Gastrodin Ameliorates the Inflammation, Oxidative Stress, and Extracellular Matrix Degradation of IL-1ß-Mediated Fibroblast-Like Synoviocytes via Suppressing the Gremlin-1/NF-κB Pathway.

BACKGROUND: Synovial inflammation plays a crucial role in osteoarthritis (OA). Gastrodin (GAS), an active ingredient derived from the Gastrodia elata Blume rhizome, possesses antioxidant and anti-inflammatory pharmacological effects. This research aimed to evaluate the function and molecular mechanism of GAS on human fibroblast-like synoviocytes of osteoarthritis (HFLS-OA) induced by interleukin (IL)-1ß. METHODS: The impact of GAS on the viability of IL-1ß-treated HFLS-OA cells was assessed using the cell counting kit-8 (CCK-8). Quantitative real-time reverse transcription PCR (qRT-PCR) was employed to detect changes in IL-8, IL-6, monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor (TNF)-α, and Gremlin-1 mRNA expression in each group. Corresponding kits were utilized to measure the catalase (CAT) and superoxide dismutase (SOD) activities, as well as the nitric oxide (NO) level. Western blot analysis was conducted to examine the expression of extracellular matrix degradation-associated proteins and nuclear factor kappa-B (NF-κB) pathway-correlated proteins in each group. RESULTS: GAS significantly promoted the proliferation of IL-1ß-induced HFLS-OA cells and concurrently down-regulated Gremlin-1 mRNA expression (p < 0.05). Through the down-regulation of Gremlin-1 expression, GAS exhibited the following effects: decreased IL-8, IL-6, and TNF-α mRNA expression, as well as NO levels (p < 0.05); increased SOD and CAT activities (p < 0.05); down-regulated matrix metallopeptidase 13 (MMP-13) and MMP-1 protein expression levels (p < 0.01); and up-regulated collagen II protein expression level (p < 0.01) in IL-1ß-treated HFLS-OA cells. Additionally, GAS decreased phospho-inhibitory kappa B (p-IκB)/IκB, phospho-inhibitory kappa B kinase (p-IKK)/IKK, and p-p65/p65 ratios in IL-1ß-induced HFLS-OA cells by inhibiting Gremlin-1 expression (p < 0.01). CONCLUSION: GAS demonstrates a positive impact on inflammation, oxidative stress, and extracellular matrix degradation in IL-1ß-mediated HFLS-OA cells. This effect is achieved by suppressing Gremlin-1 expression and reducing NF-κB pathway activity.

LncRNA SNHG3 discriminates rheumatoid arthritis from healthy individuals and regulates inflammatory response and oxidative stress via modulating miR-128-3p.

OBJECTIVES: This study evaluated the expression and significance of SNHG3 in rheumatoid arthritis (RA), aiming to explore a biomarker and regulator for RA. METHODS: The expression of SNHG3 in serum and synovial tissue was compared between RA patients and healthy individuals using polymerase chain reaction (PCR). The RA animal models were induced by the Porcine Type II collagen in Wistar rats and validated by the foot volume and arthritis index score. The human fibroblast-like synoviocytes were treated with lipopolysaccharide (LPS) to mimic the injury during RA onset, and the cell growth was assessed by cell counting kit-8 (CCK8) assay. RESULTS: SNHG3 was significantly downregulated in the serum and synovial tissue of RA patients compared with healthy individuals. Downregulated SNHG3 could discriminate RA patients from healthy individuals with high sensitivity (0.875) and specificity (0.844). Porcine Type II collagen induced increasing foot volume and arthritis index scores of rats, and SNHG3 was downregulated in RA rats. In LPS-induced human fibroblast-like synoviocytes, SNHG3 negatively regulated miR-128-3p, and the alleviated effect of SNHG3 overexpression on cellular inflammation and oxidative stress was reversed by miR-128-3p upregulation. CONCLUSIONS: Serum SNHG3 was considered a potential diagnostic biomarker for RA from healthy individuals. SNHG3 regulated inflammatory response and oxidative stress by negatively modulating miR-128-3p.

Self-actuating inflammation responsive hydrogel microsphere formulation for controlled drug release in rheumatoid arthritis (RA): Animal trials and study in human fibroblast like synoviocytes (hFLS) of RA patients.

Patients with rheumatoid arthritis (RA) often have one or more painfuljoints despite adequate medicine. Local drug delivery to the synovial cavity bids for high drug concentration with minimal systemic adverse effects. However, anti-RA drugs show short half-lives in inflamed joints after intra-articular delivery. To improve the therapeutic efficacy, it is essential to ensure that a drug is only released from the formulation when it is needed. In this work, we developed an intelligent "Self-actuating" drug delivery system where Disease-modifying anti-rheumatic Drug (DMARD) methotrexate is incorporated within a matrix intended to be injected directly into joints. This formulation has the property to sense the need and release medication only when joints are inflamed in response to inflammatory enzyme Matrix metalloproteinases (MMP). These enzymes are important proteases in RA pathology, and several MMP are present in augmented levels in synovial fluid and tissues. A high level of MMP present in synovial tissues of RA patients would facilitate the release of drugs in response and ascertain controlled drug release. The formulation is designed to be stable within the joint environment, but to dis-assemble in response to inflammation. The synthesized enzyme-responsive methotrexate (Mtx) encapsulated micron-sized polymer-lipid hybrid hydrogel microspheres (Mtx-PLHM) was physiochemically characterized and tested in synovial fluid, Human Fibroblast like synoviocytes (h-FLS) (derived from RA patients) and a rat arthritic animal model. Mtx-PLHM can self-actuate and augment the release of Mtx drug upon contact with either exogenously added MMP or endogenous MMP present in the synovial fluid of patients with RA. The drug release from the prepared formulation is significantly amplified to several folds in the presence of MMP-2 and MMP-9 enzymes. In the rat arthritic model, Mtx-PLHM showed promising therapeutic results with the significant alleviation of RA symptoms through decrease in joint inflammation, swelling, bone erosion, and joint damage examined by X-ray analysis, histopathology and immune-histology. This drug delivery system would be nontoxic as it releases more drug only during the period of exacerbation of inflammation. This will simultaneously protect patients from unwanted side effects when the disease is inactive and lower the need for repeated joint injections.

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

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

Tectoridin exhibits anti-rheumatoid arthritis activity through the inhibition of the inflammatory response and the MAPK pathway in vivo and in vitro.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by inflammation infiltration of the synovial tissues and the fibroblast-like synoviocytes. Tectoridin is a botanical active ingredient with anti-inflammatory properties. In this study, the anti-arthritic effects of tectoridin and its mechanism of action are examined in TNF-α-induced human fibroblast-like synovial cells (HFLSs cells) and complete Freund's adjuvant (CFA)-stimulated arthritic mice. Arthritis progression was evaluated via bodyweight, hind paw swelling, organ index, and synovial pathology. IL-1ß, IL-6 and other pro-inflammatory factors concentrations, and the expression of MAPK pathway proteins in HFLSs cells and arthritic mice were measured using ELISA and western blotting. Results showed that tectoridin significantly decreased the swelling of the paws and joints as well as the increased immune organ index within CFA-induced arthritic mice. Histopathological analysis showed that tectoridin alleviated the lesions of ankle joints and synovial tissues induced by CFA. Secretion of pro-inflammatory cytokines in TNF-α-induced HFLSs cells and CFA-stimulated arthritic mice were also abated by tectoridin. Similarly, the presence of tectoridin significantly inhibited the abnormal phosphorylation levels of ERK, JNK, and p38 in vivo and in vitro. All those results highlighted that tectoridin exhibits anti-arthritis effects by inhibiting MAPK-mediated inflammatory responses.

Long non-coding RNA cancer susceptibility candidate 2 regulates the function of human fibroblast-like synoviocytes via the microRNA-18a-5p/B-cell translocation gene 3 signaling axis in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a perennial inflammatory condition. Preliminary research indicated that long non-coding (lnc)RNA cancer susceptibility candidate 2 (CASC2) was downregulated in the serum of RA patients. Our study was designed to reveal the roles of lncRNA CASC2 in RA and the latent mechanisms underlying its role. Bioinformatics method (Starbase) and dual-luciferase reporter assay revealed that microRNA (miR)-18a-5p directly interacted with lncRNA CASC2. Furthermore, lncRNA CASC2 and miR-18a-5p expression in the serum samples of RA patients and healthy controls were measured via reverse transcription-quantitative PCR. Compared with the healthy subjects, lncRNA CASC2 was downregulated, whereas miR-18a-5p was upregulated in patients with RA. Overexpression of lncRNA CASC2 decreased the viability of human fibroblast-like synoviocytes (HFLSs) and induced apoptosis, as revealed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry analyses. Furthermore, the Western blotting assay suggested that Bax was upregulated and Bcl-2 was downregulated in lncRNA CASC2 up-regulated HFLSs. Downregulation of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, IL-6, matrix metalloproteinase (MMP)1, and MMP3 levels by lncRNA CASC2 up-regulation was determined using enzyme-linked immunosorbent assays (ELISAs). However, HFLSs co-transfected with miR-18a-5p mimic exhibited opposite effects compared with the case for the overexpression of lncRNA CASC2. The aforementioned methods were used to verify that a binding site exists between B-cell translocation gene 3 (BTG3) and miR-18a-5p. The effects of miR-18a-5p inhibitor on HFLSs were reversed by BTG3 silencing. Overall, lncRNA CASC2 alleviated RA by adjusting the miR-18a-5p/BTG3 signaling axis and could serve as a novel therapeutic option for RA.

Aconiti Lateralis Radix Praeparata lipid-soluble alkaloids alleviates IL-1ß-induced inflammation of human fibroblast-like synoviocytes in rheumatoid arthritis by inhibiting NF-κB and MAPKs signaling pathways and inducing apoptosis.

BACKGROUND: Fuzi lipid-soluble alkaloids (FLA) is the main bioactive components extracted from the traditional Chinese medicine Aconiti Lateralis Radix Praeparata ("Fuzi" in Chinese), which has promising analgesic and anti-inflammatory effects. However, the effects and the underlying mechanisms of FLA on rheumatoid arthritis (RA) have not been studied. The present study aimed to explore the anti-arthritic effects of FLA and its underlying mechanisms. METHODS: To standardize the FLA, UPLC-HR-MS was used for quantitative and qualitative analysis of the representative alkaloids. Cell viability was measured by MTT. The anti-inflammatory activity of FLA was examined by analyzing the expression levels of inflammatory mediators such as TNF-α, IL-6, MMP-1, MMP-3, PGE2, and COX-2 using ELISA and RT-PCR analysis. The Annexin V-FITC/PI double staining method was used to detect the apoptosis of HFLS-RA and analyzed by flow cytometry. Western blot analysis was used to analyze the expression of NF-κB, MAPKs and mitochondrial apoptosis pathway related proteins. RESULTS: FLA had a significant inhibitory effect on the proliferation of HFLS-RA induced by IL-1ß, which was accompanied by decreased expression levels of TNF-α, IL-6, MMP-1, MMP-3, COX-2 and PGE2. Remarkably, FLA inhibited the activation of NF-κB and MAPKs signaling pathways in IL-1ß-induced HFLS-RA, as well as inducing HFLS-RA apoptosis through the mitochondrial apoptosis pathway. CONCLUSIONS: FLA inhibited the expression and synthesis of inflammatory mediators by inhibiting the activation of NF-κB and MAPKs signaling pathways in HFLS-RA, and induced apoptosis of HFLS-RA via the mitochondrial apoptosis pathway.

LncRNA cardiac autophagy inhibitory factor is downregulated in rheumatoid arthritis and suppresses the apoptosis of fibroblast-like synoviocytes by promoting the maturation of miRNA-20a.

Objectives: In this study, we aimed to investigate the effects of LncRNA cardiac autophagy inhibitory factor (CAIF) and miR-20a on the apoptosis of synovial cells in rheumatoid arthritis (RA) and the regulatory mechanism. Patients and methods: Between May 2018 and March 2020, a total of 62 RA patients (24 males, 38 females; mean age: 55.2±4.9 years; range, 42 to 68 years) and 62 controls (24 males, 38 females; mean age: 55.3±4.8 years; range, 41 to 68 years) were included in this study. Plasma samples were collected from all participants. The expression levels of CAIF, mature miR-20a, and miR-20a precursor in these plasma samples were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlations were analyzed using linear regression analysis. Overexpression of CAIF was achieved in human fibroblast-like synoviocytes (HFLSs) and the expression levels of mature miR-20a and miR-20a precursor were determined using RT-qPCR. Cell apoptosis was analyzed by cell apoptosis assay. Results: The CAIF was downregulated in RA and positively correlated with the expression of mature miR-20a. In HFLSs, LPS treatment resulted in downregulation of both CAIF and miR-20a in a dose-dependent manner. In HFLSs, overexpression of CAIF did not affect the expression of miR-20a precursor, but upregulated the expression of mature miR-20a. Cell apoptosis analysis showed that overexpression of CAIF and miR-20a inhibited the apoptosis of HFLSs induced by LPS. The combination of overexpression of CAIF and miR-20a showed a stronger effect. Conclusion: The CAIF may suppress the apoptosis of HFLSs in RA by promoting the maturation of miR-20a.

MicroRNA-137-mediated inhibition of lysine-specific demethylase-1 prevents against rheumatoid arthritis in an association with the REST/mTOR axis.

BACKGROUND: It has been increasingly reported that microRNAs (miRNAs) are related to rheumatoid arthritis (RA) pathogenesis. This present research was conducted to analyze the functions of miR-137 and the underlying molecular mechanism in RA progression. METHODS: Differentially expressed miRNAs in RA patients were analyzed using microarray-based analyses. Next, experiments involving miR-137 overexpression were performed to analyze the role of miR-137 in human fibroblast-like synoviocytes-RA (HFLS-RA) using cell counting kit-8 (CCK-8) assay, EdU staining, Transwell assay and flow cytometry, respectively. The function of miR-137 in inflammation was determined using ELISA. The binding relationship between miR-137 and LSD1 was confirmed by dual-luciferase reporter gene assay and ChIP test. Besides, a rat model with RA was established for in vivo experiments. RESULTS: miR-137 was downregulated in RA tissues and cells, which was negatively correlated with inflammatory factors. Upregulated miR-137 suppressed growth, migration and invasion of HFLS-RA, but promoted apoptosis. Lysine-specific demethylase-1 (LSD1) was a target of miR-137 and could be negatively regulated by miR-137. Moreover, LSD1 could activate REST through demethylation, while the REST/mTOR pathway induced levels of pro-inflammatory factors in RA. We observed the similar results in our in vivo study. CONCLUSION: This study suggested that miR-137 reduced LSD1 expression to inhibit the activation of REST/mTOR pathway, thus preventing against inflammation and ameliorating RA development. Our research may offer new insights into treatment of RA.

Bioactive fractions and compound of Ardisia crispa roots exhibit anti-arthritic properties mediated via angiogenesis inhibition in vitro.

BACKGROUND: Ardisia crispa (Thunb.) A.DC (Primulaceae), is a medicinal herb traditionally used by Asian people as remedies to cure inflammatory related diseases, including rheumatism. The plant roots possess various pharmacological activities including antipyretic, anti-inflammation and antitumor. Previous phytochemical studies of the plant roots have identified long chain alkyl-1,4-benzoquinones as major constituents, together with other phytochemicals. Hexane fraction of the plant roots (ACRH), was previously reported with anti-angiogenic and anti-arthritic properties, while its effect on their anti-arthritic in vitro, is yet unrevealed. Considering the significance of angiogenesis inhibition in developing new anti-arthritic agent, thus we investigated the anti-arthritic potential of Ardisia crispa roots by suppressing angiogenesis, in vitro. METHODS: Ardisia crispa roots hexane extract (ACRH) was prepared from the plant roots using absolute n-hexane. ACRH was fractionated into quinone-rich fraction (QRF) and further isolated to yield benzoquinonoid compound (BQ), respectively. In vitro experiments using VEGF-induced human umbilical vein endothelial cells (HUVECs) and IL-1ß-induced human fibroblast-like synoviocytes for rheumatoid arthritis (HFLS-RA) were performed to evaluate the effects of these samples on VEGF-induced HUVECs proliferation and tube formation, and towards IL-1ß-induced HFLS-RA proliferation, invasion, and apoptosis, respectively. Therapeutic concentrations (0.05, 0.5, and 5 µg/mL) tested in this study were predetermined based on the IC50 values obtained from the MTT assay. RESULTS: ACRH, QRF, and BQ exerted concentration-independent antiproliferative effects on VEGF-induced HUVECs and IL-1ß-induced HFLS-RA, with IC50 values at 1.09 ± 0.18, 3.85 ± 0.26, and 1.34 ± 0.16 µg/mL in HUVECs; and 3.60 ± 1.38, 4.47 ± 0.34, and 1.09 ± 0.09 µg/mL in HFLS-RA, respectively. Anti-angiogenic properties of these samples were verified via significant inhibition on VEGF-induced HUVECs tube formation, in a concentration-independent manner. The invasiveness of IL-1ß-induced HFLS-RA was also significantly inhibited in a concentration-independent manner by all samples. ACRH and BQ, but not QRF, significantly enhanced the apoptosis of IL-1ß-induced HFLS-RA elicited at their highest concentration (5 µg/mL) (P < 0.05). CONCLUSIONS: These findings highlight the bioactive fractions and compound from Ardisia crispa roots as potential anti-arthritic agents by inhibiting both HUVECs and HFLS-RA's cellular functions in vitro, possibly mediated via their anti-angiogenic effects.

Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro.

BACKGROUND: Osteoarthritis (OA) is a disease of the entire joint involving synovial fibrosis and inflammation. Pathological changes to the synovium can accelerate the progression of OA. Pirfenidone (PFD) is a potent anti-fibrotic drug with additional anti-inflammatory properties. However, the influence of PFD on OA is unknown. METHODS: Proliferation of human fibroblast-like synoviocytes (FLSs) after treatment with TGF-ß1 or PFD was evaluated using a Cell Counting Kit-8 assay and their migration using a Transwell assay. The expression of fibrosis-related genes (COL1A1, TIMP-1, and ACTA-2) and those related to inflammation (IL-6 and TNF-α) was quantified by real-time quantitative PCR. The protein expression levels of COL1A1, α-SMA (coded by ACTA-2), IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. A rabbit model of OA was established and then PFD was administered by gavage. The expression of genes related to fibrosis (COL1A1, TIMP-1, and ADAM-12) and inflammation (IL-6 and TNF-α) was measured using RNA extracted from the synovium. Synovial tissue was examined histologically after staining with H&E, Masson's trichrome, and immunofluorescence. Synovitis scores, the volume fraction of collagen, and mean fluorescence intensity were calculated. Degeneration of articular cartilage was analyzed using a Safranin O-fast green stain and OARSI grading. RESULTS: The proliferation of FLSs was greatest when induced with 2.5 ng/ml TGF-ß1 although it did not promote their migration. Therefore, 2.5 ng/ml TGF-ß1 was used to stimulate the FLSs and evaluate the effects of PFD, which inhibited the migration of FLSs at concentrations as low as 1.0 mg/ml. PFD decreased the expression of COL1A1 while TGF-ß1 increased both mRNA and protein expression levels of IL-6 but had no effect on α-SMA or TNF-α expression. PFD decreased mRNA expression levels of COL1A1, IL-6, and TNF-α in vivo. H&E staining and synovitis scores indicated that PFD reduced synovial inflammation, while Masson's trichrome and immunofluorescence staining suggested that PFD decreased synovial fibrosis. Safranin O-Fast Green staining and the OARSI scores demonstrated that PFD delayed the progression of OA. CONCLUSIONS: PFD attenuated synovial fibrosis and inflammation, and postponed the progression of osteoarthritis in a modified Hulth model of OA in rabbits, which was related to its anti-fibrotic and anti-inflammatory properties.

lncRNA CASC2 downregulation participates in rheumatoid arthritis, and CASC2 overexpression promotes the apoptosis of fibroblast­like synoviocytes by downregulating IL­17.

lncRNA cancer susceptibility candidate 2 (CASC2) is a recently identified oncogenic lncRNA in different types of cancers. Our preliminary microarray data showed that lncRNA CASC2 was downregulated in the plasma of patients with rheumatoid arthritis (RA), indicating the involvement of this lncRNA in RA. In the present study, lncRNA CASC2 and IL­17 in plasma were detected by reverse transcription­-quantitative PCR and ELISA, respectively. Diagnostic analyses were performed using receiver operating characteristic curves. Flow cytometry was performed to evaluate cell apoptosis. The effects of lncRNA CASC2 on IL­17 expression were determined via western blotting. lncRNA CASC2 was found to be downregulated, while IL­17 was upregulated in the plasma of RA patients when compared with these levels in the plasma of healthy controls. Plasma levels of lncRNA CASC2 and IL­17 were significantly and inversely correlated in both RA patients and healthy controls. Altered plasma levels of lncRNA CASC2 and IL­17 were able to differentiate RA patients from healthy controls. Overexpression of lncRNA CASC2 promoted, while treatment with IL­17 inhibited the apoptosis of human fibroblast­like synoviocytes (HFLSs) isolated from RA patients. Overexpression of lncRNA CASC2 inhibited IL­17 expression in HFLS, while treatment with IL­17 did not significantly affect the expression of lncRNA CASC2. Therefore, downregulation of lncRNA CASC2 is involved in RA and lncRNA CASC2 overexpression may promote the apoptosis of HFLS by downregulating IL­17.

Activation of GPR43 suppresses TNF-α-induced inflammatory response in human fibroblast-like synoviocytes.

Although rheumatoid arthritis (RA) has long posed a major threat to global health, the mechanisms driving the development and progression of RA remain incompletely understood. In the present study, we investigated the effects of G protein-coupled receptor 43 (GPR43/FFAR2) in various aspects of the pathogenesis of RA. To our knowledge, this is the first study to demonstrate that GPR43 is expressed on human fibroblast-like synoviocytes (FLS). Furthermore, we show that GPR43 is upregulated in FLS exposed to tumor necrosis factor-α (TNF-α). Importantly, our findings demonstrate that activation of GPR43 using its specific agonist significantly suppressed expression of the following key factors of RA: cytokines, such as interleukin-6 (IL-6), IL-8, high mobility group protein 1 (HMG-1); chemokines, such as monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cellular adhesion molecule 1 (VCAM-1); markers of oxidative stress, such as production of reactive oxygen species (ROS) and 4-hydroxynoneal (4-HNE); degradative enzymes, such as matrix metalloproteinase-3 (MMP-3) and MMP-13; and activation of the nuclear factor-κB (NF-κB) inflammatory signaling pathway. These results suggest a promising potential role for GPR43 as a specific target in the treatment and prevention of RA.

Activation of Toll-Like Receptor 3 Induces Interleukin-1 Receptor Antagonist Expression by Activating the Interferon Regulatory Factor 3.

Toll-like receptor 3 (TLR3) is a sensor of endogenous cell necrosis during the process of acute inflammation. Interleukin (IL)-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine and can negatively regulate the pathogenesis of inflammation. However, whether and how activation of TLR3 can regulate IL-1Ra expression has not been clarified. Here, we show that poly(I:C) induces IL-1Ra expression in primarily cultured human fibroblast-like synoviocytes and other types of cells. Induction of IL-1Ra by poly(I:C) was dependent on TLR3, but was independent of melanoma differentiation--associated protein 5 or retinoic acid-inducible gene I. Interferon regulatory factor 3 (IRF3) directly binds to the IL-1Ra promoter and promotes IL-1Ra expression in response to poly(I:C) stimulation. Induction of IL-1Ra by poly(I:C) was abolished by the inhibition of the NF-κB signaling, attenuated by the inhibition of the PI3K-Akt signaling, enhanced by inhibition of the ERK1/2 or MSK1/2 activation, but was independent of the p38 MAPK signaling. Treatment with poly(I:C) or Sendai virus elevated the levels of serum IL-1Ra in wild-type, but not in TLR3-/- or IRF3-/- mice. Our findings may provide new insights into the intrinsic anti-inflammatory function of TLR3 and double-stranded RNA-induced IL-Ra expression by TLR3 and its regulation.

Gentiopicrin exerts anti-rheumatic effect in human fibroblast-like synoviocytes via inhibition of p38MAPK/NF-κB pathway.

To investigate the effect of gentiopicrin on the expressions of inflammatory factors in human fibroblast-like synoviocytes (HFLS) and the underlying mechanism. Human fibroblast-like synoviocytes (HFLS) were cultured in vitro at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % fetal bovine serum (FBS) in a humidified incubator containing 5 % CO2. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-tetrazolium bromide (MTT) assay, while real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expressions of interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) mRNAs. The expressions of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) were determined using Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IL-1ß and IL-6 in cell lysate. Treatment with 5-25 µM gentiopicrin did not significantly affect the number of viable cells, when compared with control group (p > 0.05). However, at 50 and 100 µM gentiopicrin, the number of viable cells were significantly increased, relative to control group (p < 0.05). Results of qRT-PCR showed that the expression levels of IL-1ß and IL-6 mRNAs were significantly higher in TNF-α group than in control group (p < 0.05). However, treatment with gentiopicrin significantly and dose-dependently decreased their expression levels compared with TNF-α group (p < 0.05). Western blotting results showed that the expressions of p-p38MAPK and NF-κB-p65 proteins were significantly upregulated in TNF-α group, when compared with control group (p < 0.05). However, treatment with gentiopicrin significantly and dose-dependently down-regulated the expression of these proteins compared with TNF-α group (p < 0.05). The levels of IL-1ß and IL-6 in cell lysate were significantly higher in TNF-α group than in control group (p < 0.05). However, treatment with gentiopicrin, and p38MAPK/NF-κB pathway inhibitors (SB203580 and BAY11-7082) significantly reduced the levels of these inflammatory factors compared with TNF-α group (p < 0.05).  Gentiopicrin has therapeutic potential for Rheumatoid arthritis (RA  ) through a mechanism involving the inhibition of p38MAPK/NF-κB pathway.

Brucine inhibits TNF-α-induced HFLS-RA cell proliferation by activating the JNK signaling pathway.

Rheumatoid arthritis (RA) is a diffuse connective tissue disease. Brucine selectively inhibits cell immunity, immune hypersensitivity and induces apoptosis. The current study aimed to investigate effects of brucine on human fibroblast-like synoviocytes (HFLS) of RA and to clarify associated molecular mechanisms. HFLS-RA were treated with tumor necrosis factor (TNF)-α prior to treatment with brucine at carrying concentrations. Cell Counting Kit-8 assays were performed to evaluate HFLS-RA proliferation. Western blot assays were employed to examine c-Jun N-terminal kinase (JNK) expression and phosphorylation in TNF-α-induced HFLS-RA. An association between brucine treatment and JNK phosphorylation was assessed by employing a linear regression analysis. The results suggested that low doses of brucine (0.125 and 0.25 mg/ml) significantly reversed proliferation effects induced by TNF-α, however, final cell viabilities were increased compared with the untreated control (P>0.05 and P<0.05, respectively). High brucine doses (≥0.5 mg/ml) significantly reversed TNF-α-induced proliferation and further inhibited viability compared with the untreated control (P<0.05). Regarding JNK expression, there were no significant differences among the brucine treatment, and between the Control and the TNF-α groups (P>0.05). Brucine treatment significantly decreased JNK phosphorylation compared with the TNF-α group (P<0.05). JNK specific inhibitor, SP600125, significantly inhibited brucine-induced cell viability enhancement compared with the brucine-treated groups without inhibitor (P<0.05). A linear regression analysis suggested that brucine was associated with JNK phosphorylation in TNF-α-treated HFLS-RA. In conclusion, brucine significantly inhibited TNF-α-induced HFLS-RA proliferation by activating the JNK signaling pathway. Therefore, brucine may have potential clinical applications in the treatment of RA.

LncRNA DILC participates in rheumatoid arthritis by inducing apoptosis of fibroblast-like synoviocytes and down-regulating IL-6.

IL-6 produced by human fibroblast-like synoviocytes (HFLS) promotes rheumatoid arthritis (RA), while lncRNA DILC regulates liver cancer stem cells by inhibiting IL-6. Therefore, lncRNA DILC may participate in RA. In the present study, we found that plasma lncRNA DILC was down-regulated, while IL-6 was up-regulated in RA patients than in healthy controls. Plasma levels of lncRNA DILC and IL-6 were significantly and inversely correlated only in RA patients. Overexpression of lncRNA DILC resulted in promoted apoptosis of HFLS isolated from RA patients, while lncRNA DILC siRNA silencing played an opposite role. In addition, overexpression of lncRNA DILC also resulted in inhibited IL-6 expression in HFLS isolated from RA patients. Therefore, lncRNA DILC may participate in RA by inducing apoptosis of HFLS and down-regulating IL-6.

Treatment effect of CDKN1A on rheumatoid arthritis by mediating proliferation and invasion of fibroblast-like synoviocytes cells.

The objective of the present study was to evaluate the role of CDKN1A in rheumatoid arthritis (RA). Related gene expression data screened from Gene Expression Omnibus (GEO) were processed with network analysis. Protein-protein interaction was analysed through string database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure mRNA and microRNA expression. Cell proliferation and cell cycle were tested by MTT assay and flow cytometry, respectively. Transwell migration and invasion assay was used to test cell migration and invasion. CDKN1A screened by bioinformatics methods showed differential expression in RA cells compared with healthy controls (HC), and was at an important position in the protein-protein interaction network of RA. Compared with the HC group, CDKN1A was down-regulated in human RA synovium tissues and human fibroblast-like synoviocytes (HFLS). Contrary to CDKN1A silencing, CDKN1A over-expression significantly inhibited the proliferation and invasion of HFLS-RA, arrested HFLS-RA in G0/G1 phase and down-regulated the expressions of tumour necrosis factor (TNF)-α and interleukin (IL)-6, while it up-regulated the expression of IL-10. CDKN1A over-expression could also suppress phosphorylated signal transducers and activators of transcription 1 (pSTAT-1) expression. MiR-146a, highly expressed in RA tissues, could regulate CDKN1A negatively. Anti-146a suppressed cell proliferation and invasion, and at the same time enhanced IL-10 expression but inhibited IL-6, TNF-α and pSTAT-1 expression. The results indicated that CDKN1A over-expression, which could be enhanced by miR-146a suppression, inhibited the proliferation of invasion in HFLS-RA. This was probably a result of suppressed pSTAT-1, IL-6 and TNF-α expression and enhanced IL-10 expression.

Protective effects of paeonol on inflammatory response in IL-1ß-induced human fibroblast-like synoviocytes and rheumatoid arthritis progression via modulating NF-κB pathway.

Various investigations have demonstrated that human fibroblast-like synoviocytes rheumatoid arthritis (HFLS-RA) take part in the chronic inflammatory responses and RA progression. Inhibition of synovium activation and inflammatory processes may represent a therapeutic target to alleviate RA. Paeonol, a major natural product, has many biological and pharmacological activities. However, its protective effects against RA considering HFLS-RA have not been explored. In this study, anti-inflammatory effects of paeonol were detected in interleukin-1ß (IL-1ß)-treated HFLS-RA. Our results demonstrated that paeonol had no effect on cell survival and IL-1ß-induced proliferation in HFLS-RA. Pretreatment with paeonol significantly suppressed the production of pro-inflammatory TNF-α, IL-6 and IL-1ß, and the expressions of matrix metalloproteinase-1/-3 in vitro and in vivo. Mice treated with paeonol (10 mg/kg) remarkablely attenuated arthritic symptoms based on clinical arthritis scores and histopathology in collagen-induced arthritis mice. Furthermore, the TLR4 expression and NF-κB p65 activation were inhibited by paeonol in vitro and in vivo. Our findings illustrated that paeonol had significantly suppressed inflammation effects in synovial tissues and RA progression. The potential mechanism might be based on the attenuation TLR4-NF-κB activation. These collective results indicated that paeonol might be a promising therapeutic agent for alleviating RA progress through inhibiting inflammations and NF-κB signalling pathway.