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.

Natural compound library screening to identify berberine as a treatment for rheumatoid arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) play a critical role on the exacerbation and deterioration of rheumatoid arthritis (RA). Aberrant activation of FLS pyroptosis signaling is responsible for the hyperplasia of synovium and destruction of cartilage of RA. This study investigated the screened traditional Chinese medicine berberine (BBR), an active alkaloid extracted from the Coptis chinensis plant, that regulates the pyroptosis of FLS and secretion of inflammatory factors in rheumatoid arthritis. METHODS: First, BBR was screened using a high-throughput drug screening strategy, and its inhibitory effect on RA-FLS was verified by in vivo and in vitro experiments. Second, BBR was intraperitoneally administrated into the collagen-induced arthritis rat model, and the clinical scores, arthritis index, and joint HE staining were evaluated. Third, synovial tissues of CIA mice were collected, and the expression of NLRP3, cleaved-caspase-1, GSDMD-N, Mst1, and YAP was detected by Western blot. RESULTS: The administration of BBR dramatically alleviated the severity of collagen-induced arthritis rat model with a decreased clinical score and inflammation reduction. In addition, BBR intervention significantly attenuates several pro-inflammatory cytokines (interleukin-1ß, interleukin-6, interleukin-17, and interleukin-18). Moreover, BBR can reduce the pyroptosis response (caspase-1, NLR family pyrin domain containing 3, and gasdermin D) of the RA-FLS in vitro, activating the Hippo signaling pathway (Mammalian sterile 20-like kinase 1, yes-associated protein, and transcriptional enhanced associate domains) so as to inhibit the pro-inflammatory effect of RA-FLS. CONCLUSION: These results support the role of BBR in RA and may have therapeutic implications by directly repressing the activation, migration of RA-FLS, which contributing to the attenuation of the progress of CIA. Therefore, targeting PU.1 might be a potential therapeutic approach for RA. Besides, BBR inhibited RA-FLS pyroptosis by downregulating of NLRP3 inflammasomes (NLRP3, caspase-1) and eased the pro-inflammatory activities via activating the Hippo signaling pathway, thereby improving the symptom of CIA.

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.

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.

Efficient delivery of the lncRNA LEF1-AS1 through the antibody LAIR-1 (CD305)-modified Zn-Adenine targets articular inflammation to enhance the treatment of rheumatoid arthritis.

BACKGROUNDS: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovial hyperplasia. Maintaining a balance between the proliferation and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs) is crucial for preventing the erosion of bone and cartilage and, ultimately, mitigating the progression of RA. We found that the lncRNA LEF1-AS1 was expressed at low levels in the RASFs and inhibited their abnormal proliferation by targeting PIK3R2 protein and regulating the PI3K/AKT signal pathway through its interaction with miR-30-5p. In this study, we fabricated a nano-drug delivery system for LEF1-AS1 using Zn-Adenine nanoparticles (NPs) as a novel therapeutic strategy against RA. METHODS: The expression levels of LEF1-AS1, miR-30-5p, PIK3R2, p-PI3K, and p-AKT were detected in the primary RASFs and a human fibroblast-like synovial cell line (HFLS). Zn-Adenine nanoparticles (NPs) were functionalized with anti-CD305 antibody to construct (Zn-Adenine)@Ab. These NPs were then loaded with LEF1-AS1 to form (Zn-Adenine)@Ab@lncRNA LEF1-AS1. Finally, the (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs were locally injected into a rat model with collagen-induced arthritis (CIA). The arthritic injuries in each group were evaluated by HE staining and other methods. RESULTS: LEF1-AS1 was expressed at low levels in the primary RASFs. High expression levels of LEF1-AS1 were detected in the HFLS cells, which corresponded to a significant downregulation of miR-30-5p. In addition, the expression level of PIK3R2 was significantly increased, and that of p-PI3K and p-AKT were significantly downregulated in these cells. The (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly inhibited the proliferation of RASFs and decreased the production of inflammatory cytokines (IL-1ß, IL-6, TNF-α). Intra-articular injection (IAI) of (Zn-Adenine)@Ab@lncRNA LEF1-AS1 NPs significantly alleviated cartilage destruction and joint injury in the CIA-modeled rats. CONCLUSIONS: LEF1-AS1 interacts with miR-30-5p to inhibit the abnormal proliferation of RASFs by regulating the PI3K/AKT signal pathway. The (Zn-Adenine)@Ab NPs achieved targeted delivery of the loaded LEF1-AS1 into the RASFs, which improved the cellular internalization rate and therapeutic effects. Thus, LEF1-AS1 is a potential target for the treatment of RA.

Myricetin ameliorates the IL-21-induced tumorigenic phenotype of adjuvant-induced arthritis FLS by modulating the choline kinase signaling cascade.

The synovial intimal lining is mainly governed by fibroblast-like synoviocytes (FLS), which portray a transformed tumor-like phenotype in rheumatoid arthritis (RA). Among the diverse cytokines that engender FLS, interleukin-21 (IL-21) was reported to stimulate hyperproliferation and perpetuate inflammation. Recently, choline kinase (ChoKα) has been reported to be an essential enzyme aiding RA-FLS hyperproliferation by altering phosphatidylcholine biosynthesis. The current study aimed to elucidate the therapeutic efficacy of myricetin, a flavonoid, in abating the IL-21-induced tumor-like phenotype of adjuvant-induced arthritis (AIA)-FLS via the ChoKα signaling cascade. Our results showed that myricetin suppressed IL-21 receptor expression and activation of the ChoKα signaling cascade (N-Ras, Ral-GDS, and PI3K) in IL-21-induced AIA-FLS. Consequently, myricetin treatment decreased ChoKα and PLD2 enzymatic activity and inhibited the proliferative, migratory, and invasive properties of AIA-FLSs. Our results demonstrated that myricetin could be a promising anti-arthritic compound by abating IL-21-induced hyperproliferation, migration, and invasive behavior of AIA-FLS by downregulating the ChoKα signaling cascade.

Moxibustion intervention improves synovitis by down-regulating NLRP3/Caspase-1/IL-1ß signaling of synovial tissue in rats with adjuvant arthritis. / 基于NLRP3/Caspase-1/IL-1ßä¿¡å·é€šè·¯æŽ¢è®¨è‰¾ç¸æ”¹å–„ä½å‰‚æ€§å ³èŠ‚ç‚Žå¤§é¼ æ»‘è†œç‚Žçš„æœºåˆ¶.

OBJECTIVES: To observe the effect of moxibustion on activities of NOD-like receptor family protein 3 (NLRP3)/cysteine aspartic acid specific protease-1 (Caspase-1)/interleukin-1ß (IL-1ß) signaling pathway in rats with adjuvant arthritis (AA), so as to explore its mechanisms underlying improvement of rheumatoid arthritis (RA). Me-thods Thirty male Wistar rats were randomly divided into normal control, AA model and moxibustion groups, with 10 rats in each group. The AA model was replicated by raising in wind, cold and damp environment combined with complete Freund's adjuvant injection. In the moxibustion group, moxibustion was applied to bilateral "Shenshu" (BL23) and "Zusanli"(ST36) for 20 min each time, once daily for 21 days. Changes of joint swelling degree (JSD) and arthritis index (AI) in each group were observed. The ultrastructural changes of synovial cells in each group were observed by transmission electron microscopy. The protein expression levels of NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, tumor necrosis factor-α (TNF-α) and IL-1ß in the synovial tissues of the knee joint were measured by Western blot. RESULTS: Compared with the normal control group, JSD, AI and the protein expressions of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß in the synovial tissues were significantly increased (P<0.01) in the model group. In comparison with the model group, JSD, AI and the protein expression levels of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß were significantly decreased (P<0.01) in the moxibustion group. Results of transmission electron microscope showed an irregular and vague nuclear membrane of synovial cells, and unclear mitochondrial membrane boundary with sparse, swelling crests in the model group, which was relatively milder in the damage degree in the moxibustion group. CONCLUSIONS: Moxibustion can relieve the inflammatory response in the synovial membrane of AA rats, which may be related to its function in down-regulating synovial NLRP3/Caspase-1/IL-1ß inflammatory signaling.

Sesamol serves as a p53 stabilizer to relieve rheumatoid arthritis progression and inhibits the growth of synovial organoids.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease known as a leading cause of disability with considerable mortality. Developing alternative drugs and targets for RA treatment is an urgent issue. Sesamol is a phenolic compound isolated from natural food sesame (Sesamum indicum L.) with various biological activities. PURPOSE: The current research intended to illuminate the bioactivity and mechanisms of sesamol in RA fibroblast-like synoviocytes (FLS), and aimed to estimate the potential clinical application value of sesamol in RA treatment. METHODS: CCK-8, EdU, and flow cytometry assays, as well as transwell tests were applied to observe the effects of sesamol on the abnormal functions of RA-FLS. Moreover, synovial organoids and a collagen-induced arthritis (CIA) mouse model were constructed to further explore the therapeutic capacity of sesamol on RA. Furthermore, RNA sequencing combined with quantitative real-time PCR assay, Western blot as well as co-immunoprecipitation were employed to clarify the mechanism of sesamol in regulating RA progression. RESULTS: Sesamol suppressed the proliferation through inhibiting DNA replication, triggering cell cycle arrest and apoptosis of RA-FLS. Besides, sesamol impaired RA-FLS migration and invasion. Interestingly, sesamol inhibited the growth of constructed synovial organoids and alleviated RA symptoms in CIA mice. Moreover, RNA sequencing further implicated p53 signaling as a downstream pathway of sesamol. Furthermore, sesamol was shown to decrease p53 ubiquitination and degradation, thereby activating p53 signaling. Finally, bioinformatics analyses also highlighted the importance of sesamol-regulated networks in the progression of RA. CONCLUSIONS: Our investigation demonstrated that sesamol served as a novel p53 stabilizer to attenuate the abnormal functions of RA-FLS via facilitating the activation of p53 signaling. Moreover, our study highlighted that sesamol might be an effective lead compound or candidate drug and p53 could be a promising target for the therapy of RA.

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.

Membrane vesicles from Lactobacillus johnsonii delay osteoarthritis progression via modulating macrophage glutamine synthetase/mTORC1 axis.

AIMS: The manipulation of macrophage recruitment and their shift in the M1/M2 ratio is a promising approach to mitigate osteoarthritis (OA). Nevertheless, the current clinical medication available for OA is only palliative and may result in undesirable outcomes. Hence, it is urgent to explore alternative disease-modifying drug supplement that are both safer and more effective in OA treatment, like probiotic and probiotic-derived membrane vesicles. METHODS: The synovial inflammation and cartilage damage in collagenase-induced OA (CIOA) mice were observed using haematoxylin and eosin, saffron O-solid green and immunohistochemical staining. Bipedal balance test and open field test were conducted to determine the effectiveness of L. johnsonii-derived membrane vesicles (LJ-MVs) in reducing joint pain of CIOA mice. Additionally, Transwell, western blot, and immunological testing were used to examine the effect of LJ-MVs on macrophage migration and reprogramming. Furthermore, a 4D label-free proteomic analysis of LJ-MVs and their parent bacterium was performed, and the glutamine synthetase (GS)/mTORC1 axis in macrophage was verified by western blot. RESULTS: L. johnsonii and its membrane vesicles, LJ-MVs, exhibit a novel ability to mitigate inflammation, cartilage damage, and pain associated with OA. This is achieved by their ability to impede macrophage migration, M1-like polarization, and inflammatory mediators secretion, while simultaneously promoting the M2/M1 ratio in synovial macrophages. The mechanism underlying this effect involves the modulation of macrophage GS/mTORC1 pathway, at least partially. SIGNIFICANCE: Owing to their probiotic derivation, LJ-MVs will be a more dependable and potent disease-modifying drugs for the prevention and therapy of OA in the long run.

Culture Media Supplemented With 10% Equine Serum Provided Chondroprotection in an In Vitro Co-Culture of Cartilage and Synovial Membrane.

No studies have evaluated the effect of culture in serum-free media (SF) vs. media supplemented with equine serum (ES) on co-culture of synovial membrane and cartilage tissue explants. The study objective was to evaluate the effects of equine serum supplementation on induced production of inflammatory and catabolic mediators from articular cartilage and synovial explants while in co-culture. Articular cartilage and synovial membrane explants were harvested from femoropatellar joints of five adult horses. Cartilage and synovial explants were harvested from the stifle of five horses, placed in co-culture, stimulated with IL-1ß (10 ng/ml) and maintained in culture for 3, 6 and 9 days in 10% ES or SF. At each time point, media was harvested for analysis of cellular viability (Lactate dehydrogenase) and elution of glycosaminoglycans (Dimethylene Blue Binding Assay). Tissue explants were harvested for histopathologic and gene expression analyses. No differences in cell viability were observed between SF and ES groups. SF culture produced an upregulation of TNF-α in synovial membrane and ADAMTS-4 and five in articular cartilage at 9 days of culture. ES produced an upregulation of aggrecan expression in cartilage at 9 days of culture. No differences in tissue viability were found between culture media, but SF media produced a higher glycosaminoglycan concentration in media at 3 days of culture. The addition of 10% ES produced a slight chondroprotective effect in an inflamed co-culture system. This effect should be considered when designing studies evaluating treatment of serum or plasma-based orthobiologic studies in vitro.

[Effect of moxibustion on the expressions of hypoxia inducible factor-1α and vascular endothelial growth factor in ankle synovial tissue of rats with adjuvant arthritis].

OBJECTIVE: To observe the effect of moxibustion on the expressions of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in ankle synovial tissue of rats with adjuvant arthritis(AA), so as to explore the mechanism of moxibustion in inhibiting synovial angiogenesis and improving joint symptoms of rheumatoid arthritis. METHODS: Sixty healthy male SD rats were randomly divided into normal group, model group, moxibustion group and medication group, with 15 rats in each group. AA rat model was established by subcutaneous injection of Freund's complete adjuvant into the right hind paw. Rats in the moxibustion group were treated with "Zusanli" (ST36), "Guanyuan" (CV4) and "Ashi" point moxibustion, 20 min each time, once a day, for consecutive 3 weeks. Rats in the medication group were given methotrexate (0.35 mg/kg) intragastric administration, twice a week, for consecutive 3 weeks. Foot plantar volume of rats was measured by toe volume mea-suring instrument. HE staining was used to observe the histopathology of ankle synovium. The protein expressions of HIF-1α and VEGF in ankle synovial tissue were detected by immunohistochemistry and Western blot. RESULTS: Compared with the normal group, the foot plantar volume and the protein expressions of HIF-1α and VEGF in synovial tissue of ankle joint were significantly increased (P<0.01) in the model group, the synovial tissue showed obvious hyperplasia and a large number of neovasculogenesis. Following the interventions, the foot plantar volume and the protein expressions of HIF-1α and VEGF in synovial tissue of ankle joint were significantly decreased (P<0.05, P<0.01) in both moxibustion and medication groups in contrast to the model group, and there was no obvious proliferation of synovial tissue, and only a few neovascularization was observed. Compared with the medication group, the foot plantar volume was decreased (P<0.05) in the moxibustion group. CONCLUSION: Moxibustion can improve joint swelling and inhibit synovial angiogenesis in AA rats, and its mechanism may be related to down-regulating of HIF-1α and VEGF protein expressions.

Increased n-6 Polyunsaturated Fatty Acids Indicate Pro- and Anti-Inflammatory Lipid Modifications in Synovial Membranes with Rheumatoid Arthritis.

Emerging evidence suggests that fatty acids (FAs) and their lipid mediator derivatives can induce both beneficial and detrimental effects on inflammatory processes and joint degradation in osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA). The present study characterized the detailed FA signatures of synovial membranes collected during knee replacement surgery of age- and gender-matched OA and RA patients (n = 8/diagnosis). The FA composition of total lipids was determined by gas chromatography and analyzed with univariate and multivariate methods supplemented with hierarchical clustering (HC), random forest (RF)-based classification of FA signatures, and FA metabolism pathway analysis. RA synovium lipids were characterized by reduced proportions of shorter-chain saturated FAs (SFAs) and elevated percentages of longer-chain SFAs and monounsaturated FAs, alkenyl chains, and C20 n-6 polyunsaturated FAs compared to OA synovium lipids. In HC, FAs and FA-derived variables clustered into distinct groups, which preserved the discriminatory power of the individual variables in predicting the RA and OA inflammatory states. In RF classification, SFAs and 20:3n-6 were among the most important FAs distinguishing RA and OA. Pathway analysis suggested that elongation reactions of particular long-chain FAs would have increased relevance in RA. The present study was able to determine the individual FAs, FA groups, and pathways that distinguished the more inflammatory RA from OA. The findings suggest modifications of FA elongation and metabolism of 20:4n-6, glycerophospholipids, sphingolipids, and plasmalogens in the chronically inflamed RA synovium. These FA alterations could have implications in lipid mediator synthesis and potential as novel diagnostic and therapeutic tools.

Naru-3 inhibits inflammation, synovial hyperplasia, and neovascularization in collagen-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Naru-3 is a prescribed formulation based on the theory of Mongolian medicine for the treatment of rheumatoid arthritis (RA). Naru-3 consists of three medicinal agents: Aconitum kusnezoffii Reichb (caowu), Terminalia chebula Retz (hezi), and Piper longum L (biba). These medicinal agents are widely distributed in the Mongolian area of China and have been used to treat rheumatism for centuries. BACKGROUND: Mongolian medicine Naru-3 is commonly prescribed to treat RA, but its mechanism of action is unknown. METHODS: A rat collagen-induced arthritis (CIA) model was established to investigate the mechanism of Naru-3. Rats were treated with Naru-3, Etanercept (ETN), and sodium carboxymethylcellulose (CMC) for four weeks. After treatment was terminated, paw thickness, ankle diameter, and arthritis index (AI) were scored. Synovial hyperplasia was evaluated using hematoxylin and eosin (H&E) staining and two-dimensional ultrasonography. Synovitis and neovascularization were assayed using power Doppler imaging (PDI) and contrast-enhanced ultrasonography (CEUS). Levels of vascular endothelial growth factor (VEGF), interleukin (IL)-1, and CD31 in the serum or synovium were detected using ELISA and immunohistochemistry analyses. RESULTS: Naru-3 and ETN alleviated the symptoms of CIA as evidenced by diminished paw thickness, ankle diameter, and AI scores. Mechanistically, Naru-3 inhibited synovial hyperplasia, synovitis, and neovascularization by diminishing systemic and local inflammation, as indicated by the relative expression of CD31, VEGF and IL-1 in the serumor synovium. After four weeks of treatment, no significant neovascularization was observed in the Naru-3 group, but neovascularization and synovitis occurred in the ETN group, as demonstrated by H&E staining, PDI, and CEUS examination. CONCLUSION: Naru-3 inhibited inflammation, synovial hyperplasia, and neovascularization and alleviates RA in our CIA rat model. No symptom recurrence was observed four weeks after drug treatment.

[Effect of moxibustion on the indicators of autophagy and apoptosis in synovium of rats with adjuvant arthritis].

OBJECTIVE: To observe the effect of moxibustion on the indicators of autophagy and apoptosis in the synovium of toes of rats with adjuvant-induced arthritis (AA), so as to explore the underlying mechanism of moxibustion in the treatment of rheumatoid arthritis. METHODS: Forty-five SD rats were randomly divided into the blank control group, model group, moxibustion group, methotrexate group and rapamycin group, with 9 rats in each group. The rat model of AA was established by injecting Freund's complete adjuvant. Rats in the moxibustion group received moxibustion treatment at "Zusanli" (ST36) and "Guanyuan" (CV4) for 20 min, once a day. The methotrexate group was given methotrexate intragastrically (0.35 mg/kg) twice a week. The rapamycin group was given rapamycin by intraperitoneal injection (1 mg/kg), once every other day. The toe volume of the left hind limb was measured by the toe volume measuring instrument after 3-day modeling and 3-week intervention respectively. The contents of interlukin(IL)-1 and tumor necrosis factor(TNF)-α in serum were detected by ELISA. The autophagosomes of synovial cells of the toe joint were observed under transmission electron microscope. The expressions of mammalian target of rapamycin(mTOR)C1, p-mTORC1, Caspase-3, Fas and FasL in synovial tissue were detected by Western blot. RESULTS: Under transmission electron microscope, the model group showed decreased autophagosomes in synovial tissues, but the moxibustion, methotrexate, and rapamycin groups showed increased autophagosomes. Compared with the blank control group, the toe volume, the contents of IL-1 and TNF-α in serum and the expression of p-mTORC1 protein in synovial tissue were significantly increased (P<0.01, P<0.001), while the expressions of Caspase-3, Fas and FasL proteins in synovial tissue were significantly decreased (P<0.05, P<0.01) in the model group. Compared with the model group, the toe volume, the contents of IL-1 and TNF-α in the serum, and expression of p-mTORC1 protein were significantly decreased (P<0.05, P<0.01, P<0.001) in the moxibustion group and the methotrexate group, while the expression of Caspase-3, Fas and FasL proteins in synovial tissue in the moxibustion group and the methotrexate group, the expression of Caspase-3 in the rapamycin group were significantly increased (P<0.05). CONCLUSION: Moxibustion can improve joint swelling in AA rats and decrease the contents of serum IL-1 and TNF-α. The mechanism may be related to regulating the expressions of p-mTORC1, Caspase-3, Fas and FasL proteins, and promoting autophagy and apoptosis of synovial cells.

Targeting Synovial Lymphatic Function as a Novel Therapeutic Intervention for Age-Related Osteoarthritis in Mice.

OBJECTIVE: The synovial lymphatic system (SLS) removes catabolic factors from the joint. Vascular endothelial growth factor C (VEGF-C) and its receptor, VEGFR-3, are crucial for lymphangiogenesis. However, their involvement in age-related osteoarthritis (OA) is unknown. This study was undertaken to determine whether the SLS and the VEGF-C/VEGFR-3 pathway contribute to the development and progression of age-related OA, using a murine model of naturally occurring joint disease. METHODS: SLS function was assessed in the knees of young (3-month-old) and aged (19-24-month-old) male and female C57BL/6J mice via a newly established in vivo IVIS-dextran imaging approach, which, in addition to histology, was used to assess the effects of VEGF-C treatment on SLS function and OA pathology in aged mice. RNA-sequencing of synovial tissue was performed to explore molecular mechanisms of the disease in the mouse knee joints. RESULTS: Results showed that aged mice had impaired SLS function, including decreases in joint clearance (mean T1/2 of signal intensity clearance, 2.8 hours in aged mice versus 0.5 hours in young mice; P < 0.0001), synovial influx (mean ± SD 1.7 ± 0.8% in aged mice versus 4.1 ± 1.9% in young mice; P = 0.0004), and lymph node draining capacity (mean ± SD epifluorescence total radiant intensity ([photons/second]/[µW/cm2 ]) 1.4 ± 0.8 in aged mice versus 3.7 ± 1.2 in young mice; P < 0.0001). RNA-sequencing of the synovial tissue showed that Vegf-c and Vegfr3 signaling genes were decreased in the synovium of aged mice. VEGF-C treatment resulted in improvements in SLS function in aged mice, including increased percentage of signal intensity joint clearance (mean ± SD 63 ± 9% in VEGF-C-treated aged mice versus 52 ± 15% in vehicle-treated aged mice; P = 0.012), increased total articular cartilage cross-sectional area (mean ± SD 0.38 ± 0.07 mm2 in VEGF-C-treated aged mice versus 0.26 ± 0.07 mm2 in vehicle-treated aged mice; P < 0.0001), and decreased percentage of matrix metallopeptidase 13-positive staining area within total synovial area in 22-month-old VEGF-C-treated mice versus 22-month-old vehicle-treated mice (mean ± SD decrease 7 ± 2% versus 4 ± 1%; P = 0.0004). CONCLUSION: SLS function is reduced in the knee joints of aged mice due to decreased VEGF-C/VEGFR-3 signaling. VEGF-C treatment attenuates OA joint damage and improves synovial lymphatic drainage in aged mice. The SLS and VEGF-C/VEGFR-3 signaling represent novel physiopathologic mechanisms that could potentially be used as therapeutic targets for age-related OA.

Combination therapy of a TRPV2 agonist with a TNF inhibitor achieves sustained suppression of disease severity and reduced joint damage.

We aimed to compare a transient receptor potential vanilloid 2 (TRPV2) agonist with a TNF inhibitor, and to test the potential of their combination in collagen-induced arthritis (CIA) as a potential future strategy for rheumatoid arthritis (RA). Following the onset of CIA DBA1/j mice were started on treatment with either vehicle, etanercept (8 mg/kg three times a week), the TRPV2 agonist O1821 (20-30 mg/kg/day), or a combination of both. Mice were scored over a 61-day period. Synovial tissues were obtained for RNA sequencing. Mice on monotherapy with either O1821 or etanercept developed milder clinical disease. The O1821 protection was observed at an earlier time-point than in the etanercept group. The combination therapy group achieved a more robust and sustained reduction in disease severity than either monotherapy group. All treatment groups had reduced scores for synovial inflammation, synovial hyperplasia, and erosive changes, compared with controls, with the combination group achieving the most significant protection. RNA sequencing and pathway analyses of synovial tissues identified pathways and processes regulated by the TRPV2 agonist, such as chemotaxis and cytokine receptor signaling, including IL6R. The combination therapy affected additional pathways not seen in the monotherapy groups. In conclusion, the TRPV2 agonist achieved an overall similar reduction in arthritis severity and histology scores as etanercept, but the combination therapy achieved a more sustained disease control and more pronounced reduction in joint damage, suggesting a potential future option for improving disease control in RA. RNA sequencing analyses identified new pathways regulated by TRPV2, and also by the combination treatment.

Huangqin Qingre Qubi Capsule inhibits RA pathology by binding FZD8 and further inhibiting the activity of Wnt/ß-catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Huangqin Qingre Qubi Capsule (HQC) is a Chinese herbal compound for the treatment of rheumatoid arthritis (RA), which is made from dry roots of Scutellaria baicalensis Georgi, dry mature seeds of Gardenia jasminoides J.Ellis, dry and mature seeds of Coix lacryma-jobi var. stenocarpa Oliv., dry mature seeds of Amygdalus persica L. and roots and rhizomes of Clematis chinensis Osbeck in the proportion of 10:9:30:5:10. HQC has a significant effect in clinical treatment of RA, which can inhibit RA inflammation, improve oxidative stress state, and effectively relieve symptoms of RA patients. AIM OF THE STUDY: The anti-arthritis effect of HQC and its mechanism, especially whether it improves RA through FZD8-Wnt/ß-catenin signal axis, were studied using adjuvant arthritis (AA) rats and FLS from RA patients. MATERIALS AND METHODS: Real time qPCR (RT-qPCR), Western blot (WB), confocal microscopy and other molecular biological methods were used to study the anti-RA effect of HQC and its mechanism. RESULTS: The expression of FZD8 was significantly up-regulated in synovium and FLS of AA rats and RA FLS. FZD8 significantly activated the Wnt/ß-catenin signaling pathway, promoted abnormal proliferation of FLS, increased the levels of inflammatory factors IL-1ß, IL-6 and IL-8, and significantly increased the expression of matrix metalloproteinase 3 (MMP3) and fibronectin. HQC has significant therapeutic effect on AA rats. Molecular docking and molecular dynamics showed that HQC had a good binding ability with FZD8. We also confirmed that HQC inhibited Wnt/ß-catenin signaling pathway by binding FZD8, and reduced the levels of the above inflammatory factors and pathological genes of RA. CONCLUSIONS: The expression of FZD8 is significantly increased in AA rats and FLS from RA patients. Clarify that HQC improves RA through the FZD8-Wnt/ß-catenin signal axis, provide a clear therapeutic mechanism for HQC to improve RA, and also provide a basis for clinical promotion of HQC.

IL-17A and TNF synergistically drive expression of proinflammatory mediators in synovial fibroblasts via IκBζ-dependent induction of ELF3.

OBJECTIVE: IL-17A and TNF act in synergy to induce proinflammatory mediators in synovial fibroblasts thus contributing to diseases associated with chronic arthritis. Many of these factors are regulated by transcription factor E74-like factor-3 (ELF3). Therefore, we sought to investigate ELF3 as a downstream target of IL-17A and TNF signalling and to characterize its role in the molecular mechanism of synergy between IL-17A and TNF. METHODS: Regulation of ELF3 expression by IL-17A and TNF was studied in synovial fibroblasts of RA and OA patients and RA synovial explants. Signalling leading to ELF3 mRNA induction and the impact of ELF3 on the response to IL-17A and TNF were studied using siRNA, transient overexpression and signalling inhibitors in synovial fibroblasts and HEK293 cells. RESULTS: ELF3 was marginally affected by IL-17A or TNF alone, but their combination resulted in high and sustained expression. ELF3 expression was regulated by the nuclear factor-κB (NF-κB) pathway and CCAAT/enhancer-binding protein ß (C/EBPß), but its induction required synthesis of the NF-κB co-factor IκB (inhibitor of NF-κB) ζ. siRNA-mediated depletion of ELF3 attenuated the induction of cytokines and matrix metalloproteinases by the combination of IL-17A and TNF. Overexpression of ELF3 or IκBζ showed synergistic effect with TNF in upregulating expression of chemokine (C-C motif) ligand 8 (CCL8), and depletion of ELF3 abrogated CCL8 mRNA induction by the combination of IκBζ overexpression and TNF. CONCLUSION: Altogether, our results establish ELF3 as an important mediator of the synergistic effect of IL-17A and TNF in synovial fibroblasts. The findings provide novel information of the pathogenic mechanisms of IL-17A in chronic arthritis and implicate ELF3 as a potential therapeutic target.

Cationic amino acid transporter-1 (CAT-1) promotes fibroblast-like synoviocyte proliferation and cytokine secretion by taking up L-arginine in rheumatoid arthritis.

BACKGROUND: Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA. METHODS: The concentrations of amino acids and cytokines in the synovial fluid of RA (n = 9) and osteoarthritis (OA, n = 9) were detected by LC-MS/MS and CBA assay, respectively. The mRNA and protein expression of cationic amino acid transporter-1 (CAT-1) were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion, and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo. RESULTS: L-rginine was upregulated in the synovial fluid of RA patients and was positively correlated with the elevation of the cytokines IL-1ß, IL-6, and IL-8. Further examination demonstrated that CAT-1 was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration, and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice. CONCLUSION: CAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.