Nuciferine alleviates collagen-induced arthritic in rats by inhibiting the proliferation and invasion of human arthritis-derived fibroblast-like synoviocytes and rectifying Th17/Treg imbalance.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder marked by persistent synovial inflammation and joint degradation, posing challenges in the development of effective treatments. Nuciferine, an alkaloid found in lotus leaf, has shown promising anti-inflammatory and anti-tumor effects, yet its efficacy in RA treatment remains unexplored. This study investigated the antiproliferative effects of nuciferine on the MH7A cell line, a human RA-derived fibroblast-like synoviocyte, revealing its ability to inhibit cell proliferation, promote apoptosis, induce apoptosis, and cause G1/S phase arrest. Additionally, nuciferine significantly reduced the migration and invasion capabilities of MH7A cells. The therapeutic potential of nuciferine was further evaluated in a collagen-induced arthritis (CIA) rat model, where it markedly alleviated joint swelling, synovial hyperplasia, cartilage injury, and inflammatory infiltration. Nuciferine also improved collagen-induced bone erosion, decreased pro-inflammatory cytokines and serum immunoglobulins (IgG, IgG1, IgG2a), and restored the balance between T helper (Th) 17 and regulatory T cells in the spleen of CIA rats. These results indicate that nuciferine may offer therapeutic advantages for RA by decreasing the proliferation and invasiveness of FLS cells and correcting the Th17/Treg cell imbalance in CIA rats.

Sophoridine exerts anti-arthritic effects on fibroblast-like synoviocytes and collagen-induced arthritis in rats.

The discovery of alternative medicines with fewer adverse effects is urgently needed for rheumatoid arthritis (RA). Sophoridine (SR), the naturally occurring quinolizidine alkaloid isolated from the leguminous sophora species, has been demonstrated to possess a wide range of pharmacological activities. However, the effect of SR on RA remains unknown. In this study, the collagen-induced arthritis (CIA) rat model and tumor necrosis factor alpha (TNFα)-induced fibroblast-like synoviocytes (FLSs) were utilized to investigate the inhibitory effect of SR on RA. The anti-arthritic effect of SR was evaluated using the CIA rat model in vivo and TNFα-stimulated FLSs in vitro. Mechanistically, potential therapeutic targets and pathways of SR in RA were analyzed through drug target databases and disease databases, and validation was carried out through immunofluorescence, immunohistochemistry, and Western blot. The in vivo results revealed that SR treatment effectively ameliorated synovial inflammation and bone erosion in rats with CIA. The in vitro studies showed that SR could significantly suppress the proliferation and migration in TNFα-induced arthritic FLSs. Mechanistically, SR treatment efficiently inhibited the activation of MAPKs (JNK and p38) and NF-κB pathways in TNFα-induced arthritic FLSs. These findings were further substantiated by Immunohistochemistry results in the CIA rat. SR exerts an anti-arthritic effect in CIA rats through inhibition of the pathogenic characteristic of arthritic FLSs via suppressing NF-κB and MAPKs (JNK and p38) signaling pathways. SR may have a great potential for development as a novel therapeutic agent for RA treatment.

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.

Salidroside suppresses cell growth and inflammatory response of fibroblast-like synoviocytes via inhibition of phosphoinositol-3 kinase/threonine kinase signaling in rheumatoid arthritis.

BACKGROUND: Salidroside (Sal) is a natural product commonly isolated from Rhodiola rosea L., which has been found to have numerous pharmacological activities (e.g., ameliorating apoptosis and inflammation, and acting as an antioxidant) in various diseases, but its concrete function in rheumatoid arthritis (RA) has not been revealed yet. Here, we aimed to explore the specific role and underlying mechanisms of Sal in RA-fibroblast-like synoviocytes (RA-FLSs). METHODS: Cell counting kit 8 (CCK-8) was used to assess the viability of normal-FLSs and RA-FLSs. Cell apoptosis in RA-FLSs was evaluated by flow cytometry. Western blotting was prepared to examine the levels of apoptosis- and signaling-related proteins. Wound-healing and Transwell assays were conducted to examine RA-FLSs migration and invasion. Enzyme-linked immunosorbent assay (ELISA) was used to assess the effect of Sal on tumor necrosis factor-alpha (TNF-α)-induced inflammation in RA-FLSs. RA animal model was established through complete Freund's adjuvant (CFA) induction, and the histopathological changes in synovial tissues of the rat model were analyzed by H&E staining. RESULTS: RA-FLSs were treated with 200 µM Sal for 24 h, and cell viability was significantly suppressed. Sal promoted RA-FLSs apoptosis. The migratory and invasive abilities of RA-FLSs were markedly inhibited by Sal. Sal incubation reduced the levels of inflammatory cytokines interleukin­8 (IL-8), IL-1ß, and IL­6 in RA-FLSs under the stimulation of TNF­α. Subsequently, Sal downregulated phosphorylated phosphatidylinositol­3 kinase (p-PI3K) and protein kinase (p-AKT) expression in RA-FLSs. After the treatment with pathway activator 740Y­P (20 µM) in RA-FLSs, the promotive effect of Sal on cell apoptosis was reversed, and inhibitory effects of it on cell viability, migration, invasion, and inflammatory response were abolished. Sal inhibited RA development in the CFA-induced rat model. CONCLUSION: Sal suppressed cell growth and inflammation in RA-FLSs by inactivating PI3K/AKT-signaling pathways.

Non-coding RNAs involved in fibroblast-like synoviocyte functioning in arthritis rheumatoid: From pathogenesis to therapy.

Rheumatoid arthritis (RA) is a polygenic autoimmune disorder with an uncertain etiology, primarily impacting the joints. Moreover, the disease may manifest beyond articular involvement, leading to extra-articular manifestations. Fibroblast-like synoviocytes (FLS) are cells of mesenchymal origin that possess crucial physiological significance within the synovium, contributing to the synthesis of specific constituents found in the synovial fluid and articular cartilage. Consequently, there has been a growing focus on FLS as a potential therapeutic target in the context of RA. Recent investigations have revealed that non-coding RNAs (ncRNAs) serve as pivotal regulators of FLS function, with their dysregulated expression patterns being detected within FLS populations. NcRNAs, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), assume essential functions as regulators of gene expression at both the post-transcriptional and transcriptional levels, and also serve as guiding molecules for chromatin-modifying complexes. Majority of these ncRNAs contribute to various FLS activities including metastasis, proliferation, and cytokine production. In the current work, we comprehensively review the existing literature on ncRNAs, which play pivotal roles in FLS activity and the pathogenesis of RA. Furthermore, this study provides a comprehensive summary and description of the lncRNA/circRNA-miRNA-mRNA regulatory axes in FLS activity, along with potential implications for the RA development. As well, in the final section, we illustrated that therapeutic agents including herbal medicine, and exosomes by modulating ncRNAs regulate FLS activity.

Tyro3 receptor tyrosine kinase contributes to pathogenic phenotypes of fibroblast-like synoviocytes in rheumatoid arthritis and disturbs immune cell balance in experimental arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by synovitis and joint damage, the underlying causes of which remain unclear. Our prior investigations revealed a notable correlation between the expression of Tyro3 Protein Tyrosine Kinase (Tyro3TK) and the progression of RA. To further elucidate the pathogenic role of Tyro3TK in RA, we analyzed the influence of Tyro3TK on pathogenic phenotypes of RA fibroblast like synoviocyte (FLS) in vitro and compared disease severity, joint damages and immunological parameters of K/BxN serum transfer arthritis (STA) in Tyro3TK-/- deficient mice and wild type controls. Our findings underscored the remarkable effectiveness of Tyro3TK blockade, as evidenced by diminished secretion of inflammatory cytokines and matrix metalloproteinases (MMPs), curtailed migration and invasiveness of RAFLS, and attenuated differentiation of pathogenic helper T cell subsets mediated by RAFLS. Correspondingly, our in vivo investigations illuminated the more favorable outcomes in Tyro3TK-deficient mice, characterized by reduced joint pathology, tempered synovial inflammation, and restored immune cell equilibrium. These data suggested that Tyro3TK might contribute to aggravated autoimmune arthritis and immunological pathology and act as a potential therapeutic target for RA.

Wnt/ß-catenin signaling pathway promotes abnormal activation of fibroblast-like synoviocytes and angiogenesis in rheumatoid arthritis and the intervention of Er Miao San.

BACKGROUND: Er Miao San (EMS) is an important herbal formula and a representative prescription for the treatment of the downwards flow of damp-heat syndrome. Clinical practice has proven that EMS can effectively treat rheumatoid arthritis (RA). Previous studies have demonstrated that EMS regulates the functions of T cells and dendritic cells and affects the polarization of macrophages. However, it is not clear whether the inhibitory effect of EMS on RA is related to the regulation of abnormal synovial activation and angiogenesis. PURPOSE: The aim of this study was to elucidate the effect and potential mechanisms of EMS on the abnormal activation and angiogenesis of fibroblast-like synoviocytes (FLSs) in RA. METHODS: The effect of EMS on rats with adjuvant arthritis (AA) and MH7A cells was examined by X-ray, haematoxylin-eosin (HE) staining, immunohistochemistry (IHC), ELISA and western blotting. Angiogenesis in AA rats was measured by a small animal ultrasound imaging system, immunofluorescence (IF) analysis and ELISA. An exchange between MH7A cells and HUVECs was induced using conditioned media that mimicked the microenvironment in vivo. CCK-8, western blotting, and scratch healing and Transwell migration assays were used to evaluate the effect of EMS on the Wnt/ß-catenin signaling pathway and angiogenesis in the inflammatory microenvironment of RA. RESULTS: Our results showed that EMS had a protective effect on AA rats. On the one hand, there was a decrease in paw swelling, the arthritis index, organ indices and proinflammatory factor levels, as well as relief of joint damage. On the other hand, blood flow, the number of immature blood vessels and proangiogenic factors were decreased. Furthermore, EMS reduced the expression of the Wnt/ß-catenin signaling pathway in the synovial tissue of AA rats and MH7A cells. In the inflammatory microenvonrment of RA, the results were consistent. CONCLUSION: This study demonstrated that EMS could protect against RA by inhibiting the abnormal activation and angiogenesis of FLSs, and the mechanism may be related to inhibiting the activation of the Wnt/ß-catenin signaling pathway.

[Effect of electroacupuncture of "Zusanli"(ST36) and "Guanyuan" (CV4) on apoptosis and expression of apoptosis-related proteins of synoviocytes in adjuvant-induced arthritis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zusanli"(ST36) and "Guanyuan"(CV4) on the apoptosis rate of synoviocytes and protein expression of Fas, FasL and Caspase-3 in synovial tissue of adjuvant-induced arthritis (AIA) rats, so as to explore its mechanisms underlying improvement of rheumatoid arthritis （RA）. METHODS: A total of 24 rats were randomly divided into normal, model, medication and EA groups, with 6 rats in each group. The AIA model was established by injection of complete Freund's adjuvant (CFA, 0.1 mL) into the left hindlimb paw. The rats in the medication group received intraperitoneal injection of 0.35 mg/kg of methotrexate, twice a week for 4 weeks. The rats in the EA group received EA stimulation of ST36 and CV4 (20 Hz/50 Hz, 1 mA) for 20 min, 6 times a week for 4 weeks. The left hind paw volume was measured using a paw volume meter, and histopathological changes of synovial tissue were observed by light microscope after H.E. staining. The serum contents of tumor necrosis factor-α（TNF-α） and interleukin-1 （IL-1） were measured by ELISA. The apoptosis of synoviocytes was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL), and the expression of apoptosis-associated proteins Fas, FasL and Caspase-3 in synovium were detected by Western blot. RESULTS: Compared with the normal group, the left hind paw volume from day 3 to 24 after administration of CFA, serum IL-1 and TNF-α contents were significantly increased (P<0.01), while the expressions of Fas, FasL and Caspase-3 proteins and apoptotic rate of synoviocytes were significantly decreased in the model group (P<0.01). In comparison with the model group, the paw volume from day 17 to 24 after modeling, the serum IL-1 and TNF-α contents were significantly reduced (P<0.01), while the apoptotic rate of synoviocytes, expressions of Fas protein in both medication and EA groups, Caspase-3 protein in the acupuncture group and FasL protein in the medication group were increased (P<0.01, P<0.05). Compared with the medication group, the expression of FasL protein was decreased in EA group (P<0.05). H.E. stain showed obvious hyperplasia of the synovial lining layer, and disordered arrangement of synovial cells, with edema and enlargement in some cells in the model group, which was relatively milder in both medication and EA groups. CONCLUSION: EA of ST36 and CV4 can promote the apoptosis of synoviocytes and the expressions of Fas and FasL proteins in AIA rats, which may contribute to its role in relieving synovitis through activating Fas/FasL signaling.

Deer Velvet Antler Extracts Exert Anti-Inflammatory and Anti-Arthritic Effects on Human Rheumatoid Arthritis Fibroblast-Like Synoviocytes and Distinct Mouse Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes joint deformity and disability. Deer velvet antler (DA), a traditional Chinese medicine, has been used to treat various types of arthritis for several thousands of years, but the underlying mechanisms are unknown. Herein, we investigated the anti-arthritic and anti-inflammatory effects of DA in vitro and in vivo. The ethyl acetate layer of DA ethanol extract (DA-EE-EA) was used to treat tumor necrosis factor (TNF)-[Formula: see text]-stimulated fibroblast-like synoviocyte MH7A cells, collagen-induced arthritis DBA/1 mice, and SKG mice with zymosan-induced arthritis. DA-EE-EA reduced nitric oxide production, prostaglandin E2 levels, and levels of pro-inflammatory cytokines including interleukin (IL)-1[Formula: see text], IL-6, and IL-8 in MH7A cells. DA-EE-EA also downregulated the phosphorylation of mitogen-activated protein kinase p38 and c-Jun N-terminal kinase and the translocation of nuclear factor kappa B p65. Intraperitoneal injection of DA-EE-EA for 3 weeks substantially reduced clinical arthritis scores in vivo models. Pathohistological images of the hind paws showed that DA-EE-EA reduced immune cell infiltration, synovial hyperplasia, and cartilage damage. The levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha, IL-1[Formula: see text], IL-6, IL-8, IL-17A, and interferon-gamma, decreased in the hind paw homogenates of DA-EE-EA-treated mice. We also identified several potential components, such as hexadecanamide, oleamide, erucamide, and lysophosphatidylcholines, that might contribute to the anti-inflammatory effects of DA-EE-EA. In conclusion, DA-EE-EA has the potential to treat RA by regulating inflammatory responses. However, the individual components of DA-EE-EA and the underlying anti-inflammatory mechanisms need further investigation in future studies.

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.

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.

Insulin Signaling in Arthritis.

Inflammatory arthritis is burdened by an increased risk of metabolic disorders. Cytokines and other mediators in inflammatory diseases lead to insulin resistance, diabetes and hyperlipidemia. Accumulating evidence in the field of immunometabolism suggests that the cause-effect relationship between arthritis and metabolic abnormalities might be bidirectional. Indeed, the immune response can be modulated by various factors such as environmental agents, bacterial products and hormones. Insulin is produced by pancreatic cells and regulates glucose, fat metabolism and cell growth. The action of insulin is mediated through the insulin receptor (IR), localized on the cellular membrane of hepatocytes, myocytes and adipocytes but also on the surface of T cells, macrophages, and dendritic cells. In murine models, the absence of IR in T-cells coincided with reduced cytokine production, proliferation, and migration. In macrophages, defective insulin signaling resulted in enhanced glycolysis affecting the responses to pathogens. In this review, we focalize on the bidirectional cause-effect relationship between impaired insulin signaling and arthritis analyzing how insulin signaling may be involved in the aberrant immune response implicated in arthritis and how inflammatory mediators affect insulin signaling. Finally, the effect of glucose-lowering agents on arthritis was summarized.

Artemisinin and artemisinin derivatives as anti-fibrotic therapeutics.

Fibrosis is a pathological reparative process that can occur in most organs and is responsible for nearly half of deaths in the developed world. Despite considerable research, few therapies have proven effective and been approved clinically for treatment of fibrosis. Artemisinin compounds are best known as antimalarial therapeutics, but they also demonstrate antiparasitic, antibacterial, anticancer, and anti-fibrotic effects. Here we summarize literature describing anti-fibrotic effects of artemisinin compounds in in vivo and in vitro models of tissue fibrosis, and we describe the likely mechanisms by which artemisinin compounds appear to inhibit cellular and tissue processes that lead to fibrosis. To consider alternative routes of administration of artemisinin for treatment of internal organ fibrosis, we also discuss the potential for more direct oral delivery of Artemisia plant material to enhance bioavailability and efficacy of artemisinin compared to administration of purified artemisinin drugs at comparable doses. It is our hope that greater understanding of the broad anti-fibrotic effects of artemisinin drugs will enable and promote their use as therapeutics for treatment of fibrotic diseases.

Alleviation of Synovial Inflammation of Juanbi-Tang on Collagen-Induced Arthritis and TNF-Tg Mice Model.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is primarily characterized by synovial inflammation. In this study, we found that a traditional Chinese decoction, Juanbi-Tang (JBT), JBT attenuated the symptoms of collagen-induced arthritis (CIA) mice and in tumor necrosis factor transgenic (TNF-Tg) mice by attenuating the arthritis index and hind paw thickness. According to histopathological staining of ankle sections, JBT significantly decreased the area of inflammation and reduced bone destruction of ankle joints in both these two types of mice. Moreover, decreased tartaric acid phosphatase-positive osteoclasts were observed in the JBT group compared with those found in the control group. We also revealed that JBT suppressed monocytes and T cells as well as the production of CCL2, CCR6, and CXCR3 ligands. We next used high-performance liquid chromatography to investigate the components and pharmacological properties of this classical herbal medicine in traditional Chinese medicine. Based on network pharmacology, we performed computational prediction simulation of the potential targets of JBT, which indicated the NF-kappa B pathway as its target, which was confirmed in vitro. JBT suppressed the production of pro-inflammatory cytokines including interleukin-6 (IL-6) and IL-8, and inhibited the expression of matrix metalloproteinase 1 in fibroblast-like synoviocytes derived from RA patients (MH7A cells). Furthermore, JBT also suppressed the phosphorylation of p38, JNK, and p65 in TNF-α-treated MH7A cells. In summary, this study proved that JBT could inhibit synovial inflammation and bone destruction, possibly by blocking the phosphorylation of NF-kappa B pathway-mediated production of proinflammatory effectors.

Photobiomodulation with 630-nm LED radiation inhibits the proliferation of human synoviocyte MH7A cells possibly via TRPV4/PI3K/AKT/mTOR signaling pathway.

Phototherapy has been used to treat postoperative pain and inflammatory response in rheumatoid arthritis. Confidence in this approach, however, is impaired by lack of understanding of the light-triggered cellular and molecular mechanisms. The purpose of this study was to characterize the response of human synoviocyte MH7A cells to visible LED red light in an attempt to elucidate the associated action mechanism. Human synoviocyte MH7A cells were treated with 630-nm LED light after stimulation of tumor necrosis factor-α (TNF-α). The effects of light radiation on cell proliferation and migration were detected by MTT assay and scratch test. The expressions of inflammatory cytokines were measured using RT-qPCR. This was followed by detection of the levels of extracellular proteins IL-6 and IL-8 after differential radiation. Furthermore, the expression levels and activation of proteins on PI3K/AKT/mTOR signaling pathway were examined with Western blot. In terms of the proliferation and migration, repeated radiation with LED red light (630 nm, 26 and 39 J/cm2) exerted an inhibitory effect on synoviocyte MH7A cells. Expression of inflammatory factors (IL-6, IL-1ß, IL-8, and MMP-3) was reduced; meanwhile, the expression of anti-inflammatory factor IL-10 was promoted. At the protein level, treatment with 39 J/cm2 of LED red light could decrease the level of extracellular protein (IL-6 and IL-8) and affect the expression and phosphorylation of proteins on TRPV4/PI3K/AKT/mTOR signaling pathway induced by TNF-α. These results demonstrated that LED red light (630 nm) inhibits proliferation and migration of MH7A cells. The growth-inhibiting effects of LED red light on human synoviocyte MH7A cells appear to be associated with regulation of the TRPV4/PI3K/AKT/mTOR signaling pathway.

Standardized Xylocarpus moluccensis fruit fraction mitigates collagen-induced arthritis in mice by regulating immune response.

OBJECTIVE: This study was undertaken to evaluate the effect of Xylocarpus moluccensis fruit fraction (F018) on the pathogenesis of collagen-induced arthritis in mice. METHODS: Arthritis was induced by intradermal injection of collagen (2 mg/ml) with complete Freund's adjuvant in DBA/1J mice. F018 was administered orally at 1, 3 and 10 mg/kg for 20 days. Disease progression and mechanism were assessed by micro-CT analysis, RT-PCR, flow cytometry assay, myeloperoxidase (MPO) and MTT assay. RESULTS: F018 at 3 and 10 mg/kg significantly reduced paw thickness, clinical score, mononuclear cell infiltration and collagen layer depletion in the knee section of collagen-induced arthritis (CIA) mice when compared with collagen-induced arthritis mice alone. Furthermore, F018 treatment in collagen-induced arthritis mice significantly recovered bone volume and trabecular number and decreased the trabecular space by modulating RANKL and OPG mRNA expression in the synovial tissue. F018 treatment in collagen-induced arthritis mice significantly attenuated spleen index, lymphocyte proliferation and paw myeloperoxidase (MPO) activity, pro-inflammatory cytokine TNFα, IL1ß, and IL6 mRNA expression and enhanced IL10 mRNA expression in paw tissue. Furthermore, F018 treatment in collagen-induced arthritis mice significantly reduced splenic dendritic cell maturation and Th17 cells. In culture, F018 significantly decreased collagen-induced arthritis-FLS proliferation and promoted apoptosis. CONCLUSION: F018 may serve as a potential curative agent for arthritis.

Apoptotic effect of Aralia echinocaulis extract on fibroblast-like synoviocytes in rats with adjuvant-induced arthritis via inhibiting the Akt/Hif-1α signaling pathway in vitro.

Aralia echinocaulis is used for the treatment of rheumatoid arthritis by Tujia Minority in China. A previous study demonstrated that A. echinocaulis had a significant anti-arthritic effect on adjuvant arthritis (AA) rats in vivo. However, it remains unclear whether A. echinocaulis can induce the apoptosis of fibroblast-like synoviocytes (FLS) from AA rats and the underlying mechanism is unknown. In this paper, CCK-8 assay, Hoechst staining and flow cytometry were used to evaluate the apoptotic effect of an A. echinocaulis ethanol extract (AEE) on AA FLS. Western blotting analysis was performed to measure the protein expression levels of Bcl-2, Bax, cleaved caspase-3, Akt, p-Akt, and Hif-1α. The results revealed that AEE could inhibit FLS proliferation in a dose and time-dependent manner. After treatment with AEE, AA FLS displayed the classical apoptotic morphology, and the apoptosis rates were significantly increased. Furthermore, we found that AEE increased the protein levels of Bax, cleaved caspase 3, and decreased the protein levels of Bcl-2, Hif-1α and p-Akt, without affecting total Akt levels. Collectively, these results suggested that the apoptosis inducing effect of AEE on AA FLS was related to the regulation of the expression of apoptosis-related proteins and the inhibition of the Akt/Hif-1α signaling pathway.

1,25-Dihydroxyvitamin D3 attenuates disease severity and induces synoviocyte apoptosis in a concentration-dependent manner in rats with adjuvant-induced arthritis by inactivating the NF-κB signaling pathway.

An aggressive proliferation of synoviocytes is the hallmark of rheumatoid arthritis (RA). Emerging evidence shows that inhibiting the NF-κB signaling pathway with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] may be a therapeutic approach for controlling inflammatory diseases. In this study, we demonstrated the protective effects of three different 1,25(OH)2D3 concentration on adjuvant-induced arthritis (AA) rats through the NF-κB signaling pathway and their pro-apoptotic roles in cultured adjuvant-induced arthritis synoviocytes (AIASs). AA rats were prepared by injecting complete Freund's adjuvant and independently given daily intraperitoneal injection of 1,25(OH)2D3 at concentrations of 50, 100, and 300 ng/day/kg. Subsequently, AIASs were isolated from the inflamed joints of AA rats to test the effects of 1,25(OH)2D3 on AIASs in vitro. Intraperitoneal injection of 1,25-(OH)2D3 was found to induce a concentration- and time-dependent improvement in relieving the symptoms of AA. We found an increased paw withdrawal thermal latency (PWTL) in the affected paw of AA rats as the concentration of 1,25-(OH)2D3 increased. 1,25-(OH)2D3 treatment reduced levels of inflammatory factors in synovial tissues of AA rats. In the case of cultured AIASs, 1,25-(OH)2D3 was shown to inhibit cell proliferation and induce cell apoptosis in a concentration-dependent manner. Additionally, 1,25-(OH)2D3 inhibited the activation of the NF-κB signaling pathway. In conclusion, our study provides evidence emphasizing that 1,25(OH)2D3 has the potential to attenuate disease severity in RA potentially due to its contributory role in synoviocyte proliferation and apoptosis. The protective role of 1,25(OH)2D3 against RA depends on the NF-κB signaling pathway.

Compound GDC, an Isocoumarin Glycoside, Protects against LPS-Induced Inflammation and Potential Mechanisms In Vitro.

Compound 3R-(4'-hydroxyl-3'-O-ß-D-glucopyranosyl phenyl)-dihydro isocoumarin (GDC) is a natural isocoumarin, recently isolated from the stems of H. paniculiflorum. However, we know little about the effects of GDC on rheumatoid arthritis (RA). This study aims to investigate the protective effects and potential mechanisms of GDC against LPS-induced inflammation in vitro. Fibroblast-like synoviocytes (FLSs) obtained from synovial tissue of rats were induced by lipopolysaccharide (LPS) and treated with GDC. Cell viability was determined by mitochondrial-respiration-dependent3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Secretion of various inflammatory mediators was analyzed by ELISA and RayBio® Rat Cytokine Antibody Array. Potential mechanisms that are associated with anti-inflammatory effect were examined by Western blot. Results showed that GDC significantly inhibited the production of tumor necrosis factor alpha (TNF-α) and interleukin- (IL-) 6 induced by LPS. GDC also reduced the expression of inducible nitric oxide synthase (iNOS), TNF-α, IL-6, and IL-1ß, as well as proinflammatory cytokines such as activin A, ciliary neurotrophic factor (CNTF), fractalkine, IFN-γ, IL-4, and TIMP-1. Moreover, GDC inhibited LPS-induced phosphorylation of extracellular regulated protein kinases (ERK1/2), p38 mitogen-activated protein kinases (p38), c-Jun N-terminal kinase (JNK), and IκB. And GDC also blocked NF-κBp65 nuclear translocation. All the results suggested that the protective effects of GDC against LPS-induced inflammation in vitro may be related with NF-κB and JNK signaling pathway.

Anti-inflammatory and anti-arthritic effects of the ethanolic extract of Aralia continentalis Kitag. in IL-1ß-stimulated human fibroblast-like synoviocytes and rodent models of polyarthritis and nociception.

BACKGROUND: Blocking the formation and invasive growth of pannus and its secretion of inflammatory cytokines and MMPs is important for treating rheumatoid arthritis. HYPOTHESIS/PURPOSE: Anti-arthritic activity of Aralia continentalis Kitag., an oriental herbal medicine, and the underlying mechanisms involved were investigated. STUDY DESIGN: Anti-inflammatory and anti-nocicpetive activities of the ethanolic extract (50% v/v) of Aralia continentalis Kitag. harvested from Imsil, Korea (ACI) were investigated in IL-1ß-stimulated human fibroblast-like synoviocyte (FLS) cells and rodent models of collagen-induced polyarthritis and carrageenan-induced acute paw pain. METHODS: In IL-1ß-stimulated FLS cells derived from rheumatoid arthritis patients, the anti-inflammatory activity of ACI was examined by analyzing the expression levels of inflammatory mediators such as TNF-α, IL-6, IL-8, MMP-1, MMP-3, MMP-13, PGE2, and COX-2 using ELISA and RT-PCR analysis. The anti-arthritic activity of ACI was investigated by measuring body weight, squeaking score, paw volume, and arthritis index in collagen-induced polyarthritis mice. The anti-nociceptive activity of ACI was examined in the paw-pressure test and Tail-flick latency test in rats. RESULTS: The ethanolic extract (50% v/v) of ACI reduced the levels of TNF-α, IL-6, IL-8, MMP-1, and MMP-13 secreted by IL-1ß-stimulated FLS cells, whereas MMP-3, COX-2, and PGE2 were not significantly affected. ACI inhibited the migration of NF-κB into the nucleus through the inhibition of ERK- and JNK-dependent MAP kinase pathways in IL-1ß-stimulated FLS cells. In collagen-induced polyarthritis mice, oral administration of ACI extract (200 mg/kg) significantly alleviated arthritic behaviors. Histological observations of arthritic mouse knees were consistent with their behaviors. The anti-arthritic and anti-inflammatory activities of 200 mg/kg ACI extract were comparable to those of 10 mg/kg prednisolone when administered to mice. However, ACI administration did not significantly affect carrageenan-induced hyperalgesia or thermal nociception in rats. CONCLUSION: These results suggest that the ethanolic extract of ACI have significant anti-inflammatory and anti-arthritic effects in a rodent arthritis model and in IL-1ß-stimulated FLS cells. Thus, ACI may be a useful candidate for developing pharmaceuticals or dietary supplements for the treatment of inflammatory arthritis.