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OBJECTIVE: To investigate the impact of IGJ on the proliferation, inflammation, and motility of rheumatoid arthritis (RA) fibroblast-like synoviocytes and elucidate the underlying mechanism. METHODS: The expression of IGJ RA fibroblast-like synoviocytes was assessed using immunoblot and qPCR. Cell growth was evaluated using CCK-8 and FCM assays. The effects on inflammatory response were determined by ELISA and immunoblot assays. Cell motility was assessed using transwell and immunoblot assays. The mechanism was further confirmed using immunoblot assays. RESULTS: IGJ expression was found to be elevated in fibroid synovial cells of RA. IGJ ablation inhibited the growth of MH7A cells and suppressed the inflammatory response. Knockdown of IGJ also blocked cell motility. Mechanically, the knockdown of IGJ suppressed the NF-κB axis in MH7A cells. CONCLUSION: IGJ suppresses RA in fibroblast-like synoviocytes via NF-κB pathway.
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Artritis Reumatoide , Movimiento Celular , Proliferación Celular , Fibroblastos , FN-kappa B , Transducción de Señal , Sinoviocitos , Humanos , Sinoviocitos/metabolismo , Sinoviocitos/patología , Sinoviocitos/efectos de los fármacos , Artritis Reumatoide/patología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , FN-kappa B/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Células Cultivadas , Línea Celular , HialuronoglucosaminidasaRESUMEN
BACKGROUND: Rheumatoid arthritis, a condition characterized by inflammation, has a substantial influence on both the worldwide economy and public health. Prior studies indicate that probiotics have the potential to enhance the composition of gut microbiota in instances of intestinal dysbiosis resulting from different disorders and contribute to the regulation of inflammation. The objective of this study is to investigate the impact of Saccharomyces boulardii on the gut microbiome in arthritis and its implications on inflammation. METHODS: The study utilized the Collagen Induced Arthritis (CIA) Sprague-Dawley (SD) rat model. After administering Saccharomyces boulardii (150 mg/kg/day) six days a week and Methotrexate (MTX) (0.2 mg/week) treatment for eight weeks, microbial DNA from the feces was sequenced using 16S rRNA. The evaluation of histopathology, bone loss, and cartilage degradation was conducted using histology, immunohistology assays, and micro-computed tomography (µCT) examinations. The enzyme-linked immunosorbent assay (ELISA) was used to analyze proinflammatory cytokines, while the western blot technique was applied to detect protein in the gut and in cell lines. The quantification of gene expression in gut,joint and cell lines was performed using real-time polymerase chain reaction. The cell lines were activated and then treated with the culture supernatant of S. boulardii for an in vitro investigation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was utilized to assess cell proliferationand viability. Cellular motility was measured in a wound healing experiment, whereas apoptotic proteins were analyzed using Western blotting. RESULTS: S. boulardii has been found to enhance bone and joint integrity, modulate gut microbiota, and mitigate proinflammatory cytokine levels in rats with arthritis. It decreases the permeability of the intestines and promotes the production of gut tight-junction proteins. The administration of S. boulardii inhibits the proliferation of T-helper-17 (Th17) and Type 3 innate lymphoid cells (ILC3). Additionally, it elicits apoptosis in MH7A cell lines and hinders their migratory activity. CONCLUSION: This study provides valuable insights into the therapeutic potential of S. boulardii for treating and preventing arthritis in rats with collagen-induced arthritis by modulating gut microbiota and inflammation.
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Artritis Experimental , Microbioma Gastrointestinal , Mucosa Intestinal , FN-kappa B , Probióticos , Ratas Sprague-Dawley , Saccharomyces boulardii , Transducción de Señal , Receptor Toll-Like 2 , Animales , Artritis Experimental/inmunología , Artritis Experimental/terapia , Probióticos/uso terapéutico , FN-kappa B/metabolismo , Mucosa Intestinal/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Ratas , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/genética , Microbioma Gastrointestinal/efectos de los fármacos , Masculino , Humanos , Citocinas/metabolismo , Línea Celular , Artritis Reumatoide/terapia , Artritis Reumatoide/inmunología , Factor 88 de Diferenciación MieloideRESUMEN
Background: Fufang Xiaohuoluo pill (FFXHL) is a commonly used prescription in clinical practice for treating rheumatoid arthritis in China, yet its specific mechanism remains unclear. This study aims to elucidate the pharmacological mechanisms of FFXHL using both in vivo and in vitro experiments. Methods: The collagen-induced arthritis (CIA) rat model was established to evaluate FFXHL's therapeutic impact. Parameters that include paw swelling, arthritis scores, and inflammatory markers were examined to assess the anti-inflammatory and analgesic effects of FFXHL. Human fibroblast-like synoviocytes (MH7A cells) is activated by tumour necrosis factor-alpha (TNF-α) were used to explore the anti-inflammatory mechanism on FFXHL. Results: Our findings indicate that FFXHL effectively reduced paw swelling, joint pain, arthritis scores, and synovial pannus hyperplasia. It also lowered serum levels of TNF-α, interleukin-1ß (IL1ß), and interleukin-6 (IL-6). Immunohistochemical analysis revealed decreased expression of nuclear factor-kappa B (NF-κB) p65 in FFXHL-treated CIA rat joints. In vitro experiments demonstrated FFXHL's ability to decrease protein secretion of IL-1ß and IL-6, suppress mRNA expression of matrix metalloproteinases (MMP) -3, -9, and -13, reduce reactive oxygen species (ROS) levels, and inhibit NF-κB p65 translocation in TNF-α stimulated MH7A cells. FFXHL also suppressed protein levels of extracellular signal-regulated kinase (ERK), c-Jun Nterminal kinase (JNK), p38 MAP kinase (p38), protein kinase B (Akt), p65, inhibitor of kappa B kinase α/ß (IKKα/ß), Toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) induced by TNF-α in MH7A cells. Conclusion: The findings imply that FFXHL exhibits significant anti-inflammatory and antiarthritic effects in both CIA rat models and TNF-α-induced MH7A cells. The potential mechanism involves the inactivation of TLR4/MyD88, mitogen-activated protein kinases (MAPKs), NF-κB, and Akt pathways by FFXHL.
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Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, pannus formation, and cartilage and bone destruction. Lysine-specific demethylase 1 (LSD1), an enzyme involved in transcriptional regulation, has an unclear role in synovial inflammation, fibroblast-like synoviocytes migration, and invasion during RA pathogenesis. In this study, we observed increased LSD1 expression in RA synovial tissues and in TNF-α-stimulated MH7A cells. SP2509, an LSD1 antagonist, directly reduced LSD1 expression and reversed the elevated levels of proteins associated with inflammation, apoptosis, proliferation, and autophagy induced by TNF-α. Furthermore, SP2509 inhibited the migratory capacity of MH7A cells, which was enhanced by TNF-α. In CIA models, SP2509 treatment ameliorated RA development, reducing the expression of pro-inflammatory cytokines and alleviating joint pathological symptoms. These findings underscore the significance of LSD1 in RA and propose the therapeutic potential of SP2509.
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Artritis Reumatoide , Histona Demetilasas , Artritis Reumatoide/metabolismo , Artritis Reumatoide/inmunología , Histona Demetilasas/metabolismo , Humanos , Animales , Ratones , Sinoviocitos/metabolismo , Sinoviocitos/patología , Sinoviocitos/inmunología , Progresión de la Enfermedad , Factor de Necrosis Tumoral alfa/metabolismo , Movimiento Celular , Línea Celular , Masculino , Membrana Sinovial/patología , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Apoptosis , Artritis Experimental/inmunología , Citocinas/metabolismo , Autofagia , Proliferación Celular/efectos de los fármacosRESUMEN
Three new biflavonoids (1-3) and two known flavonoids (4, 5) were isolated from Xylia kerrii collected in Thailand. Compounds 1-5 showed selective cytotoxicity against the rheumatoid fibroblast-like synovial MH7A cell line, and these compounds showed weak cytotoxicity against the human lung synovial fibroblast WI-38 VA13 sub 2 RA cell line. Notably, compound 1 was highly selective toward MH7A cells with an IC50 value of 6.9 µM, whereas the IC50 value for WI-38 VA13 sub 2 RA cells was > 100 µM. The western blotting analysis of MH7A cells treated with compound 1 showed increased CDKN2A /p16INK4A and caspase-8 levels.
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Artritis Reumatoide , Biflavonoides , Fibroblastos , Extractos Vegetales , Hojas de la Planta , Humanos , Fibroblastos/efectos de los fármacos , Artritis Reumatoide/tratamiento farmacológico , Extractos Vegetales/farmacología , Extractos Vegetales/química , Hojas de la Planta/química , Línea Celular , Biflavonoides/farmacología , Biflavonoides/química , Biflavonoides/aislamiento & purificación , Tailandia , Membrana Sinovial/efectos de los fármacos , Estructura MolecularRESUMEN
Orthosiphon aristatus is a well-known folkloric medicine and herb for Guangdong soup for the treatment of rheumatism in China. Eight isopimarane-type and migrated pimarane-type diterpenoids (1-8), including a new one with a rarely occurring α,ß-unsaturated diketone C-ring, were isolated from O. aristatus. Their structures were determined by spectroscopic methods and quantum chemical calculations. Furthermore, the most abundant compound, orthosiphol K, was structurally modified by modern synthetic techniques to give seven new derivatives (9-15). The anti-rheumatoid arthritis activity of these diterpenoids were evaluated on a TNF-α induced MH7A human rheumatoid fibroblast-like synoviocyte model. Compound 10 showed the most potent activity among these compounds. Based on their inhibitory effects on the release levels of IL-1ß, the preliminary structure-activity relationships were concluded. Furthermore, western blot analysis revealed that 10 could increase the expression of IκBα and decrease the expression of NF-κB p65, and the expression levels of COX-2 and NLRP3 proteins were consequently down-regulated.
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Artritis Reumatoide , Diterpenos , Orthosiphon , Humanos , Orthosiphon/química , Orthosiphon/metabolismo , Abietanos , Artritis Reumatoide/tratamiento farmacológico , Factor de Necrosis Tumoral alfa , Diterpenos/farmacología , Diterpenos/química , FN-kappa B/metabolismoRESUMEN
Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 μ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.
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Objective To study the cGAS/STING signaling pathway and investigate the potential effect of emodin(EMD)on autophagy of human rheumatoid arthritis fibroblast synovial cells(MH7A).Methods CCK-8 method was used to detect MH7A cell proliferation,and the experimental concentration of EMD was screened according to cell survival rate.Then,autophagy inhibitor 3-MA was added to further verify the effect of EMD on autophagy.Autophagy of MH7A cells was detected via the monodansylcadaverine staining method.Protein expression levels of cGAS,STING,p-STING,LC3-Ⅰ,LC3-Ⅱ,P62 and Beclin-1 were detected by Western blot.Results Monodansylcadaverine staining indicated that EMD enhanced the autophagy of MH7A cells.Western blot indicated that EMD decreased the expression of autophagy related proteins cGAS,STING,p-STING and P62,and increased that of LC3-Ⅱ and Beclin-1 in MH7A cells.After addition of the autophagy inhibitor 3-MA,the expression of P62 protein in MH7A cells increased,while that of LC3-Ⅱ and Beclin-1 decreased.Conclusions EMD may accelerate autophagy and inhibit MH7A cell proliferation by down-regulating cGAS/STING signaling pathway proteins.
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BACKGROUND: Rheumatoid arthritis (RA) is a common disease with joint cartilage destruction. BUB1 Mitotic Checkpoint Serine/Threonine Kinase (BUB1) is abnormally expressed in synovial tissues of RA patients, but its effect on RA remains unclear. In this study, we explored the role of BUB1 in RA. METHODS: An RA cell model was constructed by treating MH7A cells with tumor necrosis factor-α (TNF-α). The levels of BUB1, GAPDH, phosphorylated phosphatidylinositol 3 kinase (p-PI3K)/PI3K, and phosphorylated serine/threonine kinase (p-Akt)/Akt in MH7A cells were examined by Western blot. The MH7A cell proliferation was examined by colony formation assay. Wound healing assay and transwell assay were carried out to detect MH7A cell migration and invasion. The mRNA levels of proinflammatory cytokines were assessed by quantitative reverse transcription polymerase chain reaction. RESULTS: The results showed that knockdown BUB1 inhibited TNF-α-induced MH7A cell proliferation, migration, and invasion. Silencing BUB1 repressed the PI3K/Akt pathway in TNF-α-induced MH7A cells. We also found that the TNF-α-induced MH7A cell proliferation, migration, and invasion were repressed by si-BUB1 transfection, whereas these effects were attenuated by 740Y-P (an activator of the PI3K pathway) co-treatment. Knockdown of BUB1 reduced the expression of the proinflammatory cytokines. CONCLUSION: Knockdown BUB1 repressed TNF-α-induced MH7A cell proliferation, migration and invasion through the PI3K/Akt pathway.
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Artritis Reumatoide , Factor de Necrosis Tumoral alfa , Humanos , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/farmacología , Transducción de Señal , Artritis Reumatoide/metabolismo , Citocinas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/farmacología , Proliferación Celular , Fibroblastos/metabolismo , Serina/metabolismo , Serina/farmacologíaRESUMEN
Introduction: This study aimed to investigate the structure characterization and antiinflammatory activity of a novel polysaccharide, PKP2-1, from the rhizomes of Polygonatum kingianum Coll. and Hemsl. Methods: We isolated a novel polysaccharide, PKP2-1, from the rhizomes of Polygonatum kingianum Coll. and Hemsl. for the first time, which was then successively purified through hot-water extraction, 80% alcohol precipitation, anion exchange and gel permeation chromatography. The in vitro anti-inflammatory activity of PKP2-1 in MH7A cells was assessed using a CCK-8 kit assay. Results: Monosaccharide composition assay revealed that PKP2-1 was mainly composed of glucose, galactose, mannose, and glucuronic acid at an approximate molar ratio of 6:2:2:1. It had a molecular weight of approximately 17.34 kDa. Structural investigation revealed that the backbone of PKP2-1 consisted of (â2, 3)-α-D-Galp(4â, â2)-α-D-Manp(3â, â2)-ß-D-Glcp(4â) and α-D-Glcp(3â) residues with side chains (â2)-ß-D-Glcp(4â, â1)-α-D-Galp(4â) and α-D-Glcp(3â) branches located at O-3 position of (â2, 3)-α-D-Galp(4â). The in vitro anti-inflammatory activity of PKP2-1 in MH7A cells revealed that PKP2-1 could reduce the expression of IL-11ß and IL-6, increase the expression of IL-10 and induce apoptosis of synovial fibroblasts. Conclusion: The PKP2-1 could inhibit MH7A cell growth and potentially be exploited as an anti-inflammatory agent.
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Rheumatoid arthritis (RA) is now widely recognized as a chronic systemic inflammatory autoimmune disease characterized by swelling, pain and stiffness, which are often disabling. Although the number of drugs available for the treatment of RA has increased in recent years, they are generally expensive, leave patients prone to relapse and can result in severe effects when discontinued. Thus, there is a need for an inexpensive drug with fewer side effects that can be adhered to relieve pain and slow down the progression of the disease. Strychnine, a traditional Chinese medicine, was often used in ancient times to treat swollen and painful joints; however, because of its somewhat toxic nature, it is often combined with Atractylodes macrocephala to reduce its toxicity for safer therapeutic action. The present study performed high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) analysis to confirm whether the use of strychnine with Atractylodes macrocephala had the effect of reducing strychnine content. MH7A cells were induced using IL-1ß to study the effect of strychnine with Atractylodes macrocephala on the Toll-like receptor 4 (TLR4)/NF-κB/NLR family pyrin domain-containing 3 (NLRP3) pathway in order to verify its role in the treatment of RA. The results indicated that the combined application of HPLC-MS/MS strychnine and Atractylodes macrocephala had a reducing effect on the strychnine content. From the subsequent experimental results, it can be inferred that Strychnine combined with Atractylodes macrocephala extract could promote the apoptosis of synovial cells, and could inhibit the expression levels of TLR4, NF-κB and NLRP3 in the cells as well as reducing the MH7A-positive cells. The expression levels of TLR4, IκB kinase ß, NF-κB and NLRP3 were significantly reduced after treatment with each administration group, resulting in a decrease in the phosphorylation levels of TLR4 and NF-κB, indicating that the combination potently inhibited their phosphorylation. The combination of strychnine and atractylenolide II was also revealed to be the main active ingredient in the treatment of RA.
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Rheumatoid arthritis (RA) is characterized by systemic inflammation and synovial hyperplasia. Pristimerin, a natural triterpenoid isolated from plants belonging to the Celastraceae and Hippocrateaceae families, has been reported to exhibit anti-inflammation and anti-proliferation activities. Our study aims to reveal the antiarthritic effects of pristimerin and explore its potential mechanism using in vitro, in silico, and in vivo methods. In the present study, pristimerin treatment led to a dose-dependent decrease in cell viability and migration in TNF-α stimulated human rheumatoid arthritis fibroblast-like synoviocytes MH7A. Moreover, UPLC-LTQ-Orbitrap-based cell metabolomics analysis demonstrated that phospholipid biosynthesis, fatty acid biosynthesis, glutathione metabolism and amino acid metabolic pathways were involved in TNF-α induced MH7A cells after pristimerin treatment. In addition, the adjuvant-induced arthritis (AIA) rat model was employed, and the results exhibited that pristimerin could effectively relieve arthritis symptoms and histopathological damage as well as reduce serum levels of TNF-α, NO and synovial expressions of p-Akt and p-Erk in AIA rats. Furthermore, network pharmacology analysis was performed to visualize crucial protein targets of pristimerin for RA treatment, which showed that the effects were mediated through the MAPK/Erk1/2, PI3K/Akt pathways and directing binding with TNF-α. Taken together, our study not only offered new insights into the biochemical mechanism of natural compounds for RA treatment, but also provided a strategy that integrated in vitro, in silico and in vivo studies to facilitate screening of new anti-RA drugs.
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The present study investigated the pharmacodynamic material basis of Laportea bulbifera in the treatment of rheumatoid arthritis. Firstly, human rheumatoid arthritis fibroblast-like synoviocyte line MH7A was cultured in vitro and treated with tumor necrosis factor alpha(TNF-α, 50 ng·mL~(-1)). The proliferation and the levels of inflammatory cytokines such as prostaglandin E2(PGE2), interleukin-1ß(IL-1ß), and interleukin-6(IL-6) of the MH7A cells exposed to the serum containing L. bulbifera were determined to evaluate the anti-rheumatoid arthritis effects of the serum. Furthermore, the ultra-performance liquid chromatography tandem mass spectrometry fingerprints of the L. bulbifera crude extract, the drug-containing serum, and the drug-free serum were compared to identify the compounds newly generated in the serum after oral administration of the extract. According to the peak areas of common peaks and the results of anti-rheumatoid arthritis effect test, the active components were identified. The serum containing L. bulbifera significantly inhibited the proliferation of the MH7A cells activated by TNF-α and the expression of PGE2, IL-6, and IL-1ß. Thirty newly generated compounds were detected in the drug-containing serum. Among them, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, rutin, isoquercitrin, luteoloside, kaempferol-3-O-rutinoside, and quercitrin were also present in the crude extract. Twelve characteristic peaks(3, 7, 8, 14, 18, 19, 21, 23, 24, m6, m7, and m15) were significantly correlated with the pharmaceutical effect. According to the correlations, neochlorogenic acid, cryptochlorogenic acid, and chlorogenic acid had great contributions to the anti-rheumatoid arthritis activity. This study preliminarily clarified the potential pharmacodynamic substances of L. bulbifera in the treatment of rheumatoid arthritis, which laid a theoretical and experimental foundation for further development and application of the medicinal plant.
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Artritis Experimental , Artritis Reumatoide , Urticaceae , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Ácido Clorogénico/análogos & derivados , Citocinas/metabolismo , Dinoprostona , Humanos , Interleucina-1beta/genética , Interleucina-6 , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Ácido Quínico/análogos & derivados , Rutina , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Urticaceae/químicaRESUMEN
Backgrounds: Rheumatoid arthritis synovial fibroblasts (RASFs) are the primary cells responsible for destruction of marginal cartilage in rheumatoid arthritis (RA). G1dP3, a bioactive peptide derived from galectin-1 domain, possesses potent anti-inflammatory and anti-proliferation properties in RASFs. This study aimed to determine the effects of G1dP3 ferroptosis induction in RASFs and to further clarify the possible mechanisms. Methods: TNF-α was used to establish a RA model in MH7A cells. Cell Counting Kit-8 assays were employed to detect MH7A cell viability with different treatments. The occurrence of ferroptosis was examined by Lipid ROS assay, cellular labile iron pool measurement, reduced glutathione/oxidized glutathione activity, Gpx4 expression and transmission electron microscopy (TEM) morphology observation. Lentiviral-mediated siRNA interference was used to determine the downstream pathway. Results: G1dP3 markedly suppressed MH7A cell viability induced by TNF-α. G1dP3-treated MH7A cells presented the morphological features of ferroptosis. Moreover, G1dP3 triggered ferroptosis in MH7A cells by promoting the accumulation of lipid peroxides as well as iron deposition. Inhibition of ferroptosis alleviated G1dP3-mediated suppression of MH7A cell viability. Furthermore, G1dP3 increased p53 expression, which in turn transcriptionally suppressed SLC7A11, a key component of system Xc - essential for ferroptosis. Knockdown of p53 abrogated the ferroptotic effects of G1dP3 on MH7A cells. Conclusion: Our findings reveal that the bioactive peptide G1dP3 promotes RASFs ferroptosis cell death via a p53/SLC7A11 axis-dependent mechanism, suggesting its potential role in the treatment of RA.
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BACKGROUND: As a prolonged autoimmune disorder, rheumatoid arthritis (RA) is characterised by synovial hyperplasia and the erosion of bone and cartilage. Magnoflorine (MAG) is the main component purified from Clematis manshurica Rupr. Recent studies have shown that MAG has anti-inflammatory, antioxidant, and immunosuppressive effects, which are relevant to anti-RA activities. OBJECTIVE: The current investigation was conducted to explore the anti-RA effects of MAG and to discover the possible molecular mechanisms. METHODS: In vitro experiments, CCK-8, wound healing, and transwell assays were utilized to evaluate the anti-proliferative, anti-migratory, and anti-invasive activities of MAG, respectively. The rate of cell distribution and cell apoptosis were evaluated by flow cytometry. ROS generation was detected by DCFH-DA staining. Western blotting, quantitative real-time polymerase chain reaction assay, and immunofluorescent staining were employed to test the anti-RA effect of MAG as well as to explore the potential mechanisms by evaluating related gene and protein expression. For in vivo experiments, an adjuvant-induced arthritis (AIA) rat model was established. The related parameters were measured in rats. Then, rats were sacrificed, and ankle joints were collected for histopathological analysis and observation. RESULTS: MAG significantly decreased the proliferation, migration, invasion, and reactive oxygen species levels in IL-1ß-treated MH7A cells. Furthermore, MAG promoted cell apoptosis by increasing Bax levels and decreasing Bcl-2 levels. MAG also induced cell cycle arrest. Inflammatory cytokines (iNOS, COX-2, IL-6, and IL-8) and MMPs (MMP-1, 2, 3, 9, and 13) were reduced by MAG treatment. Molecular analysis revealed that MAG exerted anti-RA effects by partly inhibiting the PI3K/Akt/NF-κB signalling axis and activating the Keap1-Nrf2/HO-1 signalling pathway. In vivo studies have revealed that MAG treatment substantially improved severe symptoms in AIA rats, and these curative effects were linked to the attenuation of inflammatory responses. CONCLUSION: These results first suggested that MAG exhibits anti-arthritic effects in IL-1ß-treated MH7A cells and AIA rat models. Thus, MAG may be used as a new drug to treat RA clinically.
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Artritis Reumatoide , FN-kappa B , Animales , Apoptosis , Aporfinas , Artritis Reumatoide/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , RatasRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) is an chronic autoimmune disease and characterized by high incidence. However, there is no effective therapies for RA. Therefore, it is urgent to discover new drugs for RA treatment. Nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) can effectively protect against arthritic inflammatory diseases through diverse stages, such as regulating redox balance, detoxification, metabolism and inflammation. Dimethyl fumarate (DMF), targets the Nrf2 pathway, was approved by FDA for the clinical treatment of multiple sclerosis (MS), which is another autoimmune disease. The latest report shown that DMF ameliorates complete Freund's adjuvant-induced arthritis in rats through activation of the Nrf2/HO-1 signaling pathway. Hence, Nrf2 serves as an important target for inflammation interference and oxidative stress of macrophages and RASFs in RA; therefore, it can be adopted as an effective therapeutic approach in the future. Rheumatoid arthritis synovial fibroblasts (RASFs) play crucial roles in the RA pathogenesis. Our results revealed that 7-deacetyl-gedunin (7-d-GDN), derived from fruits of Toona sinensis (A. Juss.) Roem, significantly inhibited RASFs proliferation in dose- and time- dependent manners and inhibited cell viability in MH7A cells, which is a kind of immortal cell line from joints of patients with RA. Additionally, 7-d-GDN remarkably down-regulated MMP-1/3/9/13 in RASFs, IL-6 and IL-33 in MH7A cells. Besides, 7-d-GDN sharply inhibited reactive oxygen species (ROS) in RASFs. Further mechanistic study demonstrated that 7-d-GDN induced heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1(NQO1), which all participated in suppressing of oxidative stress. Additionally, 7-d-GDN increased sequestosome 1 (SQSTM1, p62), causing down-regulating Kelch-like ECH-associated protein 1 (Keap1), which resulting in NF-E2-related factor 2 (Nrf2) cytoplasm accumulation and subsequently translocation into nucleus. Collectively, 7-d-GDN exerts the anti-inflammatory effect through regulating anti-oxidative enzymes via p62/ Nrf2/ARE signaling. All suggest that the potential of 7-d-GDN in suppression of inflammation, especially antagonizing RA severity. Our works support for drugs discovery in RA treatment.
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Artritis Reumatoide , Factor 2 Relacionado con NF-E2 , Animales , Humanos , Ratas , Artritis Reumatoide/metabolismo , Proliferación Celular , Fibroblastos , Inflamación/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Limoninas , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Transducción de SeñalRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The dried aboveground part of Geranium Wilfordii Maxim. (G. Wilfordii) is a traditional Chinese herbal medicine named lao-guan-cao. It has long been used for dispelling wind-dampness, unblocking meridians, and stopping diarrhea and dysentery. Previous investigations have revealed that 50% ethanolic extract of G. Wilfordii has anti-inflammatory and anti-proliferation activities on TNF-α induced murine fibrosarcoma L929 cells. Corilagin (COR) is a main compound in G. Wilfordii with the content up to 1.69 mg/g. Pharmacology study showed that COR has anti-inflammatory, anti-tumor, anti-microorganism, anti-oxidant, and hepatoprotective effects. However, there is no any investigation on its anti-proliferation and anti-inflammation effects in rheumatoid arthritis (RA). AIM OF THE STUDY: The present study aimed to evaluate the potential pharmacological mechanisms of anti-proliferation and anti-inflammation effects of COR in RA. MATERIALS AND METHODS: In vitro, MH7A cells model induced by IL-1ß was used. The anti-proliferation activity of COR was assessed by Cell Counting Kit-8 (CCK-8) assay, and the anti-migration and anti-invasion activity of COR was determined by wound healing assay and transwell assay, respectively. Furthermore, apoptosis assay by flow cytometer was used to measure the pro-apoptotic effect of COR. The mRNA expressions of Bax, Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS were measured by qRT-PCR, and related protein were further verified by ELISA kits or Western blot. Moreover, protein levels associated with NF-κB and MAPK signaling pathways of p65, P-p65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK1/2/3, p38, and P-p38 were determined by Western blot. The nuclear translocation of NF-κB-p65 was detected by immunofluorescent staining. In vivo, adjuvant-induced arthritis (AIA) rat model was used, and the body weight, paw swelling, and arthritis score during the entire period were measured. Histopathological analysis of joints of synovial tissues was also determined. The expression of pro-inflammatory cytokines in serum including IL-6, TNF-α, IL-1ß, and IL-17 were measured. RESULTS: The in vitro results showed that COR could dose-dependently inhibit the proliferation, migration, and invasion of IL-1ß-induced MH7A cells, as well as promote its apoptosis. Moreover, it also suppressed the over-expression of Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS while up-regulated the level of Bax. Besides, the ratios of P-p65/p65, P-IκBα/IκBα, P-ERK/ERK, P-JNK/JNK, and P-p38/p38 were decreased, and the nuclear translocation of p65 induced by IL-1ß was blocked by COR. In vivo results indicated that COR significantly reduced the paw swelling and arthritis score in AIA rats, and inhibited synovial tissue hyperplasia and erosion, as well as inflammatory cells infiltration. It also decreased the serum pro-inflammatory cytokines (IL-6, TNF-α, IL-1ß, and IL-17) production. CONCLUSION: These results revealed that COR exerted anti-rheumatoid arthritis effect, and its underlying mechanisms may be related to inhibiting the proliferation, migration, and invasion of synovial fibroblasts, enhancing cell apoptosis, and suppressing inflammatory responses via downregulating NF-κB and MAPK signaling pathways.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Glucósidos/uso terapéutico , Taninos Hidrolizables/uso terapéutico , Inflamación/inducido químicamente , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Glucósidos/química , Humanos , Taninos Hidrolizables/química , Inflamación/tratamiento farmacológico , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Estructura Molecular , FN-kappa B , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero , Ratas , Ratas WistarRESUMEN
Dehydroevodiamine (DHE) is an effective natural active substance extracted from Euodiae Fructus, which is a widely used herbal drug in traditional Chinese medicine. The focus of this study was to test the possibility of using DHE in the treatment of rheumatoid arthritis (RA) diseases. A rat model of adjuvant-induced arthritis (AIA) was generated using Complete Freund's Adjuvant (CFA). Body weight changes, arthritis scores, ankle pathology, tumor necrosis factor-alpha (TNF-α), interleukin-1ß(IL-1ß), interleukin-6 (IL-6), and interleukin-17 (IL-17) secretion, as well as matrix metalloproteinase (MMP) expression in joint tissue, were measured as indicators of viability of DHE medicated AIA rats. Human fibroblast-like synoviocytes (MH7A cells) were connected to check these impacts. The results confirmed that DHE administration had an excellent therapeutic impact on the AIA rat model, substantially relieving joint swelling, inhibiting synovial pannus hyperplasia, and decreasing joint scores. In addition, the serum enzyme-linked immunosorbent assay (ELISA) showed that DHE treatment reduced the expression of pro-inflammatory factors in AIA rats. The immunohistochemical results showed that DHE treatment could reduce the synthesis of MMPs such as matrix metalloproteinase-1(MMP-1) and matrix metalloproteinase-3 (MMP-3) in the ankle tissue of AIA rats. In vitro, DHE inhibited cell proliferation, mRNA transcription, protein synthesis of proinflammatory factors such as IL-1ßand IL-6, and matrix metalloproteinases such as MMP-1 and MMP-3. Furthermore, DHE inhibited the phosphorylation levels of p38, JNK, and ERK proteins in TNF-α-treated MH7A cells.This work assessed the effect of DHE in AIA rats and revealed its mechanism in vitro.
Asunto(s)
Alcaloides/administración & dosificación , Antiinflamatorios/administración & dosificación , Artritis Experimental/tratamiento farmacológico , Adyuvante de Freund/efectos adversos , Sinoviocitos/citología , Alcaloides/farmacología , Animales , Antiinflamatorios/farmacología , Artritis Experimental/inducido químicamente , Artritis Experimental/genética , Artritis Experimental/inmunología , Peso Corporal/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metaloproteinasas de la Matriz/metabolismo , Ratas , Sinoviocitos/efectos de los fármacos , Sinoviocitos/inmunologíaRESUMEN
ObjectiveTo observe the effects of Fuzitang (FZT) on the proliferation of MH7A cells, the human rheumatoid arthritis synovial fibroblasts, and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group, high- (25 g·L-1) and low-dose (12.5 g·L-1) FZT groups, and a positive drug group (hydroxychloroquine, 0.006 25 g·L-1). The cell proliferation was detected by cell counting kit-8(CCK-8) method, and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes, including SH2 domain-containing inositol 5-phosphatase-1(SHIP-1), protein kinase B 3(Akt3), and mammalian target of rapamycin(mTOR), was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR), and the protein expression of phosphatidylinositol 3-kinase (PI3K), Akt3, and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group, the FZT groups showed increased proportions of cells in the G2/M phase (P<0.05), and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase (P<0.05). The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group, the FZT groups showed down-regulated miR-155 and mTOR mRNA expression (P<0.05), and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression (P<0.05). Compared with the blank group, the FZT groups showed reduced protein expression of PI3K, Akt3, and mTOR (P<0.05). ConclusionFZT can significantly inhibit the proliferation of MH7A cells, and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.
RESUMEN
Rheumatoid arthritis (RA) is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes (FLS). The up-regulated cellular membrane expression of G protein coupled receptor kinase 2 (GRK2) of FLS plays a critical role in RA progression, the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor (EP4) signaling and FLS abnormal proliferation. Recently, although our group found that paeoniflorin-6'-O-benzene sulfonate (CP-25), a novel compound, could reverse FLS dysfunction via GRK2, little is known as to how GRK2 translocation activity is suppressed. Our findings revealed that GRK2 expression up-regulated and EP4 expression down-regulated in synovial tissues of RA patients and collagen-induced arthritis (CIA) rats, and prostaglandin E2 (PGE2) level increased in arthritis. CP-25 could down-regulate GRK2 expression, up-regulate EP4 expression, and improve synovitis of CIA rats. CP-25 and GRK2 inhibitors (paroxetine or GSK180736A) inhibited the abnormal proliferation of FLS in RA patients and CIA rats by down-regulating GRK2 translocation to EP4 receptor. The results of microscale thermophoresis (MST), cellular thermal shift assay, and inhibition of kinase activity assay indicated that CP-25 could directly target GRK2, increase the protein stability of GRK2 in cells, and inhibit GRK2 kinase activity. The docking of CP-25 and GRK2 suggested that the kinase domain of GRK2 might be an important active pocket for CP-25. G201, K220, K230, A321, and D335 in kinase domain of GRK2 might form hydrogen bonds with CP-25. Site-directed mutagenesis and co-immunoprecipitation assay further revealed that CP-25 down-regulated the interaction of GRK2 and EP4 via controlling the key amino acid residue of Ala321 of GRK2. Our data demonstrate that FLS proliferation is regulated by GRK2 translocation to EP4. Targeted inhibition of GRK2 kinase domain by CP-25 improves FLS function and represents an innovative drug for the treatment of RA by targeting GRK2.