Anti-inflammatory and anti-rheumatic effects of Phoenix dactylifera L. (date palm) seed by controlling cytokines and inhibiting JAK1/STAT3 pathway on CFA-induced arthritis rat and its phytochemical profiling.

ETHNOPHARMACOLOGICAL RELEVANCE: Phoenix dactylifera L. (date palm) seed is widely used in Arabian traditional medicine to alleviate several health problems including inflammatory conditions. The herbal tea of date palm seed has been consumed by rheumatoid patients to relief their symptoms. AIM OF THE STUDY: The purpose of this study was to investigate the claimed beneficial use of P. dactylifera L. (Sewy variety) seed (PDS) in the treatment of rheumatoid arthritis (RA) and its mechanism of action as well as to study its phytoconstituents. MATERIALS AND METHODS: The anti-inflammatory and anti-oxidative properties of the non-polar and the polar extracts of PDS were studied using Complete Freund's adjuvant (CFA)-induced arthritis rat model. Paw edema, body weight, total nitrate/nitrite NOX content and cytokine markers were evaluated to monitor the progress of arthritis. Also, histological examination and thermal analysis were conducted. The phytoconstituent profiles of non-polar and polar extracts of PDS were investigated using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The multiple reactions monitoring mode (MRM) of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used to quantify phenolic phytoconstituents in both extracts. RESULTS: According to the findings, the polar and non-polar PDS extracts kept body weight comparable to those of healthy individuals while considerably lowering paw swelling, edema, and neutrophil infiltration. It also reduced the levels of Nuclear Factor Kappa B (NF-κB), Tumor Necrosis Factor Alpha (TNF-α), Interleukin 22, Interleukin 23, Interferon (IFN), Interleukin 17, Interleukin 1ß, Interleukin 6, Interleukin 36, Janus Kinase 1 (JAK1), and Signal Transducer and Activator of Transcription 3 (STAT3). They also reduced the degenerative alterations caused by RA. Thermal research gave additional support for these findings. 83 phytoconstituents were identified in the non-polar PDS extract and 86 phytoconstituents were identified in the polar PDS extract. 74 of the identified phytoconstituents were common in both extracts. 33 phytoconstituents were identified here from P. dactylifera for the first time as far as we know. In MRM-LC-ESI-MS/MS analysis, the major phenolics in both extracts were chlorogenic acid, naringenin, and vanillin. Catechin was only detected in the non-polar PDS extract. On the other hand, apigenin, kaempferol, and hesperetin were only detected in the polar PDS extract. Generally, the polar PDS extract showed higher concentrations of the identified phenolics than the non-polar extract. CONCLUSIONS: The PDS extracts especially the non-polar extract showed significant anti-inflammatory and anti-oxidative properties in the CFA-induced arthritis rat model. PDS might be used to produce RA medicines.

6-O-angeloylplenolin inhibits osteoclastogenesis in vitro via suppressing c-Src/NF-κB/NFATc1 pathways and ameliorates bone resorption in collagen-induced arthritis mouse model.

One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 µM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion.

Fangji Huangqi Decoction alleviates rheumatoid arthritis through regulating HIF-1α mediated the angiogenesis and the balance between autophagy and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Fangji Huangqi Decoction (FHD) is frequently prescribed for the clinical treatment of wind-cold and wind-dampness pathogenic superficial deficiency syndrome. It also has a notable curative effect on rheumatoid arthritis (RA). AIM OF THE STUDY: The study aimed to explore the possible mechanism of FHD against RA and provided a theoretical basis for alternative therapies for RA. MATERIALS AND METHODS: We used UPLC-Q-TOF-MS to analysis the ingredients and absorbed blood components of FHD. At the same time, the collagen-induced arthritis (CIA) rat model was established to estimate the therapeutic effects on FHD by considering body weight, arthritis score, paw swelling, autonomous movement ability, and synovial microvessel counts. Subsequently, immunofluorescence, immunohistochemistry, and Western blot were employed to detect the anti-angiogenic capacity of FHD in vivo, as well as the levels of apoptosis and autophagy in the synovial tissue. In addition, flow cytometry and Western blot were used to assess the effects of FHD on apoptosis and autophagy in MH7A cells. The effects of FHD on the proliferation and migration of MH7A cells were measured by CCK8 assay, cell migration and, invasion experiments. Finally, a tube formation assay was performed to evaluate the angiogenic capacity of FHD in co-cultures of MH7A cells and HUVEC cells. RESULTS: Through testing of FHD's original formula, a total of 26 active ingredients have been identified, with 17 of them being absorbed into the bloodstream. FHD significantly improved the pathological symptoms and synovial hyperplasia of CIA rats. FHD could suppress the expression of HIF-1α, promote apoptosis in CIA rat synovial tissue, and suppress autophagy and angiogenesis. In vitro experiments showed that serum containing FHD inhibited the proliferation, migration, and invasion of MH7A cells, and also suppressed the expression of autophagy-related proteins while promoting apoptosis. FHD markedly repressed the expression of HIF-1α protein in TNF-α-stimulated MH7A cells and inhibited the tube formation capacity induced by MH7A cells in HUVEC cells. CONCLUSIONS: The study had proven that FHD played an excellent anti-RA role, which may be attributed to its potential mechanism of regulating the balance between autophagy and apoptosis in RA FLS by suppressing the HIF-1α, thus contributing to its anti-angiogenic activities.

Co-Delivery of Aceclofenac and Methotrexate Nanoparticles Presents an Effective Treatment for Rheumatoid Arthritis.

Background: Rheumatoid arthritis (RA) is a common acute inflammatory autoimmune connective tissue arthropathy. The genetic studies, tissue analyses, experimental animal models, and clinical investigations have confirmed that stromal tissue damage and pathology driven by RA mounts the chronic inflammation and dysregulated immune events. Methods: We developed methotrexate (MTX)-loaded lipid-polymer hybrid nanoparticles (MTX-LPHNPs) and aceclofenac (ACE)-loaded nanostructured lipid carriers (ACE-NLCs) for the efficient co-delivery of MTX and ACE via intravenous and transdermal routes, respectively. Bio-assays were performed using ex-vivo skin permeation and transport, macrophage model of inflammation (MMI) (LPS-stimulated THP-1 macrophages), Wistar rats with experimental RA (induction of arthritis with Complete Freund's adjuvant; CFA and BCG), and programmed death of RA affected cells. In addition, gene transcription profiling and serum estimation of inflammatory, signaling, and cell death markers were performed on the blood samples collected from patients with RA. Results: Higher permeation of ACE-NLCs/CE across skin layers confirming the greater "therapeutic index" of ACE. The systemic delivery of MTX-loaded LPHNPs via the parenteral (intravenous) route is shown to modulate the RA-induced inflammation and other immune events. The regulated immunological and signaling pathway(s) influence the immunological axis to program the death of inflamed cells in the MMI and the animals with the experimental RA. Our data suggested the CD40-mediated and Akt1 controlled cell death along with the inhibited autophagy in vitro. Moreover, the ex vivo gene transcription profiling in drug-treated PBMCs and serum analysis of immune/signalling markers confirmed the therapeutic role co-delivery of drug nanoparticles to treat RA. The animals with experimental RA receiving drug treatment were shown to regain the structure of paw bones and joints similar to the control and were comparable with the market formulations. Conclusion: Our findings confirmed the use of co-delivery of drug nanoformulations as the "combination drug regimen" to treat RA.

Bub1 suppresses inflammatory arthritis-associated bone loss in mice through inhibition of TNFα-mediated osteoclastogenesis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis, bone and cartilage destruction, and increased fracture risk with bone loss. Although disease-modifying antirheumatic drugs have dramatically improved clinical outcomes, these therapies are not universally effective in all patients because of the heterogeneity of RA pathogenesis. Therefore, it is necessary to elucidate the molecular mechanisms underlying RA pathogenesis, including associated bone loss, in order to identify novel therapeutic targets. In this study, we found that Budding uninhibited by benzimidazoles 1 (BUB1) was highly expressed in RA patients' synovium and murine ankle tissue with arthritis. As CD45+CD11b+ myeloid cells are a Bub1 highly expressing population among synovial cells in mice, myeloid cell-specific Bub1 conditional knockout (Bub1ΔLysM) mice were generated. Bub1ΔLysM mice exhibited reduced femoral bone mineral density when compared with control (Ctrl) mice under K/BxN serum-transfer arthritis, with no significant differences in joint inflammation or bone erosion based on a semi-quantitative erosion score and histological analysis. Bone histomorphometry revealed that femoral bone mass of Bub1ΔLysM under arthritis was reduced by increased osteoclastic bone resorption. RNA-seq and subsequent Gene Set Enrichment Analysis demonstrated a significantly enriched nuclear factor-kappa B pathway among upregulated genes in receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated bone marrow-derived macrophages (BMMs) obtained from Bub1ΔLysM mice. Indeed, osteoclastogenesis using BMMs derived from Bub1ΔLysM was enhanced by RANKL and tumor necrosis factor-α or RANKL and IL-1ß treatment compared with Ctrl. Finally, osteoclastogenesis was increased by Bub1 inhibitor BAY1816032 treatment in BMMs derived from wildtype mice. These data suggest that Bub1 expressed in macrophages plays a protective role against inflammatory arthritis-associated bone loss through inhibition of inflammation-mediated osteoclastogenesis.

Rheumatoid arthritis (RA) is a disease caused by an abnormal immune system, resulting in inflammation, swelling, and bone destruction in the joints, along with systemic bone loss. While new medications have dramatically improved treatment efficacy, these therapies are not universally effective for all patients. Therefore, we need to understand the regulatory mechanisms behind RA, including associated bone loss, to develop better therapies. In this study, we found that Budding uninhibited by benzimidazoles 1 (Bub1) was highly expressed in inflamed joints, especially in myeloid cells, which are a type of immune cells. To explore its role, we created myeloid cell­specific Bub1 conditional knockout (cKO) mice and induced arthritis to analyze its role during arthritis. The cKO mice exhibited lower bone mineral density when compared with control mice under inflammatory arthritis because of increased osteoclastic bone resorption, without significant differences in joint inflammation or bone erosion. Further investigation showed that Bub1 prevents excessive osteoclast differentiation induced by inflammation in bone marrow macrophages. These data suggest that Bub1 in macrophages protects against bone loss caused by inflammatory arthritis, offering potential insights for developing treatments that focus on bone health.

Lipid mediators obtained from docosahexaenoic acid by soybean lipoxygenase attenuate RANKL-induced osteoclast differentiation and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by persistent inflammation and joint destruction. A lipid mediator (LM, namely, 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA, exhibits anti-inflammatory activity. In this study, we determined the effect of LM on collagen antibody-induced arthritis (CAIA) in mice and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in RAW264.7 cells. LM effectively downregulated the expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K, inhibited osteoclast formation, and suppressed the NF-κB signaling pathway in vitro. In vivo, LM at 10 µg/kg/day significantly decreased paw swelling and inhibited progression of arthritis in CAIA mice. Moreover, proinflammatory cytokine (tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, IL-17, and interferon-γ) levels in serum were decreased, whereas IL-10 levels were increased following LM treatment. Furthermore, LM alleviated joint inflammation, cartilage erosion, and bone destruction in the ankles, which may be related to matrix metalloproteinase and Janus kinase (JAK)-signal transducer and activators of transcription (STAT) signaling pathway. Our findings suggest that LM attenuates arthritis severity, restores serum imbalances, and modifies joint damage. Thus, LM represents a promising therapy for relieving RA symptoms.

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.

Isorhamnetin Downregulates MMP2 and MMP9 to Inhibit Development of Rheumatoid Arthritis through SRC/ERK/CREB Pathway.

OBJECTIVE: To investigate the effect of isorhamnetin on the pathology of rheumatoid arthritis (RA). METHODS: Tumor necrosis factor (TNF)- α -induced fibroblast-like synoviocytes (FLS) was exposed to additional isorhamnetin (10, 20 and 40 µ mol/L). Overexpression vectors for matrix metalloproteinase-2 (MMP2) or MMP9 or SRC were transfected to explore their roles in isorhamnetin-mediated RA-FLS function. RA-FLS viability, migration, and invasion were evaluated. Moreover, a collagen-induced arthritis (CIA) rat model was established. Rats were randomly divided to sham, CIA, low-, medium-, and high-dosage groups using a random number table (n=5 in each group) and administed with normal saline or additional isorhamnetin [2, 10, and 20 mg/(kg·day)] for 4 weeks, respectively. Arthritis index was calculated and synovial tissue inflammation was determined in CIA rats. The levels of MMP2, MMP9, TNF-α, interleukin-6 (IL-6), and IL-1 ß, as well as the phosphorylation levels of SRC, extracellular regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding (CREB), were detected in RA-FLS and synovial tissue. Molecular docking was also used to analyze the binding of isorhamnetin to SRC. RESULTS: In in vitro studies, isorhamnetin inhibited RA-FLS viability, migration and invasion (P<0.05). Isorhamnetin downregulated the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß in RA-FLS (P<0.05). The overexpression of either MMP2 or MMP9 reversed isorhamnetin-inhibited RA-FLS migration and invasion, as well as the levels of TNF-α, IL-6, and IL-1 ß (P<0.05). Furthermore, isorhamnetin bound to SRC and reduced the phosphorylation of SRC, ERK, and CREB (P<0.05). SRC overexpression reversed the inhibitory effect of isorhamnetin on RA-FLS viability, migration and invasion, as well as the negative regulation of MMP2 and MMP9 (P<0.05). In in vivo studies, isorhamnetin decreased arthritis index scores (P<0.05) and alleviated synovial inflammation. Isorhamnetin reduced the levels of MMP2, MMP9, TNF-α, IL-6, and IL-1 ß, as well as the phosphorylation of SRC, ERK, and CREB in synovial tissue (P<0.05). Notably, the inhibitory effect of isorhamnetin was more pronounced at higher concentrations (P<0.05). CONCLUSION: Isorhamnetin exhibited anti-RA effects through modulating SRC/ERK/CREB and MMP2/MMP9 signaling pathways, suggesting that isorhamnetin may be a potential therapeutic agent for RA.

L-arginine metabolism inhibits arthritis and inflammatory bone loss.

OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.

Antiarthritic activity of Physalis peruviana fruit extract via inhibition of inflammatory mediators: Integrated in vitro, in vivo and in silico study.

ETHNOPHARMACOLOGICAL RELEVANCE: P. peruviana fruit, native to Andean region, is cultivated worldwide for its adaptability to various soil natures and climatic conditions. It is increasingly consumed for its high nutritional profile and history of ethnomedical uses including treatment of arthritis. Little pharmacological evidences support this folk use except for previous in vitro study that reported significant inhibition of protein denaturation. AIM OF THE STUDY: The study aims at providing new in vivo evidence on antiarthritic activity of P. peruviana fruits in vivo that justifies its traditional use through mechanism-based experiment. MATERIAL AND METHODS: Inhibition of inflammatory mediators is considered one of the key treatments to alleviate painful symptoms of rheumatoid arthritis (RA). Anti-inflammatory activity was assessed against COX-1 and COX-2 activity in vitro. Serum TNFα, IL-1ß and IL-6 were traced using in vivo model of adjuvant-induced arthritis. Gross/inflammatory changes in rat paw, relative mass indices of spleen and liver were further investigated together with joint tissue histoarchitecture. Seven metabolites from different phytochemical classes, that were previously reported in P. peruviana fruit, were evaluated in silico against TNF-α target protein (PDB ID: 2AZ5) to assess their inhibitory effect. This was followed by assessment of their drug-likeness based on Lipinski's rule according to their physicochemical and pharmacokinetic properties. RESULTS: High dose of extract (E-1000 mg) improved adjuvant-induced cachexia and attenuated immune-inflammatory responses in paw and serum parameters, with equipotent effect to MTX, in addition to minimal side effect profile on spleen and liver. Histopathological study of knee joint tissues confirmed dose-dependent improvement in arthritic groups treated with P. peruviana fruit extracts. The insilico study recommended steroidal lactones withaperuvin E/C and hydroxywithanolide E as promising lead compounds for inhibiting TNF enzyme as evidenced by docking scores of 6.301, 5.488 and 5.763 kcal/mol, respectively, fitting as well the Lipinski's rule of drug likeness. CONCLUSION: The study provided novel approach that rationalize folk use of P. peruviana fruit in treatment of arthritis.

Abelmoschus manihot (L.) medik. seeds alleviate rheumatoid arthritis by modulating JAK2/STAT3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Abelmoschus manihot (L.) Medik. Seeds (AMS, སོ་མ་ར་ཛ།), a Tibetan classical herbal in China, are rich in flavonoids and phenolic glycosides compounds, such as quercetin and its derivatives. Moreover, it has been found to possess anti-rheumatoid arthritis (RA) effects. Nonetheless, its anti-RA mechanism is yet unknown. AIM OF THE STUDY: This research aimed to examine the active ingredients of AMS as well as potential pharmacological mechanisms in AMS on RA. MATERIALS AND METHODS: The ultra-performance liquid chromatography-electrospray ionization-tandem multistage mass spectrometry (UPLC-ESI-IT-MSn) technique was used to determine the primary chemical components of AMS that were responsible for the therapeutic effects on RA. In addition, 36 male Wistar rats weighing between 200 and 220 g were classified at random into six groups [normal control group, collagen-induced arthritis (CIA) group, methotrexate group (positive control, 1.05 mg/kg), AMS group (157.5 mg/kg, 315 mg/kg, 630 mg/kg)]. CIA rats were given AMS extract by intragastric administration for 28 days, and their ankles were photographed to observe the degree of swelling. Further, the arthritis score, paws swelling, and body weight changes of CIA rats were determined to observe whether AMS has any effect on RA, and synovial and cartilage tissue injuries were identified by histopathology. Besides, the levels of IL-10, TNF-α, IL-1ß, INF-γ, etc. in serum were estimated by ELISA. Western blot experiments were implemented to identify the expression levels of protein involved in the JAK2/STAT3 signaling pathway in the CIA rats' synovial tissues. Moreover, the mechanisms and targets of active ingredient therapy of AMS for RA were predicted using network pharmacology and then verified using molecular docking. RESULT: In the present study, 12 compounds were detected by UPLC-ESI-IT-MSn, such as quercetin and its derivative which could be potential active ingredients that contribute to the anti-RA properties of AMS. Our in vivo studies on CIA rats revealed that an AMS-H dose of 630 mg/kg significantly improved joint damage while decreasing the arthritic index and paw swelling. Furthermore, AMS inhibited the INF-γ, IL-6, IL-17, IL-1ß, and TNF-α, levels while upregulating the expression of anti-inflammatory cytokines IL-10 and IL-4 in serum. Besides, AMS inhibited the protein Bcl-2/Bax, STAT3, and JAK2 levels, and promoted the expression of Caspase3, SOCS1, and SOCS3 in the JAK2/STAT3 pathway. Additionally, the JAK/STAT signaling pathway was found to perform a remarkable function in the AMS therapy of RA as evidenced by enrichment in GO terms and KEGG pathways. Meanwhile, data from molecular docking experiments indicated that the core targets of PIK3CA, JAK2, and SRC bound stably to the active ingredients of mimuone, 4'-methoxy-bavachromanol, and quercetin. CONCLUSION: According to these findings, the AMS could improve joint inflammation in CIA rats, and its underlying mechanism could be linked to the regulation of the JAK2/STAT3 pathway. Therefore, AMS might become a promising agent for alleviating inflammation in RA patients.

Danggui Buxue Decoction exerts its therapeutic effect on rheumatoid arthritis through the inhibition of Wnt/ß-catenin signaling pathway.

BACKGROUND: Danggui Buxue Decoction (DBD) is a traditional Chinese medicine prescription, which has the functions of benefiting Qi, generating blood and regulating the immune system. At present, various clinical reports suggest that DBD has some efficacy in Rheumatoid arthritis (RA), but its mechanism of action is still unclear. Thus, the present study explored mechanism of this preparation on RA. METHODS: The effect of DBD was evaluated by tumor necrosis factor (TNF)-α-induced Human fibroblast-like synoviocyte of rheumatoid arthritis (HFLS-RA) cell model and collagen-induced arthritis (CIA) rat model, respectively. Inflammatory factors including TNF-ɑ, IL-1ß, IL-6 and IL-10 in the culture supernatants or rat serum were measured using ELISA. The related indexes including fur luster, mental state and activity of rat and the symptoms including swelling and deformation of toes and ankles were also measured. RESULTS: In vitro results showed that DBD cannot only inhibit the proliferation of HFLS-RA cells but also reduce the levels of pro-inflammatory factors while increasing the level of anti-inflammatory factors. Similar results were obtained from in vivo experiments. Rats receiving DBD showed a decrease in the severity of rheumatoid arthritis in rat models. Moreover, the protein levels of c-myc and ß-catenin decreased significantly, while the protein level of SFRP4 increased, which indicated that DBD might inhibit the inflammatory reaction by regulating Wnt/ß-catenin signaling pathway, thus alleviating the symptoms of RA. CONCLUSION: Our findings not only provide insights for understanding the molecular mechanism of DBD in treating RA, but also provide the theoretical basis for further clinical prevention and treatment.

Amelioration of Rheumatoid Arthritis by Fragaria nubicola (Wild Strawberry) via Attenuation of Inflammatory Mediators in Sprague Dawley Rats.

Background and Objectives: Fragaria nubicola has never been evaluated scientifically for its anti-arthritic potential despite its use in folkloric systems of medicine. The research was conducted to assess the potential of F. nubicola against rheumatoid arthritis. Materials and Methods: The current study provided scientific evidence by evaluating the effects of plants using an in vivo CFA-induced model of arthritic rats and subsequent microscopic histopathological evaluation of ankle joints along with the determination of paw edema using a digital water displacement plethysmometer. The study also gave insight by determining levels of pro-inflammatory cytokines, matrix metalloproteinase enzymes (MMPs), prostaglandin E2 (PGE2), nuclear factor kappa B (NF-κB), vascular endothelial growth factor (VEGF), and biochemical and hematological parameters. GCMS analysis was also conducted for the identification of possible anti-inflammatory plant constituents. Results: The data showed that F. nubicola-treated groups attenuated the progression of arthritis and paw edema. Microscopic histopathological evaluation validated the anti-arthritic potential by showing amelioration of bone erosion, infiltration of inflammatory cells, and pannus formation. RT-PCR analysis displayed that treatment with F. nubicola down-regulated IL1ß, IL6, TNFα, NF-κB, VEGF, MMP2, MMP3, and MMP9 levels. Moreover, ELISA exhibited a reduction in levels of PGE2 levels in treatment groups. The levels of RBCs, platelets, WBCs, and Hb content were found to be nearly similar to negative control in the treated group. Statistically, a non-significant difference was found when all groups were compared for urea, creatinine, ALT, and AST analysis, indicating the safety of plant extract and fractions at test doses. GCMS analysis of extract and fractions showed the existence of many anti-inflammatory and antioxidant phytochemicals. Conclusion: In conclusion, F. nubicola possessed anti-arthritic properties that might be attributed to the amelioration of MMPs and pro-inflammatory cytokines.

Attenuation of CFA-induced arthritis through regulation of inflammatory cytokines and antioxidant mechanisms by Solanum nigrum L. leaves extracts.

Solanum nigrum L. is a popular traditional medicine for various inflammatory conditions including rheumatism and joint pain. The current study aimed to evaluate the anti-arthritic mechanism of Solanum nigrum L. Four extracts were prepared using n-hexane, methanol, chloroform, and water. The anti-nociceptive and anti-inflammatory activity was carried out with 100, 200, and 300 mg/kg body wt. PO of each extract by the hot plate and carrageenan-induced paw oedema methods, respectively. The anti-arthritic study was performed with chloroform and aqueous extracts (300 mg/kg) in complete Freund's adjuvant (CFA)-induced arthritis. Paw size (mm), ankle joint diameter (mm), and latency time (sec) were recorded on day 0 and every 4th day till 28 days. The hematological, inflammatory, and oxidative biomarkers were estimated. Results showed that significant analgesia (p < 0.05) and reduction in paw inflammation were achieved with all extracts. The highest percent inhibition in Carrageenan-induced inflammation was achieved with 300 mg/kg of chloroform (72.19%) and aqueous (71.30%) extracts, respectively. In the CFA model, both extracts showed a significant reduction in paw size and ankle joint diameter (p < 0.05). The RT-qPCR analysis revealed the upregulation of interleukin-4 and interleukin-10, and down-expression of interleukin-1ß, interleukin-6, tumor necrosis factor-α, cycloxygenase-2, nuclear factor-κB, prostaglandin E synthase 2, and interferon-γ. A significant increase in superoxide dismutase, catalase, and glutathione levels was observed. Hence, it is concluded that Solanum nigrum L. leaf extracts regulate the expression of inflammatory markers and improve oxidative stress resulting in the attenuation of CFA-induced arthritis.

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

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

A newly developed PLD1 inhibitor ameliorates rheumatoid arthritis by regulating pathogenic T and B cells and inhibiting osteoclast differentiation.

Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays multiple roles in inflammation. We investigated the therapeutic effects of the newly developed PLD1 inhibitors A2998, A3000, and A3773 in vitro and in vivo rheumatoid arthritis (RA) model. A3373 reduced the levels of LPS-induced TNF-α, IL-6, and IgG in murine splenocytes in vitro. A3373 also decreased the levels of IFN-γ and IL-17 and the frequencies of Th1, Th17 cells and germinal-center B cells, in splenocytes in vitro. A3373 ameliorated the severity of collagen-induced arthritis (CIA) and suppressed infiltration of inflammatory cells into the joint tissues of mice with CIA compared with vehicle-treated mice. Moreover, A3373 prevented systemic bone demineralization in mice with CIA and suppressed osteoclast differentiation and the mRNA levels of osteoclastogenesis markers in vitro. These results suggest that A3373 has therapeutic potential for RA.

Appraisal of anti-inflammatory and immunomodulatory potential of ramipril against Freund's adjuvant-provoked arthritic rat model.

Because of evident role of renin-angiotensin system in the etiology of rheumatoid arthritis, the current study's objective was to assess the anti-arthritic efficacy of ramipril through CFA-instigated arthritic model. The drug has been shown to have anti-inflammatory potential. CFA-instigated arthritic model assessed the anti-arthritic efficacy of ramipril by estimating different parameters, including paw volume, arthritic index scoring, haematological and biochemical attributes, histological and radiographic analyses, and various cytokines level. Ramipril significantly (p < 0.001) reduced paw volume and the arthritic index especially at the dose of 4mg/kg. The biochemical and haematological changes were likewise restored to normal by ramipril administration with an increase in anti-inflammatory cytokines while reducing pro-inflammatory cytokines level. Ramipril's ability to prevent arthritis by preserving the normal architecture of arthritis-induced joints is further supported by radiographic and histological investigation. The study's findings demonstrated ramipril's considerable anti-arthritic activity. To identify the precise mechanism of action, however, thorough mechanistic studies are still needed.

Ganoderma lucidum polysaccharide peptide (GLPP) attenuates rheumatic arthritis in rats through inactivating NF-κB and MAPK signaling pathways.

BACKGROUND: Not many drugs with fewer side effects are available for the treatment of rheumatoid arthritis (RA). Ganoderma lucidum polysaccharide peptide (GLPP) has good immunomodulatory effects, but whether it is effective in managing RA is not clear. PURPOSE: This study was conducted to examine the anti-RA activity and possible mechanisms of GLPP in collagen-induced arthritis (CIA) rats. METHODS: Male Wistar rats were intradermally injected with bovine type II collagen in the tail base to establish the CIA model and were orally administered 100 or 200 mg/kg GLPP for 35 days. Paw thickness, clinical arthritis scores, gait analysis, organ index determination, blood cell counts, micro-CT imaging and pathological staining were performed on the rats. Liver and kidney function were measured by commercial kits, and antibody levels were measured by ELISA kits. RA-related protein levels were detected by Western blotting. RESULTS: GLPP effectively alleviated CIA symptoms and reduced immune organ indexes, antibody levels and systemic organ injury. GLPP decreased the protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, matrix metalloproteinase (MMP)2, MMP9, MMP13, BCL-2, OPN, ß-Catenin, and hypoxia inducible factor (HIF)-1α and increased the protein expression of BAX in the joint tissues of CIA rats. Moreover, GLPP decreased the phosphorylation levels of p65, IκB-α and ERK1/2. CONCLUSION: GLPP effectively alleviated RA symptoms in CIA rats by inhibiting the NF-κB and MAPK pathways. This study suggests a promising therapeutic effect of mushroom-derived polysaccharide peptides on RA.

Zinc and L-carnitine combination with or without methotrexate prevents intestinal toxicity during arthritis treatment via Nrf2/Sirt1/Foxo3 pathways: an In vivo and molecular docking approach.

Methotrexate (MTX) is an antifolate that is inescapable and widely used to treat autoimmune diseases and is the gold standard medicine for the arthritic condition. Despite its importance, it is more prone to gastrointestinal toxicity, which is most common in arthritis patients during MTX treatment. Combination therapies are required to ensure MTX's antiarthritic activity while providing gastrointestinal protection. Zinc (Zn) and L-carnitine (Lc) are well-known potent antioxidants and anti-inflammatory supplements with promising results in pre-clinical studies. Arthritis was induced in Wistar rat's ankles with Freund's adjuvant and treated with either MTX (2.5 mg/kg b.w per week for two weeks) or Zn (18 mg/kg b.w. per day) Lc (200 mg/kg b.w. per day) individually or in combination (MTX + Zn Lc). The antiarthritic effects were evaluated by body weight, paw volume, ankle tissue, and joint histopathology. At the same time, anti-toxicity/gastrointestinal protective activity was examined by tissue oxidative stress markers, antioxidants, mitochondrial function, inflammatory mediators, and antioxidant signaling proteins and their binding mechanism. Repercussions of MTX intoxication induced upregulation of oxidative stress markers, antioxidant depletion, ATP depletion, decreased expression of Nrf2/Sirt1/Foxo3, and the overexpression of inflammatory mediators attenuated by co-treatment with Zn Lc. Zn Lc markedly mitigated MTX-instigated intestinal injury by activating antioxidant signaling mechanisms Nrf2/Sirt1/Foxo3 signaling and tissue architectural anomalies and exhibited an enhanced antiarthritic effect. In conclusion, we report that Zn Lc and MTX combination could presumably protect the intestine from low-dose MTX which managed arthritis but induced severe intestinal damage with increased inflammation and downregulated Nrf2/Sirt1/Foxo3 pathway.

Cynanchum komarovii extract for the treatment of rheumatoid arthritis by acting on synovial cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Cynanchum komarovii (CK), the northwest Chinese region's common medicinal herb, was traditionally utilized to treat arthritis, toothache, bald sores and cholecystitis. Various forms of arthritis can be treated with CK, based on "Medicinal Plants of Chinese Desert Areas". However, the exact mechanism of action in rheumatoid arthritis (RA) is uncertain. AIM OF THE STUDY: To evaluate the in vitro and in vivo effects of CK extracts on RA and to preliminarily investigate its anti-RA mechanism of action. MATERIALS AND METHODS: The main components of CK extract were analyzed by HPLC method. The effects of CK on the proliferation and apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA) cells and the expression of apoptosis-related proteins in HFLS-RA cells were evaluated by CCK8 assay, flow cytometry and WB assay. To verify the anti-RA effect of CK extracts in vivo, a collagen-induced arthritis (CIA) rat model was established. The rats were divided into six groups: normal group, model group, CK high-dose group (1000 mg/kg, CK-H), CK medium-dose group (500 mg/kg, CK-M), CK low-dose group (250 mg/kg, CK-L) and methotrexate-positive drug group (MTX); the drug was administered continuously for 28 days. Body weight changes, joint swelling, arthritis index, bone density, ankle lesions, immune organ index, splenic lesions and inflammatory factor expression were used to evaluate the in vivo anti-RA activity of the extract. RESULTS: The findings of in vitro experiments showed that 10% CK-containing serum decreased the expression level of Bcl-2, increased the expression levels of Bax and Cleaved Caspase-3 in synovial cells, and prevented TNF-α induced aberrant proliferation and apoptotic antagonism in HFLS-RA cells. According to in vivo studies, CK extract at doses above 250 mg/kg was effective in controlling the levels of inflammatory factors, lowering the arthritis index, and improving foot swelling in CIA rats. When administered at doses up to 1000 mg/kg, CK extract significantly improved synovial lesions, increased bone density, and decreased abnormally elevated immune organ index in CIA rats. CONCLUSIONS: CK has significant anti-RA activity, and its anti-RA mechanism of action may be related to the regulation of the expression levels of apoptosis related proteins and the promotion of apoptosis in synovial cells.