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.

Coptisine inhibits aggressive and proliferative actions of fibroblast like synoviocytes and exerts a therapeutic potential for rheumatoid arthritis.

OBJECTIVE: Coptisine, a natural bioactive small molecular compound extracted from traditional Chinese herb Coptis chinensis, has been shown to exhibit anti-tumor effect. However, its contribution to autoimmune diseases such as rheumatoid arthritis (RA) is unknown. Here, we evaluate the effect of coptisine in controlling fibroblast-like synoviocytes (FLS)-mediated synovial proliferation and aggression in RA and further explore its underlying mechanism(s). METHODS: FLS were separated from synovial tissues obtained from patients with RA. Protein expression was measured by Western blot or immunohistochemistry. Gene expression was detected by quantitative RT-PCR. The EdU incorporation was used to measure cell proliferation. Migration and invasion were determined by Boyden chamber assay. RNA sequencing analysis was used to seek for the target of coptisine. The in vivo effect of coptisine was evaluated in collagen-induced arthritis (CIA) model. RESULTS: Treatment with coptisine reduced the proliferation, migration, and invasion, but not apoptosis of RA FLS. Mechanistically, we identified PSAT1, an enzyme that catalyzes serine/one-carbon/glycine biosynthesis, as a novel targeting gene of coptisine in RA FLS. PSAT1 expression was increased in FLS and synovial tissues from patients with RA compared to healthy control subjects. Coptisine treatment or PSAT1 knockdown reduced the TNF-α-induced phosphorylation of p38, ERK1/2, and JNK MAPK pathway. Interestingly, coptisine administration improved the severity of arthritis and reduced synovial PSAT1 expression in mice with CIA. CONCLUSIONS: Our data demonstrate that coptisine treatment suppresses aggressive and proliferative actions of RA FLS by targeting PSAT1 and sequential inhibition of phosphorylated p38, ERK1/2, and JNK MAPK pathway. Our findings suggest that coptisine might control FLS-mediated rheumatoid synovial proliferation and aggression, and be a novel potential agent for RA treatment.

Reduction of Edema and Pain in Transcutaneous Electrical Nerve Stimulation Treated-Arthritic Rat.

Transcutaneous electrical nerve stimulation (TENS) has been used to reduce pain or improve motor function in musculoskeletal and neurological disorders in the clinic. Although some studies have suggested electrotherapy as an intervention for edema, the effects and mechanisms of TENS on inflammation-induced edema remain unclear. Thus, we aimed to investigate the effects of TENS on arthritic pain with edema. 1% carrageenan was injected into the right tibiofemoral joint of 69 male Sprague-Dawley rats (200-250 g). After the development of arthritic pain, low-frequency (4-Hz, Low-TENS, n = 25) and high-frequency (100-Hz, High-TENS, n = 25) TENS with sub-motor threshold or placebo-TENS (n = 19) was applied for 20-min to medio-lateral part of the ipsilateral side. Weight bearing and knee-bend tests were used to assess pain-like behaviors. Also, we examined the size of edema and measured tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) levels in the synovium by western blot. Eight rats in each of the two TENS groups were injected with Naloxone. Edema was reduced in the low- and high-frequency TENS groups at 6-h. TENS-treated rats showed reduced pain in the knee-bend test at 6-h. We observed decreased weight load shifts on the ipsilateral side in TENS groups. Naloxone reduced these effects. TNF-α and IL-1ß expression decreased in the synovial membrane at 6-h. These results suggest that low- and high-frequency TENS have acutely positive effects on inflammatory edema, with the management of arthritic pain and reduction in pro-inflammatory mediators. Therefore, Low-TENS and High-TENS may be useful in treating acute inflammatory pain and edema.

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-α was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Lycium ruthenicum Murr. anthocyanins inhibit hyperproliferation of synovial fibroblasts from rheumatoid patients and the mechanism study powered by network pharmacology.

BACKGROUND: Rheumatoid arthritis (RA), is a typical autoimmune disease affecting nearly 1% of the world's population. The dysfunctional hyperproliferation of synovial fibroblast (SF) in articular cartilage of RA patients is considered as the essential etiology. Traditional chemotherapeutic agents for RA treatment are imperfect for their high cost and unpredictable side-effects. L. ruthenicum anthocyanins (LRAC) is a natural product that of potential for therapeutic application against RA. METHODS: LRAC was characterized by UPLC-MS/MS. Bioinformatics analyses based on network pharmacology were applied to predict the potential targets of LRAC, and to select DEGs (differentially expressed genes) caused by RA pathogenesis from GSE77298. Interactions between LRAC and the predicted targets were evaluated by molecular docking. Effects of LRAC on SFs from RA patients were examined by in vitro assays, which were analyzed by flow cytometry and western blotting (WB). RESULTS: LRAC was able to inhibit the abnormal proliferation and aggressive invasion of SFs from RA patients. LRAC was mainly constituted by petunidin (82.7%), with small amount of delphinidin (12.9%) and malvidin (4.4%) in terms of anthocyanidin. Bioinformatics analyses showed that in 3738 RA-related DEGs, 58 of them were collectively targeted by delphinidin, malvidin and delphinidin. AR, CDK2, CHEK1, HIF1A, CXCR4, MMP2 and MMP9, the seven hub genes constructed a central network mediating the signal transduction. Molecular docking confirmed the high affinities between the LRAC ligands and the protein receptors encoded by the hub genes. The in vitro assays validated that LRAC repressed the growth of RASF by cell cycle arresting and cell invasion paralyzing (c-Myc/p21/CDK2), initiating cell apoptosis (HIF-1α/CXCR4/Bax/Bcl-2), and inducing pyroptosis via ROS-dependent pathway (NOX4/ROS/NLRP3/IL-1ß/Caspase-1). CONCLUSION: LRAC can selectively inhibit the proliferation of RASFs, without side-effecting immunosuppression that usually occurred for RA treatment using MTX (methotrexate). These findings demonstrate the potential application of LRAC as a phytomedicine for RA treatment, and provide a valid approach for exploring natural remedies against autoimmune diseases.

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.

Alcohol extracts from Anemone flaccida Fr. Schmidt treat rheumatoid arthritis via inhibition of synovial hyperplasia and angiogenesis.

Anemone flaccida Fr. Schmidt, a Traditional Chinese Medicine, has been used in the treatment of rheumatoid arthritis (RA) for numerous years. However, the specific mechanisms remain to be elucidated. Thus, the present study aimed to investigate the main chemical constituents and potential mechanisms of Anemone flaccida Fr. Schmidt. The ethanol extract obtained from Anemone flaccida Fr. Schmidt (EAF) was analyzed using mass spectrometry to determine the main components and the therapeutic effects of EAF on RA were verified using a collagen­induced arthritis (CIA) rat model. Results of the present study demonstrated that synovial hyperplasia and pannus of the model rats were significantly improved following EAF treatment. Moreover, the protein expression levels of VEGF and CD31­labeled neovascularization were significantly reduced in the synovium of CIA rats following treatment with EAF, compared with those of the untreated model group. Subsequently, in vitro experiments were carried out to verify the impact of EAF on synovial proliferation and angiogenesis. Results of the western blot analysis revealed that EAF inhibited the PI3K signaling pathway in endothelial cells, which is associated with anti­angiogenesis. In conclusion, results of the present study demonstrated the therapeutic effects of Anemone flaccida Fr. Schmidt on RA and preliminarily revealed the mechanisms of this drug in the treatment of RA.

Triptolide improves chondrocyte proliferation and secretion via down-regulation of miR-221 in synovial cell exosomes.

BACKGROUND: Rheumatoid arthritis (RA), the most common type of inflammatory arthritis, can cause bone damage and disability. Triptolide, a prominent treatment for RA, has satisfactory anti-inflammatory effects. However, the mechanism of action of triptolide in RA remains unknown. PURPOSE: This study aimed to explore the molecular mechanisms underlying triptolide-mediated improvements in RA and identify the miRNA pathway responsible for these effects. METHODS: We identified various dysregulated miRNAs associated with RA by mining previously described microarray data and verified and screened these candidates using RT-qPCR. Hematoxylin-eosin staining was then applied to identify pathological changes in the affected joints, and cell counting kit-8 analysis and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. Extracted exosomes were verified using transmission electron microscopy. RESULTS: Our results revealed that the legs of rats with collagen-induced arthritis presented with obvious swelling and bone damage, a high degree of inflammatory cell infiltration into the synovium, and structural changes to the cartilage. Data mining identified 39 dysregulated miRNAs in these tissues, and RT-qPCR further refined these observations to highlight miR-221 as a potential RA biomarker. Subsequent evaluations revealed that fibroblast-like synovial (FLS) cells secrete Exs carrying dysregulated miR-221 in vitro. These Exs mediate miR-221 levels, inflammation, and TLR4/MyD88 signaling via their fusion with chondrocytes, leading to changes in chondrocyte growth and metabolic factor levels. Additionally, the addition of triptolide impaired miR-221 expression, cell proliferation, inflammatory factors, and the protein levels of TLR4/MyD88 in RA-FLS and promoted the apoptosis of FLS. The therapeutic effect of triptolide on miR-221 Exs was reversed by miR-221 inhibitor in both normal and RA FLS. CONCLUSION: Our research shows that effective treatment with triptolide is mediated by its regulation of growth and secretory functions of chondrocytes via the inhibition of miR-221 secretion by FLS, providing a new target and natural medicinal candidate for future RA treatments.

Transcriptomic profiling revealed the role of apigenin-4'-O-α-L-rhamnoside in inhibiting the activation of rheumatoid arthritis fibroblast-like synoviocytes via MAPK signaling pathway.

BACKGROUND: Activated fibroblast-like synoviocyte (FLS) played a significant role in the pathogenesis and progression of rheumatoid arthritis (RA). Apigenin-4'-O-α-L-rhamnoside showed remarkable effects against RA, however, no relevant studies on pharmacology of apigenin-4'-O-α-L-rhamnoside yet, the effects and underlying molecular mechanism of apigenin-4'-O-α-L-rhamnoside on RA are still unclear. PURPOSE: This study aimed to investigate the therapeutic effects and mechanisms of apigenin-4'-O-α-L-rhamnoside on RA-FLS cells by transcriptomic analysis. METHODS: In vitro, RA-FLS cell viability and migration were measured by CCK-8 and scratch assays, respectively. The effects of apigenin-4'-O-α-L-rhamnoside on inflammatory levels of MMP-1, MMP-3, RANKL and TNF-α in RA-FLS cells were detected using ELISA kits. High-throughput transcriptome analysis was performed to screen the key genes and related pathways of apigenin-4'-O-α-L-rhamnoside inhibit RA-FLSs, and the result of which were validated by RT-qPCR and western blot. Furthermore, in vivo, we also evaluated the effects of apigenin-4'-O-α-L-rhamnoside in rat with CIA. RESULTS: Apigenin-4'-O-α-L-rhamnoside significantly suppressed RA-FLS migration, exerted remarkable inhibiting effects on the expression levels on MMP-1, MMP3, RANKL and TNF-α in RA-FLS cells. It seemed that MAPK signaling pathway might be closely related to the pathogenesis of RA by down-regulated relevant core targets (MAPK1, HRAS, ATF-2, p38 and JNK). Moreover, apigenin-4'-O-α-L-rhamnoside attenuated the severity of arthritis in CIA rat. CONCLUSION: Apigenin-4'-O-α-L-rhamnoside inhibited pro-inflammatory cytokine, chemokine and MMPs factors production of RA-FLS by targeting the MAPK signaling pathway, which provided a scientific basis for potential application in the treatment of RA.

Which part of the blood in the knee joint is responsible for its detrimental effects? (An experimental study on the knee joint in rabbits).

AIM: This study aims to determine the potential causative elements which are responsible for the cartilage damage in case of frequent intra-articular bleeding and to evaluate the effects of intra-articular free iron and chelation of iron in the knee joint. METHODS: Thirty-five New Zealand rabbits were randomly divided into five groups according to substances injected into their knee joints. Plasma (group I) and cellular components (group II) of the blood harvested from the rabbits, iron (ferric hydroxide sucrose) (group III), iron&chelator (group IV) and only chelator (deferoxamine mesylate) (group V) were injected into their right knees three times a week for 12 weeks. The joint surface was examined histologically according to the classification system modified from Colombo et al. The changes in the synovial tissue were evaluated according to the scoring system modified from Madhok et al. RESULTS: Cartilage and synovial abnormality scores were significantly higher in all study groups when compared to their own controls (p < 0.0001). Cartilage scores of groups I and V were significantly lower when compared to groups III and IV (p = 0.002 for group I and p = 0.003 for group V). Synovial abnormality score of group I was significantly lower than scores of groups III and IV (p = 0.001); and of group V lower than groups III and IV (p = 0.003 and p = 0.001, respectively). CONCLUSIONS: All substances tested in this study caused a certain amount of damage in the cartilage tissue and led to synovial abnormalities. Both iron and iron&chelator caused more damage in the cartilage and led to more advanced synovial changes when compared to the plasma component of blood and chelator itself. Influence of iron and iron&chelators were found to be similar showing that chelation was inadequate in antagonizing the detrimental effects of iron.

Lactate metabolism in rheumatoid arthritis: Pathogenic mechanisms and therapeutic intervention with natural compounds.

BACKGROUND: Rheumatoid arthritis (RA) is a common chronic and systemic autoimmune disease characterized by persistent inflammation and hyperplasia of the synovial membrane, the degradation of cartilage, and the erosion of bones in diarthrodial joints. The inflamed joints of patients with RA have been recognized to be a site of hypoxic microenvironment which results in an imbalance of lactate metabolism and the accumulation of lactate. Lactate is no longer considered solely a metabolic waste product of glycolysis, but also a combustion aid in the progression of RA from the early stages of inflammation to the late stages of bone destruction. PURPOSE: To review the pathogenic mechanisms of lactate metabolism in RA and investigate the potential of natural compounds for treating RA linked to the regulation of imbalance in lactate metabolism. METHODS: Research advances in our understanding of lactate metabolism in the pathogenesis of RA and novel pharmacological approaches of natural compounds by targeting lactate metabolic signaling were comprehensively reviewed and deeply discussed. RESULTS: Lactate produced by RA synovial fibroblasts (RASFs) acts on targeted cells such as T cells, macrophages, dendritic cells and osteoclasts, and affects their differentiation, activation and function to accelerate the development of RA. Many natural compounds show therapeutic potential for RA by regulating glycolytic rate-limiting enzymes to limit lactate production, and affecting monocarboxylate transporter and acetyl-CoA carboxylase to inhibit lactate transport and conversion. CONCLUSION: Regulation of imbalance in lactate metabolism offers novel therapeutic approaches for RA, and natural compounds capable of targeting lactate metabolic signaling constitute potential candidates for development of drugs RA.

Wilforlide A ameliorates the progression of rheumatoid arthritis by inhibiting M1 macrophage polarization.

Rheumatoid arthritis (RA) is an autoimmune disease with increased M1 macrophages. The classical activated M1 macrophages produce various cytokines to control inflammation. Wilforlide A is a natural product that displays anti-inflammatory activities. However, the effect of Wilforlide A on RA progression and the potential mechanisms are unclear. Herein, the collagen-induced arthritis (CIA) mouse was used as an experimental model of RA. The administration of Wilforlide A reduced clinical scores, joint swelling and histological damage in ankle joints of RA mice. The secreted pro-inflammatory factors (MCP1, GM-CSF and M-CSF) and M1 biomarker iNOS in synovium were inhibited by Wilforlide A. In vitro, macrophages deriving from THP-1 cells were stimulated with LPS/IFN-γ to mimic M1 polarization. Similarly, Wilforlide A blocked macrophages polarizing towards M1 subsets. The in vitro results demonstrated that Wilforlide A suppressed LPS/IFN-γ-induced TLR4 upregulation, IκBα degradation and NF-κB p65 activation. In addition, TAK242 (a TLR4 inhibitor) treatment caused a similar inhibitory effect on M1 polarization with Wilforlide A, whereas it was less than the combination of TAK242 and Wilforlide A. Therefore, this work supports that Wilforlide A ameliorates M1 macrophage polarization in RA, which is partially mediated by TLR4/NF-κB signaling pathway inactivation.

[Sidaxue, a traditional Guizhou Miao herbal medicine formula, reduces necroptosis and synovial vascular proliferation in rats with collagen-induced arthritis by inhibiting the RIPK1/RIPK3/MLKL pathway].

OBJECTIVE: To explore the inhibitory effect of Sidaxue (SX), a traditional Guizhou Miao herbal medicine formula, on necrotic apoptosis and synovial angiogenesis in rats with collagen-induced arthritis (CIA) and the role of the RIPK1/RIPK3/MLKL pathway in mediating this effect. METHODS: Forty-two SD rats were randomized into 6 groups (n=7), including a normal control group, a CIA model group, 3 SX treatment groups at low (10 g/kg), moderate (20 g/kg) and high (40 g/kg) doses, and a GTW treatment group. CIA rat models were established by subcutaneous injections of bovine type II collagen, and the treatments were administered daily by gavage for 21 days. The rats were observed for swelling of the hind limb joints, which was rated using the arthritis index (AI) score on a weekly basis. Serum levels of TNF-α, IL-1ß and IL-17 in the rats were detected using ELISA, and the pathological changes in the synovium were observed with HE staining. Real-time PCR was performed to detect the mRNA expression levels of VEGF, MMP-9, Ang-1, RIPK1, RIPK3, and caspase-8 in the synovial tissues, and the protein expressions of VEGF, MMP9, Ang-1, Stat-3, RIPK1, RIPK3, MLKLl, p-MLKL and caspase-8 were detected using Western blotting. RESULTS: Compared with those in CIA model group, the rats receiving treatment with GTW and SX showed milder swelling of the hind limb joints with significantly lower AI scores (P < 0.05). In CIA model group, a large number of inflammatory cells were observed in the synovium with obvious damages of the tissue structure. In the drug treatment groups, inflammatory cell infiltration, synovial angiogenesis and synovial hyperplasia were alleviated, and the therapeutic effects were obviously enhanced as SX dose increased. Compared with those in the model group, the rats treated with GTW and high-dose SX showed significantly decreased serum levels of IL-1ß, IL-17 and TNF-α (P < 0.05), lower mRNA and protein expressions of RIPK1, RIPK3, VEGF, Ang-1, and MMP9 (P < 0.05), higher expressions of caspase-8 (P < 0.01), and obviously lowered expression of Stat-3 protein and phosphorylation level of MLKL (P < 0.05). CONCLUSION: SX can improve synovial angiogenesis in CIA rats possibly by inhibiting the activation of RIP1/RIP3/MLKL signaling pathway and down-regulating the expression of the vascular growth factors VEGF, Ang-1, MMP9, and Stat-3.

Inhibition of SYK and cSrc kinases can protect bone and cartilage in preclinical models of osteoarthritis and rheumatoid arthritis.

The pathophysiology of osteoarthritis (OA) includes the destruction of subchondral bone tissue and inflammation of the synovium. Thus, an effective disease-modifying treatment should act on both of these pathogenetic components. It is known that cSrc kinase is involved in bone and cartilage remodeling, and SYK kinase is associated with the inflammatory component. Thus the aim of this study was to characterize the mechanism of action and efficacy of a small molecule multikinase inhibitor MT-SYK-03 targeting SYK and cSrc kinases among others in different in vitro and in vivo arthritis models. The selectivity of MT-SYK-03 kinase inhibition was assayed on a panel of 341 kinases. The compound was evaluated in a set of in vitro models of OA and in vivo OA and RA models: surgically-induced arthritis (SIA), monosodium iodoacetate-induced arthritis (MIA), collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA). MT-SYK-03 inhibited cSrc and SYK with IC50 of 14.2 and 23 nM respectively. Only five kinases were inhibited > 90% at 500 nM of MT-SYK-03. In in vitro OA models MT-SYK-03 reduced hypertrophic changes of chondrocytes, bone resorption, and inhibited SYK-mediated inflammatory signaling. MT-SYK-03 showed preferential distribution to joint and bone tissue (in rats) and revealed disease-modifying activity in vivo by halving the depth of cartilage erosion in rat SIA model, and increasing the pain threshold in rat MIA model. Chondroprotective and antiresorptive effects were shown in a monotherapy regime and in combination with methotrexate (MTX) in murine and rat CIA models; an immune-mediated inflammation in rat AIA model was decreased. The obtained preclinical data support inhibition of cSrc and SYK as a viable strategy for disease-modifying treatment of OA. A Phase 2 clinical study of MT-SYK-03 is to be started.

Dissecting the Molecular Mechanism of Wang-Bi Capsule in the Treatment of Experimental Rheumatoid Arthritis Based on Synovial Tissue Proteomic Analysis.

Wang-Bi capsule (WB) is a traditional Chinese medicine formula and has been applied for rheumatoid arthritis (RA) treatment for many years. However, its underlying molecular mechanisms still remain unclear. In this study, collagen-induced arthritis (CIA) rats were used to observe the therapeutic effect of WB used at different time points, and the proteomic analysis of synovial tissue was applied to reveal its basic molecular mechanisms. The results demonstrated that WB not only effectively ameliorated the symptoms and synovitis, but also downregulated the serum levels of inflammatory cytokines/chemokines in CIA rats. Furthermore, the proteomic analysis of synovial tissue showed that WB could regulate several signaling pathways associated with inflammation or cell migration, such as "IL-1 signaling," "IL-8 signaling," and "CXCR4 signaling." The expression levels of proteins including matrix metalloproteinase 3 (MMP3), MMP19, lipopolysaccharide-binding protein (LBP), serine/threonine kinase interleukin-1 receptor-associated kinase 4 (IRAK4), and actin-related protein 2/3 complex subunit 5 (ARPC5) in these pathways were downregulated significantly by WB when compared with the model group. In sum, this study indicated that WB had obvious inhibitory effects on synovitis of CIA rats, and the mechanisms of which may be involved in downregulating the expression levels of several key proteins including MMP3, MMP19, LBP, IRAK4, and ARPC5.

Nitidine chloride inhibits fibroblast like synoviocytes-mediated rheumatoid synovial inflammation and joint destruction by targeting KCNH1.

OBJECTIVE: Nitidine chloride (NC), a natural small molecular compound from traditional Chinese herbal medicine zanthoxylum nitidum, has been shown to exhibit anti-tumor effect. However, its role in autoimmune diseases such as rheumatoid arthritis (RA) is unknown. Here, we investigate the effect of NC in controlling fibroblast-like synoviocytes (FLS)-mediated synovial inflammation and joint destruction in RA and further explore its underlying mechanism(s). METHODS: FLSs were separated from synovial tissues obtained from patients with RA. Protein expression was analyzed by Western blot or immunohistochemistry. Gene expression was measured using quantitative RT-PCR. ELISA was used to measure the levels of cytokines and MMPs. Cell proliferation was detected using EdU incorporation. Migration and invasion were evaluated by Boyden chamber assay. RNA sequencing analysis was used to identify the target of NC. Collagen-induced arthritis (CIA) model was used to evaluate the in vivo effect of NC. RESULTS: NC treatment reduced the proliferation, migration, invasion, and lamellipodia formation but not apoptosis of RA FLSs. We also demonstrated the inhibitory effect of NC on TNF-α-induced expression and secretion of IL-6, IL-8, CCL-2, MMP-1 and MMP-13. Furthermore, we identified KCNH1, a gene that encodes ether-à-go-go-1 channel, as a novel targeting gene of NC in RA FLSs. KCNH1 expression was increased in FLSs and synovial tissues from patients with RA compared to healthy controls. KCNH1 knockdown or NC treatment decreased the TNF-α-induced phosphorylation of AKT. Interestingly, NC treatment ameliorated the severity of arthritis and reduced synovial KCNH1 expression in mice with CIA. CONCLUSIONS: Our data demonstrate that NC treatment inhibits aggressive and inflammatory actions of RA FLSs by targeting KCNH1 and sequential inhibition of AKT phosphorylation. Our findings suggest that NC might control FLS-mediated rheumatoid synovial inflammation and joint destruction, and be a novel therapeutic agent for RA.

Critical Role of Synovial Tissue-Resident Macrophage and Fibroblast Subsets in the Persistence of Joint Inflammation.

Rheumatoid arthritis (RA) is a chronic prototypic immune-mediated inflammatory disease which is characterized by persistent synovial inflammation, leading to progressive joint destruction. Whilst the introduction of targeted biological drugs has led to a step change in the management of RA, 30-40% of patients do not respond adequately to these treatments, regardless of the mechanism of action of the drug used (ceiling of therapeutic response). In addition, many patients who acheive clinical remission, quickly relapse following the withdrawal of treatment. These observations suggest the existence of additional pathways of disease persistence that remain to be identified and targeted therapeutically. A major barrier for the identification of therapeutic targets and successful clinical translation is the limited understanding of the cellular mechanisms that operate within the synovial microenvironment to sustain joint inflammation. Recent insights into the heterogeneity of tissue resident synovial cells, including macropahges and fibroblasts has revealed distinct subsets of these cells that differentially regulate specific aspects of inflammatory joint pathology, paving the way for targeted interventions to specifically modulate the behaviour of these cells. In this review, we will discuss the phenotypic and functional heterogeneity of tissue resident synovial cells and how this cellular diversity contributes to joint inflammation. We discuss how critical interactions between tissue resident cell types regulate the disease state by establishing critical cellular checkpoints within the synovium designed to suppress inflammation and restore joint homeostasis. We propose that failure of these cellular checkpoints leads to the emergence of imprinted pathogenic fibroblast cell states that drive the persistence of joint inflammation. Finally, we discuss therapeutic strategies that could be employed to specifically target pathogenic subsets of fibroblasts in RA.

Human umbilical cord mesenchymal stem cell-derived small extracellular vesicles ameliorate collagen-induced arthritis via immunomodulatory T lymphocytes.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease for which there are currently no effective therapies. Although mesenchymal stem cells (MSCs) can prevent arthritis through immunomodulatory mechanisms, there are several associated risks. Alternatively, MSC-derived small extracellular vesicles (sEVs) can mimic the effects of MSCs, while reducing the risk of adverse events. However, few studies have examined sEVs in the context of RA. Here, we evaluate the immunomodulatory effects of human umbilical cord MSC (hUCMSC)-derived sEVs on T lymphocytes in a collagen-induced arthritis (CIA) rat model to elucidate the possible mechanism of sEVs in RA treatment. We then compare these mechanisms to those of MSCs and methotrexate (MTX). METHODS: The arthritis index and synovial pathology were assessed. T lymphocyte proliferation and apoptosis, Th17 and Treg proportions, and interleukin (IL)-17, IL-10, and transforming growth factor (TGF)-ß expression were detected using flow cytometry. Retinoic acid receptor-related orphan receptor gamma t (RORγt) and forkhead box P3 (FOXP3), which are master transcriptional regulators of Th17 and Treg differentiation, were also assessed using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: sEV treatment ameliorated arthritis and inhibited synovial hyperplasia in a dose-dependent manner. These effects were mediated by inhibiting T lymphocyte proliferation and promoting their apoptosis, while decreasing Th17 cell proportion and increasing that of Treg cells in the spleen, resulting in decreased serum IL-17, and enhanced IL-10 and TGF-ß expression. Transcriptionally, sEVs decreased RORγt and increased FOXP3 expression in the spleen, and decreased RORγt and FOXP3 expression in the joints. In some aspects sEVs were more effective than MSCs and MTX in treating CIA. CONCLUSIONS: hUCMSC-derived sEVs ameliorate CIA via immunomodulatory T lymphocytes, and might serve as a new therapy for RA.

ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses.

Zinc is a trace element that is essential for immune responses. Therefore, changes in cellular zinc levels in specific immune cells may influence inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). However, the regulation of zinc mobilization in immune cells and its role in the pathogenesis of RA are not fully understood. Thus, we investigated the roles of zinc transporters in RA pathogenesis. We demonstrated that ZIP8 was specifically upregulated in CD4+ T cells that infiltrated the inflamed joint and that ZIP8 deficiency in CD4+ T cells abrogated collagen-induced arthritis. ZIP8 deficiency dramatically affected zinc influx in effector T cells and profoundly reduced T cell receptor (TCR)-mediated signaling, including NF-κB and MAPK signaling, which are pathways that are involved in T helper (Th) 17 cell differentiation. Taken together, our findings suggest that ZIP8 depletion in CD4+ T cells attenuates TCR signaling due to insufficient cellular zinc, thereby reducing the function of effector CD4+ T cells, including Th17 cells. Our results also suggest that targeting ZIP8 may be a useful strategy to inhibit RA development and pathogenesis.

Effects of rhizome of Atractylodes koreana (Nakai) Kitam on intestinal flora and metabolites in rats with rheumatoid arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodis rhizoma in Chinese Pharmacopoeia are Atractylodes lancea (Thunb.) DC and Atractylodes chinensis (DC.) Koidz. Atractylodes koreana (Nakai) Kitam has not been included in Chinese Pharmacopoeia, however, in the 'dictionary of traditional Chinese medicine', Atractylodes koreana (Nakai) Kitam is often used as Atractylodis rhizoma in the north of China. According to 'Chinese traditional medicine resources', Atractylodes koreana (Nakai) Kitam has the function of drying dampness and strengthening the spleen, dispelling wind and eliminating dampness. AIM OF THIS STUDY: The study was to explore the effect and mechanism of Atractylodes koreana (Nakai) Kitam on rheumatoid arthritis(RA) through intestinal flora and its metabolites(short chain fatty acids). MATERIALS AND METHODS: 36 male SD rats were randomly divided into 6 groups. The Freund's complete adjuvant method was used to reproduce RA model. The contents of inflammatory factors in the plasma of rats were monitored by ELISA method. The pathological changes of synovium were observed. 16SrDNA high-throughput sequence method was used to study the composition and structure of intestinal microflora in each group of rats. Gas Chromatography and Mass Spectrum(GC-MS) method was used to determine the content of short chain fatty acids(SCFAs) in colon of rats of each group. RESULTS: After oral administration of Atractylodes koreana (Nakai) Kitam, the synovial infiltration and vascular proliferation in RA rats were alleviated, the level of TNF - α, IL-1, IL-1 ß, IL-2, IL-6, hs-CRP in the plasma of RA rats were declined. RA could cause the disturbance of intestinal flora and SCFAs, Atractylodes koreana (Nakai) Kitam could regulate 8 genera of intestinal flora and improve the disorder of SCFAs. CONCLUSIONS: Atractylodes koreana (Nakai) Kitam has a therapeutic effect on RA, the therapeutic mechanism may be related to down-regulating inflammatory factors and improving the imbalance of intestinal flora and SCFAs.