RESUMO
The aggressive phenotype exhibited by fibroblast-like synoviocytes (FLSs) is critical for the progression of joint destruction in rheumatoid arthritis (RA). Long noncoding RNAs (lncRNAs) have crucial roles in the pathogenesis of diverse disorders; however, few have been identified that might be able to control the joint damage in RA. In this study, we identified an lncRNA, ENST00000509194, which was expressed at abnormally high levels in FLSs and synovial tissues from patients with RA. ENST00000509194 positively modulates the migration and invasion of FLSs by interacting with human Ag R (HuR, also called ELAVL1), an RNA-binding protein that mainly stabilizes mRNAs. ENST00000509194 binds directly to HuR in the cytoplasm to form a complex that promotes the expression of the endocytic adaptor protein APPL2 by stabilizing APPL2 mRNA. Knockdown of HuR or APPL2 impaired the migration and invasion of RA FLSs. Given its close association with HuR and FLS migration, we named ENST00000509194 as HAFML (HuR-associated fibroblast migratory lncRNA). Our findings suggest that an increase in synovial HAFML might contribute to FLS-mediated rheumatoid synovial aggression and joint destruction, and that the lncRNA HAFML might be a potential therapeutic target for dysregulated fibroblasts in a wide range of diseases.
Assuntos
Artrite Reumatoide , RNA Longo não Codificante , Sinoviócitos , Humanos , Sinoviócitos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Membrana Sinovial/patologia , Artrite Reumatoide/patologia , Movimento Celular/genética , Fibroblastos/metabolismo , Células Cultivadas , Proliferação de CélulasRESUMO
BACKGROUND: Given the traditional acceptance of higher central venous pressure (CVP) levels, clinicians ignore the incidence of acute kidney injury (AKI). The objective of this study was to assess whether elevated CVP is associated with increased AKI in critically ill patients with multiple comorbidities. METHODS: This was a retrospective observational cohort study using data collected from the Medical Information Mart for Intensive Care (MIMIC)-III open-source clinical database (version 1.4). Critically ill adult patients with CVP and serum creatinine measurement records were included. Linear and multivariable logistic regression were performed to determine the association between elevated CVP and AKI. RESULTS: A total of 11,135 patients were enrolled in our study. Critically ill patients in higher quartiles of mean CVP presented greater KDIGO AKI severity stages at 2 and 7 days. Linear regression showed that the CVP quartile was positively correlated with the incidence of AKI within 2 (R2 = 0.991, P = 0.004) and 7 days (R2 = 0.990, P = 0.005). Furthermore, patients in the highest quartile of mean CVP exhibited an increased risk of AKI at 7 days than those in the lowest quartile of mean CVP with an odds ratio of 2.80 (95% confidence interval: 2.32-3.37) after adjusting for demographics, treatments and comorbidities. The adjusted odds of AKI were 1.10 (95% confidence interval: 1.08-1.12) per 1 mmHg increase in mean CVP. CONCLUSIONS: Elevated CVP is associated with an increased risk of AKI in critically ill patients with multiple comorbidities. The optimal CVP should be personalized and maintained at a low level to avoid AKI in critical care settings.
Assuntos
Injúria Renal Aguda/complicações , Injúria Renal Aguda/fisiopatologia , Pressão Venosa Central , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Estado Terminal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Rheumatoid arthritis (RA) is featured by erosive cartilage and bone destruction. The enhancing aggressive property of fibroblast-like synoviocytes (FLSs) plays a critical role in this process. Small ubiquitin-like modifier (SUMO) proteins, including SUMO-1, SUMO-2, SUMO-3 and SUMO-4, participate in regulating many cellular events such as survival, migration and signal transduction in some cell lines. However, their roles in the pathogenesis of RA are not well established. Therefore, we evaluated the role of SUMO proteins in RA FLSs migration and invasion. We found that expression of both SUMO-1 and SUMO-2 was elevated in FLSs and synovial tissues (STs) from patients with RA. SUMO-1 suppression by small interference RNA (siRNA) reduced migration and invasion as well as MMP-1 and MMP-3 expression in RA FLSs. We also demonstrated that SUMO-1 regulated lamellipodium formation during cell migration. To explore further into molecular mechanisms, we evaluated the effect of SUMO-1 knockdown on the activation of Rac1/PAK1, a critical signaling pathway that controls cell motility. Our results indicated that SUMO-1-mediated SUMOylation controlled Rac1 activation and modulated downstream PAK1 activity. Inhibition of Rac1 or PAK1 also decreased migration and invasion of RA FLSs. Our findings suggest that SUMO-1 suppression could be protective against joint destruction in RA by inhibiting aggressive behavior of RA FLSs.
Assuntos
Artrite Reumatoide/genética , Movimento Celular/genética , Invasividade Neoplásica/genética , Proteína SUMO-1/genética , Artrite Reumatoide/patologia , Proliferação de Células/genética , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Invasividade Neoplásica/patologia , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Ubiquitinas/genética , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
OBJECTIVES: Recent studies have indicated that piperlongumine (PLM) may exert anti-inflammatory effects. In the present study, we determined the effect of PLM on the proliferation, apoptosis, migration and invasion of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) (referred to herein as RA FLS). We further explored the mechanisms by which the studied compound inhibits the functions of RA FLS. METHODS: RA FLS viability and apoptosis were tested using MTT and Annexin V/PI assays, respectively. We performed an EDU assay to examine the proliferation of RA FLS. The migration and invasion of these cells were measured using a transwell chamber method and wound closure assay. The MMP-1, MMP-3, and MMP-13 levels in the culture supernatants of RA FLS were detected using a Luminex Assay kit. The intracellular ROS levels were detected using DCFH-DA. The expression levels of signal transduction proteins were measured using western blot. RESULTS: We found that PLM induced apoptosis in RA FLS at concentrations of 15 and 20 µM. The proliferation of RA FLS was downregulated by PLM at concentrations of 1, 5 and 10 µM. Migration and invasion of RA FLS were reduced by PLM at concentrations of 1, 5 and 10 µM. PLM also inhibited cytoskeletal reorganization in migrating RA FLS and decreased TNF-α-induced intracellular ROS production. Moreover, we demonstrated the inhibitory effect of PLM on activation of the p38, JNK, NF-κB and STAT3 pathways. CONCLUSIONS: Our findings suggest that PLM can inhibit proliferation, migration and invasion of RA FLS. Moreover, these data suggests that PLM might have therapeutic potential for the treatment of RA.
Assuntos
Dioxolanos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sinoviócitos/efeitos dos fármacos , Idoso , Apoptose/efeitos dos fármacos , Artrite Reumatoide/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinoviócitos/metabolismo , Sinoviócitos/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Piperlongumine (PLM) is a natural product from the plant Piper longum that inhibits platelet aggregation, atherosclerosis plaque formation, and tumor cell growth. It has potential value in immunomodulation and the management of autoimmune diseases. In this study, we investigated the role of PLM in regulating the differentiation and maturation of dendritic cells (DCs), a critical regulator of immune tolerance, and evaluated its clinical effects in a rheumatoid arthritis mouse model. We found that PLM treatment reduced LPS-induced murine bone marrow-derived DC maturation, characterized by reduced expression of CD80/86, secretion of MCP-1, IL-12p70, IL-6, TNFα, IFN-γ, and IL-23, and reduced alloproliferation of T cells; however, PLM does not affect cell differentiation. Furthermore, PLM reduced intracellular reactive oxygen species (ROS) production by DCs and inhibited the activation of p38, JNK, NF-κB, and PI3K/Akt signaling pathways. Conversely, PLM increased the expression of GSTP1 and carbonyl reductase 1, two enzymes that counteract ROS effects. ROS inhibition by exogenous N-acetyl-l-cysteine suppressed DC maturation. PLM treatment improved the severity of arthritis and reduced in vivo splenic DC maturation, collagen-specific CD4(+) T cell responses, and ROS production in mice with collagen-induced arthritis. Taken together, these results suggest that PLM inhibits DC maturation by reducing intracellular ROS production and has potential as a therapeutic agent for rheumatoid arthritis.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Dioxolanos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Oxirredutases do Álcool/genética , Animais , Artrite Experimental/tratamento farmacológico , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno/administração & dosagem , Citocinas/imunologia , Citocinas/metabolismo , Dioxolanos/administração & dosagem , Modelos Animais de Doenças , Glutationa S-Transferase pi/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) is a critical factor of cartilage destruction in rheumatoid arthritis (RA). Increased FLSs migration and subsequent degradation of the extracellular matrix are essential to the pathology of RA. Protein inhibitor of activated STAT (PIAS), whose family members include PIAS1, PIAS2 (PIASx), PIAS3, and PIAS4 (PIASy), play important roles in regulating various cellular events, such as cell survival, migration, and signal transduction in many cell types. However, whether PIAS proteins have a role in the pathogenesis of RA is unclear. In this study, we evaluated the role of PIAS proteins in FLSs migration, invasion, and matrix metalloproteinases (MMPs) expression in RA. We observed increased expression of PIAS3, but not PIAS1, PIAS2, or PIAS4, in FLSs and synovial tissues from patients with RA. We found that PIAS3 knockdown by short hairpin RNA reduced migration, invasion, and MMP-3, MMP-9, and MMP-13 expression in FLSs. In addition, we demonstrated that PIAS3 regulated lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of PIAS3 knockdown on Rac1/PAK1 and JNK activation. Our results indicated that PIAS3-mediated SUMOylation of Rac1 controlled its activation and modulated the Rac1 downstream activity of PAK1 and JNK. Furthermore, inhibition of Rac1, PAK1, or JNK decreased migration and invasion of RA FLSs. Thus, our observations suggest that PIAS3 suppression may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, and activation.
Assuntos
Artrite Reumatoide/patologia , Movimento Celular , Fibroblastos/patologia , Chaperonas Moleculares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Membrana Sinovial/patologia , Adulto , Idoso , Artrite Reumatoide/metabolismo , Western Blotting , Feminino , Fibroblastos/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Membrana Sinovial/metabolismoRESUMO
OBJECTIVES: To evaluate the inhibition of indirubin in FLSs migration, invasion, activation, and proliferation in RA FLSs. METHODS: The levels of IL-6 and IL-8 in cultural supernatants were measured by ELISA. RA FLS migration and invasion in vitro were measured by the Boyden chamber method and the scratch assay. Signal transduction protein expression was measured by western blot. FLS proliferation was detected by Edu incorporation. F-actin was measured by immunofluorescence staining. RESULTS: We found that indirubin reduced migration, invasion, inflammation, and proliferation in RA FLSs. In addition, we demonstrated that indirubin inhibited lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of indirubin on PAK1 and MAPK activation. Our results indicated that indirubin inhibited the activity of PAK1 and MAPK. CONCLUSIONS: Our observations suggest that indirubin may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, activation, and proliferation.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Reumatoide/metabolismo , Sinoviócitos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Humanos , Indóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA Mensageiro/metabolismo , Sinoviócitos/fisiologia , Quinases Ativadas por p21/metabolismoRESUMO
OBJECTIVE: To explore the roles of the bromodomain (Brd) and extra-terminal domain (BET) of chromatin adaptors in regulating synovial inflammation in RA. METHODS: Fibroblast-like synoviocytes (FLSs) were isolated from synovial tissue from RA patients. A specific BET inhibitor, JQ1, and short hairpin RNA (shRNA) for Brd2 or Brd4 were used to evaluate the role of the BET Brd in inflammatory responses. Protein expression was measured by western blot or immunofluorescence staining. Nuclear factor kappa B (NF-κB) gene activity was detected by luciferase assay. The secretion and gene expression of cytokines and MMPs were evaluated by ELISA and real-time PCR, respectively. FLS proliferation was detected by BrdU incorporation. RESULTS: Four Brd proteins, including Brd2, Brd3, Brd4 and Brdt, were expressed in FLSs from patients with RA and OA; however, the expression of Brd2 and Brd4 was increased in RA compared with that in OA. Treatment with JQ1, Brd2 shRNA or Brd4 shRNA decreased the production of pro-inflammatory cytokines (TNFα, IL-1ß, IL-6 and IL-8), MMPs expression (MMP-1, MMP-3 and MMP-13) and proliferation by RA FLSs. BET inhibition downregulated TNFα-induced NF-κB-dependent transcription and expression of the NF-κB target genes. JQ1 suppressed the phosphorylation of IκB kinaseß and IκBα, and nuclear translocation of p65. Intraperitoneal injection of JQ1 in mice with collagen-induced arthritis reduced synovial inflammation, joint destruction and serum levels of the anti-CII antibodies TNFα and IL-6. CONCLUSION: This study implicates BET Brds as important regulators of IκB kinase/NF-κB-mediated synovial inflammation of RA and identifies BET proteins as novel therapeutic targets in inflammatory arthritis.
Assuntos
Artrite Reumatoide/genética , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Membrana Sinovial/metabolismo , Fatores de Transcrição/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Proteínas de Ciclo Celular , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Membrana Sinovial/patologia , Fatores de Transcrição/biossínteseRESUMO
OBJECTIVES: This study evaluated the anti-inflammatory effect of niclosamide in tumor necrosis factor (TNF)-α-stimulated human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and inhibitory effects on migration and invasion in RA FLS and investigated the signal mechanism, and further explored the treatment activity of niclosamide on collagen-induced arthritis (CIA). METHODS: The levels of interleukin (IL)-1ß, IL-6, IL-8, IL-10,IL-17A and interferon (IFN)-γ in cultural supernatants were measured by multiplex cytokine assay kits. RA FLS migration and invasion in vitro were measured by the Boyden chamber method and the scratch assay. Signal transduction proteins expression was measured by western blot. The in vivo suppressive effects of niclosamide were elucidated on CIA in a mouse model. RESULTS: Niclosamide reduced the secretion of IL-1ß, IL-6, IL-8, IL-17A and IFN-γ from TNF-α-induced RA FLS in a dose-dependent manner. Niclosamide inhibits FBS-induced migration and invasion and exhibits F-actin alterations in RA FLS. Niclosamide decreased the phosphorylation of c-Jun N-terminal kinase and ERK in TNF-α-stimulated RA FLS and blocked TNF-α-induced IKK, IκBα phosphorylation and translocation of p65. Niclosamide treatments reduced the severity of CIA model. CONCLUSIONS: Our data suggest for the first time that niclosamide posses the anti-inflammatory effect in RA both in vitro and in vivo.
Assuntos
Artrite Reumatoide/prevenção & controle , Movimento Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Inflamação/prevenção & controle , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Membrana Sinovial/efeitos dos fármacos , Animais , Anticestoides/farmacologia , Anticestoides/uso terapêutico , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Experimental/prevenção & controle , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Técnicas In Vitro , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: Niclosamide is known to have anti-cancer and anti-inflammatory activities; however, its therapeutic mechanism has not been defined. In this study, to explain the therapeutic mechanism of niclosamide, we examined the effect of niclosamide on endothelial cell activation,leukocyte integration, proliferation, migration and angiogenesis in vitro. METHODS: Endothelia-leukocyte adhesion assays were used to assess primary cultures of human umbilical vein endothelial cells' (HUVECs) activation following TNF-α treatment. Each step of angiogenesis was evaluatedin vitro, including endothelial cell proliferation, migration and tube formation. Proliferation was examined using EdU assays, while wound migration assays and transwell assays were used to evaluate cell migration; cord like structure formation assays on Matrigel were used to assess tube formation. In vivo matrigel plug assay was used to assess angiogenesis. The protein expression was measured using western blot. RESULTS: Niclosamide reduced the adhesion of human monocyte cells to HUVECs. Niclosamide also reduced protein expression of VCAM-1 and ICAM1 in HUVECs.Niclosamide significantly inhibited HUVEC proliferation,migration and cord-like structure formation. Niclosamide also suppresses VEGF-induced angiogenesis in vivo.Niclosamide attenuated IKK-mediated activation of NF-κB pathway in TNFα-induced endothelial cells. Niclosamide also suppresses VEGF-induced endothelial VEGFR2 activation and downstream P-AKT, P-mTOR and P-p70S6K. CONCLUSIONS: Niclosamide exerted a potent effect on HUVECs activation, suggesting that it might function via an endothelia-based mechanism in the treatment of various diseases, including rheumatoid arthritis and cancer.
Assuntos
Indutores da Angiogênese/farmacologia , Anti-Inflamatórios/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Neovascularização Patológica/prevenção & controle , Niclosamida/farmacologia , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Leucócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
OBJECTIVE: Increasing evidence indicates that the cytoskeletal protein ezrin may play a critical role in cell motility. This study aims to investigate the role of ezrin in regulating the migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with RA. METHODS: Synovial tissues were obtained from 12 patients with RA and 6 with OA, and then FLSs were separated from synovial tissues. The expression of ezrin and phosphorylated ezrin (p-ezrin) was examined by Western blotting or IF staining. A specific inhibitor of ezrin phosphorylation and small interference RNA-mediated ezrin knockdown were used to inhibit the phosphorylation of ezrin. Migration and invasion of FLSs in vitro were measured by the Boyden chamber assay. RESULTS: Increased expression of p-ezrin protein was found in synovial tissue and FLSs in patients with RA compared with patients with OA. Stimulation with TNF-α and IL-1ß increased ezrin phosphorylation in RA FLSs. Inhibition of p-ezrin protein by a specific inhibitor of phosphorylation of ezrin and small interfering RNA-mediated knockdown reduced in vitro migration and invasion, as well as actin stress fibre formation in RA FLS. Furthermore, rho kinase and p38 mitogen-activated protein kinase (MAPK) signal pathways were involved in the phosphorylation of ezrin and invasion of RA FLSs. CONCLUSION: Increased expression of p-ezrin may contribute to aberrant aggressive behaviours of RA FLSs, which are mediated by rho kinase and the p38 MAPK pathway. This suggests a novel strategy targeting phosphorylation of ezrin to prevent synovial invasiveness and joint destruction in RA.
Assuntos
Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/patologia , Membrana Sinovial/patologia , Adulto , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Interleucina-1beta/farmacologia , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
AIMS: To explore the synovial expression of mucin 1 (MUC1) and its role in rheumatoid arthritis (RA), as well as the possible downstream mechanisms. METHODS: Patients with qualified synovium samples were recruited from a RA cohort. Synovium from patients diagnosed as non-inflammatory orthopaedic arthropathies was obtained as control. The expression and localization of MUC1 in synovium and fibroblast-like synoviocytes were assessed by immunohistochemistry and immunofluorescence. Small interfering RNA and MUC1 inhibitor GO-203 were adopted for inhibition of MUC1. Lysophosphatidic acid (LPA) was used as an activator of Rho-associated pathway. Expression of inflammatory cytokines, cell migration, and invasion were evaluated using quantitative real-time polymerase chain reaction (PCR) and Transwell chamber assay. RESULTS: A total of 63 RA patients and ten controls were included. Expression of MUC1 was observed in both the synovial lining and sublining layer. The percentage of MUC1+ cells in the lining layer of synovium was significantly higher in RA than that in control, and positively correlated to joint destruction scores of RA. Meanwhile, MUC1+ cells in the sublining layer were positively correlated to the Krenn subscore of inflammatory infiltration. Knockdown of MUC1, rather than GO-203 treatment, ameliorated the expression of proinflammatory cytokines, cell migration, and invasion of rheumatoid synoviocytes. Knockdown of MUC1 decreased expression of RhoA, Cdc42, and Rac1. Treatment with LPA compromised the inhibition of migration and invasion, but not inflammation, of synoviocytes by MUC1 knockdown. CONCLUSION: Upregulated MUC1 promotes the aggression of rheumatoid synoviocytes via Rho guanosine triphosphatases (GTPases), thereby facilitating synovitis and joint destruction during the pathological process of RA.Cite this article: Bone Joint Res 2022;11(9):639-651.
RESUMO
BACKGROUND: Fibroblast-like synoviocytes (FLSs), which can migrate and directly invade the cartilage and the bone, are crucial players in joint damage in rheumatoid arthritis (RA). Nevertheless, the detailed mechanisms underlying the aberrant activation of RA FLSs remain unclear. Several studies have attempted to explore the relationship between long non-coding RNAs (lncRNAs) and RA pathology; however, the role of lncRNAs in RA is unknown. The present study aimed to determine the functions of tumor necrosis factor-α and heterogeneous nuclear ribonucleoprotein L-related immunoregulatory lincRNA (THRIL) in RA FLSs migration and invasion. METHODS: Small interfering RNA targeting THRIL or lentivirus overexpressing THRIL was used to knockdown or overexpress THRIL. Quantitative reverse transcription polymerase chain reaction (PCR) was employed for the detection of RNA expression. The proliferation rate of RA FLSs was measured using a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Migration and invasion were detected using a transwell chamber. Downstream targets were identified using a human cell cycle real-time PCR array and a human cell motility real-time PCR array. RESULTS: A significant decrease in THRIL expression was found in RA FLSs compared with cells from healthy control (HC)patients. THRIL is mainly localized in the nucleus. Knockdown of THRIL increased the proliferation, migration, and invasion of RA FLSs. In contrast, THRIL overexpression had the opposite effect. THRIL knockdown increased interleukin-1ß (IL-1ß)-triggered expression of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13. THRIL overexpression led to a significant decrease in MMP-13 expression in response to stimulation with IL-1ß. Furthermore, we observed that the expression levels of cyclin-dependent kinase 1 (CDK1) and G2 and S phase-expressed-1 (GTSE1), both of which are associated with cellular mobility and proliferation, were downregulated with THRIL overexpression. CONCLUSIONS: Reduced expression of lncRNA THRIL represses the proliferation, migration, and invasion of RA FLSs, suggesting that lncRNA THRIL might be a potential target for RA therapy.
RESUMO
Synovium fibroblast-like synoviocytes (FLSs) are important participants in the pathogenesis of synovitis and joint destruction in rheumatoid arthritis (RA). Pyroptosis is a pro-inflammatory and cell lytic programmed cell death mechanism mediated by gasdermin (GSDM) family proteins. In this study, we demonstrated the increased expression of GSDME and increased levels of GSDME-mediated pyroptosis in RA synovial tissues. In vitro, stimulation with TNF-α plus hypoxia mimicking the inflammatory and hypoxic environment in RA synovium induced GSDME-mediated pyroptosis in RA-FLSs in combination with the promotion of migration and invasion abilities and the release of inflammatory cytokines (IL-6, IL-8). Moreover, knockdown of GSDME significantly inhibited the proliferation rate, migration/invasion effects and cytokines released through the reduction of GSDME-mediated pyroptosis. The immunohistochemistry results showed that RA patients with high GSDME N-terminal (GSDME-NT) expression, which is the active form of GSDME, showed higher IL-6 expression in both lining and sublining layer of synovium than that in patients with low GSDME-NT expression, osteoarthritis and non-inflammatory orthopedic arthropathies. Our findings revealed a novel mechanism regulating cell proliferation, migration, invasion and inflammatory cytokines release during the process of GSDME mediated pyroptosis in RA.
RESUMO
OBJECTIVE: Nitidine chloride (NC), a natural small molecular compound from traditional Chinese herbal medicine zanthoxylum nitidum, has been shown to exhibit anti-tumor effect. However, its role in autoimmune diseases such as rheumatoid arthritis (RA) is unknown. Here, we investigate the effect of NC in controlling fibroblast-like synoviocytes (FLS)-mediated synovial inflammation and joint destruction in RA and further explore its underlying mechanism(s). METHODS: FLSs were separated from synovial tissues obtained from patients with RA. Protein expression was analyzed by Western blot or immunohistochemistry. Gene expression was measured using quantitative RT-PCR. ELISA was used to measure the levels of cytokines and MMPs. Cell proliferation was detected using EdU incorporation. Migration and invasion were evaluated by Boyden chamber assay. RNA sequencing analysis was used to identify the target of NC. Collagen-induced arthritis (CIA) model was used to evaluate the in vivo effect of NC. RESULTS: NC treatment reduced the proliferation, migration, invasion, and lamellipodia formation but not apoptosis of RA FLSs. We also demonstrated the inhibitory effect of NC on TNF-α-induced expression and secretion of IL-6, IL-8, CCL-2, MMP-1 and MMP-13. Furthermore, we identified KCNH1, a gene that encodes ether-à-go-go-1 channel, as a novel targeting gene of NC in RA FLSs. KCNH1 expression was increased in FLSs and synovial tissues from patients with RA compared to healthy controls. KCNH1 knockdown or NC treatment decreased the TNF-α-induced phosphorylation of AKT. Interestingly, NC treatment ameliorated the severity of arthritis and reduced synovial KCNH1 expression in mice with CIA. CONCLUSIONS: Our data demonstrate that NC treatment inhibits aggressive and inflammatory actions of RA FLSs by targeting KCNH1 and sequential inhibition of AKT phosphorylation. Our findings suggest that NC might control FLS-mediated rheumatoid synovial inflammation and joint destruction, and be a novel therapeutic agent for RA.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Benzofenantridinas/farmacologia , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Membrana Sinovial/efeitos dos fármacos , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Benzofenantridinas/uso terapêutico , Células Cultivadas , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Cultura Primária de Células , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/imunologiaRESUMO
Objectives: This study aims to investigate if addition of fibroblast-stromal cell markers to a classification of synovial pathotypes improves their predictive value on clinical outcomes in rheumatoid arthritis (RA). Methods: Active RA patients with a knee needle synovial biopsy at baseline and finished 1-year follow-up were recruited from a real-world prospective cohort. Positive staining for CD20, CD38, CD3, CD68, CD31, and CD90 were scored semiquantitatively (0-4). The primary outcome was radiographic progression defined as a minimum increase of 0.5 units of the modified total Sharp score from baseline to 1 year. Results: Among 150 recruited RA patients, 123 (82%) had qualified synovial tissue. Higher scores of CD20+ B cells, sublining CD68+ macrophages, CD31+ endothelial cells, and CD90+ fibroblasts were associated with less decrease in disease activity and greater increase in radiographic progression. A new fibroblast-based classification of synovial pathotypes giving more priority to myeloid and stromal cells classified samples as myeloid-stromal (57.7%, 71/123), lymphoid (31.7%, 39/123), and paucicellular pathotypes (10.6%, 13/123). RA patients with myeloid-stromal pathotype showed the highest rate of radiographic progression (43.7% vs. 23.1% vs. 7.7%, p = 0.011), together with the lowest rate of Boolean remission at 3, 6, and 12 months. Baseline synovial myeloid-stromal pathotype independently predicted radiographic progression at 1 year (adjusted OR: 3.199, 95% confidence interval (95% CI): 1.278, 8.010). Similar results were obtained in a subgroup analysis of treatment-naive RA. Conclusions: This novel fibroblast-based myeloid-stromal pathotype could predict radiographic progression at 1 year in active RA patients which may contribute to the shift of therapeutic decision in RA.
Assuntos
Antígenos CD/análise , Artrite Reumatoide/imunologia , Fibroblastos/imunologia , Imuno-Histoquímica , Articulação do Joelho/imunologia , Células Estromais/imunologia , Membrana Sinovial/imunologia , Adulto , Antirreumáticos/uso terapêutico , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Biomarcadores/análise , Biópsia por Agulha , Progressão da Doença , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/patologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Indução de Remissão , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Membrana Sinovial/diagnóstico por imagem , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Autoimmune diseases are a group of heterogeneous disorders characterized by damage to various organs caused by abnormal innate and adaptive immune responses. The pathogenesis of autoimmune diseases is extremely complicated and has not yet been fully elucidated. Long noncoding RNAs (lncRNAs), which are defined as transcripts containing more than 200 nucleotides with no protein-coding capacity, are emerging as important regulators of gene expression via epigenetic modification, transcriptional regulation and posttranscriptional regulation. Accumulating evidence has demonstrated that lncRNAs play a key role in the regulation of immunological functions and autoimmunity. In this review, we discuss various molecular mechanisms by which lncRNAs regulate gene expression and recent findings regarding the involvement of lncRNAs in many human autoimmune diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), idiopathic inflammatory myopathy (IIM), systemic sclerosis (SSc) and Sjögren's syndrome (pSS).
RESUMO
Fibroblast-like synoviocytes (FLSs) are critical to joint inflammation and destruction in rheumatoid arthritis (RA). Increased glycolysis in RA FLSs contributes to persistent joint damage. SUMOylation, a posttranslational modification of proteins, plays an important role in initiation and development of many diseases. However, the role of small ubiquitin-like modifier-activating (SUMO-activating) enzyme 1 (SAE1)/ubiquitin like modifier activating enzyme 2 (UBA2) in regulating the pathogenic FLS behaviors is unknown. Here, we found an increased expression of SAE1 and UBA2 in FLSs and synovial tissues from patients with RA. SAE1 or UBA2 knockdown by siRNA and treatment with GA, an inhibitor of SAE1/UBA2-mediated SUMOylation, resulted in reduced glycolysis, aggressive phenotype, and inflammation. SAE1/UBA2-mediated SUMOylation of pyruvate kinase M2 (PKM2) promoted its phosphorylation and nuclear translocation and decreased PK activity. Moreover, inhibition of PKM2 phosphorylation increased PK activity and suppressed glycolysis, aggressive phenotype, and inflammation. We further demonstrated that STAT5A mediated SUMOylated PKM2-induced glycolysis and biological behaviors. Interestingly, GA treatment attenuated the severity of arthritis in mice with collagen-induced arthritis and human TNF-α transgenic mice. These findings suggest that an increase in synovial SAE1/UBA2 may contribute to synovial glycolysis and joint inflammation in RA and that targeting SAE1/UBA2 may have therapeutic potential in patients with RA.
Assuntos
Artrite Reumatoide/patologia , Fibroblastos/patologia , Glicólise , Proteína SUMO-1/metabolismo , Sinoviócitos/patologia , Enzimas Ativadoras de Ubiquitina/metabolismo , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fosforilação , Proteína SUMO-1/genética , Transdução de Sinais , Sinoviócitos/metabolismo , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
OBJECTIVE: To determine the prognostic effect of adjuvant radiation and clinicopathological variables in surgically treated patients with small cell carcinoma of the cervix (SCCC). METHODS: Clinical data of SCCC patients with International Federation of Gynaecology and Obstetrics (FIGO) stage I-II underwent radical surgery from May 2000 to August 2014 at Sun Yat-sen Memorial Hospital were retrospectively reviewed. Forty-three patients with SCCC were included to this study. Chi-square test or Fisher's exact test, Student's t test or Mann-Whitney U test, Kaplan-Meier method and multivariate analysis of Cox proportional hazards regression were used for statistical analysis. P < 0.05 was considered to be statistically significant. RESULTS: Among 43 patients (median age, 49 years old) recruited, 25(58.1%) had stage I, 18(41.9%) had stage II disease. The 5-year overall survival (OS) rate was 39.54%, and the 5-year disease free survival (DFS) was 27.91%. Distant metastasis was the main cause of treatment failure (71.9%). Patients with adjuvant chemoradiation displayed lower rate of local recurrence than those with adjuvant chemotherapy (10.7% vs 60.0%, P < 0.0001). Multivariable analysis identified lymph node metastasis as a significant prognostic factor for both DFS and OS (P = 0.001, 0.004 respectively). Age was also an independent predictor of OS (P = 0.004). Adjuvant radiation appeared to significantly improve DFS (HR = 0.383, 95% CI, 0.185-0.791), but not OS. CONCLUSIONS: Adjuvant radiotherapy could improve the local control and prolong DFS in surgically treated SCCC. However, a large prospective clinical trial is needed to confirm this.
Assuntos
Carcinoma de Células Pequenas/radioterapia , Carcinoma de Células Pequenas/cirurgia , Neoplasias do Colo do Útero/radioterapia , Neoplasias do Colo do Útero/cirurgia , Adulto , Terapia Combinada , Feminino , Humanos , Histerectomia , Estimativa de Kaplan-Meier , Linfonodos/patologia , Metástase Linfática , Pessoa de Meia-Idade , Análise Multivariada , Metástase Neoplásica , Recidiva Local de Neoplasia/patologia , Período Pós-Operatório , Prognóstico , Modelos de Riscos Proporcionais , Radioterapia Adjuvante , Recidiva , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The accumulation of cytosolic dsDNA plays important roles in the regulation of cellular processes. However, whether cytosolic dsDNA is involved in the pathogenesis of rheumatoid arthritis (RA) is not clear. Therefore, the present study investigated the roles of cytosolic dsDNA in the modulation of inflammatory responses of fibroblast-like synoviocytes (FLS) in patients with RA. FLS were obtained from active RA patients. dsDNA accumulation in the cytosol was detected by immunofluorescence staining and the Qubit® dsDNA HS Assay. Immunohistochemistry was employed to detect the dsDNA and cGMP-AMP synthase (cGAS) expression in the synovium. Short hairpin RNA (shRNA) was used to knockdown the expression of cGAS and stimulator of interferon genes (STING). Protein expression was detected by Western blotting and immunofluorescence staining. We observed increased cytosolic dsDNA and cGAS expression in FLS and synovium from RA patients. dsDNA and cGAS expression correlated with the severity of rheumatoid synovitis. Transfection of dsDNA into the cytosol of RA FLS promoted pro-inflammatory cytokines production. DNaseII overexpression downregulated cytosolic dsDNA expression and inhibited dsDNA-induced cytokines secretion. We also found that dsDNA and TNF-α enhanced cGAS and STING expression, and dsDNA-induced cytokine secretion was reduced by cGAS or STING knockdown. Furthermore, we determined that the dsDNA-induced phosphorylation of IRF3 and NF-κBp65 was decreased by DNaseII overexpression or cGAS/STING knockdown. Overall, our findings show that increased cytosolic dsDNA level promoted inflammatory responses via the cGAS/STING pathway in RA FLS, which suggests that cytosolic dsDNA accumulation is an important contributor to FLS-mediated rheumatoid synovial inflammation.