hippo signaling pathway, and the effects and intrinsic mechanisms of related proteins. Dual luciferase was used to investigate the binding of miR-21 to YOD1 deubiquitinase (YOD1) and yes-associated protein (YAP). Immunofluorescence was used to investigate the localization of YAP within the nucleus and cytoplasm. RESULTS: Treatment with LE significantly inhibited joint swelling, bone damage, synovial inflammation, and proteoglycan loss in the CIA mice. It also reduced the proliferation, cell colonization, migration/invasion, and inflammation levels of RA-FLSs, and promoted miR-21 expression in vitro. The effects of LE on RA-FLSs were enhanced by an miR-21 mimic and reversed by an miR-21 inhibitor. The dual luciferase investigation confirmed that both YOD1 and YAP are direct targets of miR-21. Treatment with LE activated the Hippo signaling pathway, and promoted the downregulation and dephosphorylation of MST1 and LATS1 in RA, while inhibiting the activation of YOD1 and YAP. Regulation of the therapeutic effects of LE by miR-21 was counteracted by YOD1 overexpression, which caused the phosphorylation of YAP and prevented its nuclear ectopic position, thereby reducing LE effect on pro-proliferation-inhibiting apoptosis target genes. CONCLUSION: LE regulates the Hippo signaling pathway through the miR-21/YOD1/YAP axis to reduce joint inflammation and bone destruction in CIA mice, thereby inhibiting the growth and inflammation of RA-FLSs. LE has potential for the treatment of RA.

Bisdemethoxycurcumin, a curcumin derivative, ameliorates adjuvant-induced arthritis by suppressing inflammatory reactions and macrophage migration.

Rheumatoid arthritis (RA) is a highly prevalent and chronic inflammatory synovial joint disease manifested by hyperplasia and continuous inflammation. Curcumin (Cur) has been studied for alleviating RA. However, poor stability and oral bioavailability restrict its therapeutic value. Bisdemethoxycurcumin (BDMC), a curcumin (Cur) derivative, exerts better stability and oral bioavailability than Cur. However, the efficacy of BDMC on RA has not been fully clarified. The aim of the study was to investigate the therapeutic effects and underlying mechanisms of BDMC on RA. The in-vivo anti-arthritic activity of BDMC was determined via adjuvant-induced arthritis (AIA) rat model. Paw swelling, body weight, arthritic index, and histopathological assessments were performed. RAW264.7 cell was stimulated by lipopolysaccharides (LPS) in vitro. The cell viability were determined by CCK8 assay, while the migration ability was determined using cell wound healing and transwell assays. Furthermore, in-vivo and in-vitro levels of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) were assayed by ELISA, and that of IκBα, p-NF-κB, NF-κB, and COX-2 were assessed via Western blot or immunofluorescence. In AIA rat model, it suggested a higher anti-arthritic activity of BDMC than Cur, including amelioration of swelling in hind paws, reduced arthritic index, and alleviated histopathological injury in rats. Furthermore, BDMC also substantially decreased the levels of the aforementioned pro-inflammatory cytokines in both in-vivo and in-vitro, inhibited the IκBα degradation, down-regulated the COX-2 levels and p-NF-κB/NF-κB ratio in AIA rats and LPS-stimulated RAW264.7 cells. Additionally, BDMC showed an inhibitory effect on the migration of LPS-stimulated RAW264.7 cells. BDMC could effectively ameliorate RA by suppressing inflammatory reactions and inhibiting macrophage migration, more potentially than Cur.

DKK-1 and Its Influences on Bone Destruction: A Comparative Study in Collagen-Induced Arthritis Mice and Rheumatoid Arthritis Patients.

Dickkopf-1 (DKK-1) has been considered a master regulator of bone remodeling. As precursors of osteoclasts (OCs), myeloid-derived suppressor cells (MDSCs) were previously shown to participate in the process of bone destruction in rheumatoid arthritis (RA). However, the role of DKK-1 and MDSCs in RA is not yet fully understood. We investigated the relevance between the level of DKK-1 and the expression of MDSCs in different tissues and joint destruction in RA patients and collagen-induced arthritis (CIA) mouse models. Furthermore, the CIA mice were administered recombinant DKK-1 protein. The arthritis scores, bone destruction, and the percentage of MDSCs in the peripheral blood and spleen were monitored. In vitro, the differentiation of MDSCs into OCs was intervened with recombinant protein and inhibitor of DKK-1. The number of OCs differentiated and the protein expression of the Wnt/ß-catenin signaling pathway were explored. The level of DKK-1 positively correlates with the frequency of MDSCs and bone erosion in RA patients and CIA mice. Strikingly, recombinant DKK-1 intervention significantly exacerbated arthritis scores and bone destruction, increasing the percentage of MDSCs in the peripheral blood and spleen in CIA mice. In vitro experiments showed that recombinant DKK-1 promoted the differentiation of MDSCs into OCs, reducing the expression of ß-catenin and TCF4 and increasing the expression of CyclinD1. In contrast, the DKK-1 inhibitor had the opposite effect. Our findings highlight that DKK-1 promoted MDSCs expansion in RA and enhanced the differentiation of MDSCs into OCs via targeting the Wnt/ß-catenin pathway, aggravating the bone destruction in RA.

Exploring the efficacy and mechanism of Glycyrrhizae Radix et Rhizoma in improving collagen-induced arthritis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA), a chronic auto-immune disease, will cause serious joint damage and disability. Glycyrrhizae Radix et Rhizoma (GRR) is commonly included in many anti-RA formulas used in the clinical practice in China. AIM OF THE STUDY: To elucidate the alleviation of GRR and its active compounds on RA and the possible engaged mechanism. MATERIALS AND METHODS: The clinical score, paw swelling degree and pain threshold were detected in the collagen-induced arthritis (CIA) in DBA/1 mice. The ankle joints of mice were observed by using X-Ray, hematoxylin-eosin (H&E), masson's trichrome (Masson), and safranin O and fast green (Safranin O) staining. The potential targets of GRR were predicted by network pharmacology and further verified by using enzyme-linked immunosorbent assay (ELISA) and western-blot. Real-time polymerase chain reaction (Real-time PCR) and wound healing assay were conducted in synovial MH7A cells. The interaction between active compounds and potential targets predicted by molecular docking was confirmed by using cellular thermal shift assay (CETSA). RESULTS: GRR (615 mg/kg) obviously alleviated CIA in mice. Network pharmacology implied that GRR might affect angiogenesis and inflammation, among which vascular endothelial growth factor-A (VEGF-A), tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß), IL-6 and phosphorylated protein kinase B (AKT) might be the key targets involved in this process. GRR decreased AKT phosphorylation and reduced the elevated levels of TNFα, VEGF-A, IL-1ß and IL-6. Next, in vitro results demonstrated that glycyrrhetinic acid (GA) and isoliquiritigenin (ISL) were two active compounds that inhibited TNFα-induced synovial cell angiogenesis and inflammation. Moreover, GA and ISL actually improved RA in CIA mice. The results of molecular docking and CETSA displayed that ISL and GA might interact with TNF receptor-1 (TNFR1), toll-like receptor-4 (TLR4) and VEGF receptor-2 (VEGFR2), thereby contributing to their inhibition on angiogenesis and inflammation. CONCLUSION: GRR and two active compounds, including ISL and GA, alleviated RA via inhibiting angiogenesis and inflammation.

Discovery of a First-in-Class GPR183 Antagonist for the Potential Treatment of Rheumatoid Arthritis.

GPR183 is required for humoral immune responses, and its polymorphisms have been associated with inflammatory autoimmune diseases. Despite increasing attention to GPR183 as a potential therapeutic target for autoimmune diseases, relatively few antagonists have been reported, and none of them have progressed to the clinical stage. In this study, we discovered a highly potent GPR183 antagonist, compound 32, with good aqueous solubility, excellent selectivity, and pharmacokinetic properties. Meanwhile, compound 32 showed exceptional efficacy for rheumatoid arthritis (RA) disease in a mouse collagen-induced arthritis (CIA) model, with an efficacious dose of 0.1 mg/kg. Functionally, compound 32 significantly reduced the swelling of paws and joints, the gene expression of proinflammatory cytokines, MCP-1, MMPs, and VEGF, inflammatory cell infiltration, cartilage damage, pannus formation, and bone erosion in the joints of CIA mice in a dose-dependent manner. Hence, these findings suggest compound 32 as a valuable molecule for further development.