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1.
BMC Biol ; 17(1): 89, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31722692

RESUMO

BACKGROUND: Cardiac differentiation from human pluripotent stem cells provides a unique opportunity to study human heart development in vitro and offers a potential cell source for cardiac regeneration. Compared to the large body of studies investigating cardiac maturation and cardiomyocyte subtype-specific induction, molecular events underlying cardiac lineage commitment from pluripotent stem cells at early stage remain poorly characterized. RESULTS: In order to uncover key molecular events and regulators controlling cardiac lineage commitment from a pluripotent state during differentiation, we performed single-cell RNA-Seq sequencing and obtained high-quality data for 6879 cells collected from 6 stages during cardiac differentiation from human embryonic stem cells and identified multiple cell subpopulations with distinct molecular features. Through constructing developmental trajectory of cardiac differentiation and putative ligand-receptor interactions, we revealed crosstalk between cardiac progenitor cells and endoderm cells, which could potentially provide a cellular microenvironment supporting cardiac lineage commitment at day 5. In addition, computational analyses of single-cell RNA-Seq data unveiled ETS1 (ETS Proto-Oncogene 1) activation as an important downstream event induced by crosstalk between cardiac progenitor cells and endoderm cells. Consistent with the findings from single-cell analysis, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) against ETS1 revealed genomic occupancy of ETS1 at cardiac structural genes at day 9 and day 14, whereas ETS1 depletion dramatically compromised cardiac differentiation. CONCLUSION: Together, our study not only characterized the molecular features of different cell types and identified ETS1 as a crucial factor induced by cell-cell crosstalk contributing to cardiac lineage commitment from a pluripotent state, but may also have important implications for understanding human heart development at early embryonic stage, as well as directed manipulation of cardiac differentiation in regenerative medicine.

2.
Int Immunopharmacol ; 76: 105791, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31472320

RESUMO

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.

3.
Exp Cell Res ; 375(1): 52-61, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30562482

RESUMO

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.

4.
Clin Rheumatol ; 2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30488367

RESUMO

OBJECTIVES: The standard strategy for treating lupus nephritis comprises glucocorticoids together with either intravenous cyclophosphamide or oral mycophenolate mofetil, but the low remission rate is still a challenge in practice. This study was aimed to seek higher remission rate of lupus nephritis using a combined strategy. METHOD: A 24-week trial was conducted in 17 rheumatology or nephrology centers in China. A total of 191 lupus nephritis patients were randomized to follow a combined immunosuppressive treatment (CIST) with intravenous cyclophosphamide, an oral immunosuppressive agent, namely mycophenolate mofetil, azathioprine or leflunomide, and hydroxychloroquine (n = 95), or receive intravenous cyclophosphamide alone (n = 96) for 24 weeks. Glucocorticoid was given to both groups. The primary end point was a complete remission with a most stringent standard as proteinuria < 150 mg per 24 h, normal urinary sediment, serum albumin, and renal function at 24 weeks. The secondary end point was treatment failure at 24 weeks. RESULTS: At week 24, both the rate of complete remission (39.5%) and total response (87.2%) was higher in the combined group, compared with CYC group (20.8% and 68.8%, p < 0.05). The cumulative probability of complete remission was also higher in the combined group (p = 0.013). In addition, the combined treatment was superior to routine CYC with less treatment failure (12.8% vs.31.2%, p < 0.001). No difference was found between the incidences of severe adverse events in the two arms: 3.2% (3/95 combined group) vs.4.2% (4/96 CYC group). CONCLUSION: Treatment with a combined immunosuppressive agent is superior to routine CYC only therapy in lupus nephritis.

5.
Nat Commun ; 9(1): 4291, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327464

RESUMO

Cell-free DNA (cfDNA) released from damaged or dead cells can activate DNA sensors that exacerbate the pathogenesis of rheumatoid arthritis (RA). Here we show that ~40 nm cationic nanoparticles (cNP) can scavenge cfDNA derived from RA patients and inhibit the activation of primary synovial fluid monocytes and fibroblast-like synoviocytes. Using clinical scoring, micro-CT images, MRI, and histology, we show that intravenous injection of cNP into a CpG-induced mouse model or collagen-induced arthritis rat model can relieve RA symptoms including ankle and tissue swelling, and bone and cartilage damage. This culminates in the manifestation of partial mobility recovery of the treated rats in a rotational cage test. Mechanistic studies on intracellular trafficking and biodistribution of cNP, as well as measurement of cytokine expression in the joints and cfDNA levels in systemic circulation and inflamed joints also correlate with therapeutic outcomes. This work suggests a new direction of nanomedicine in treating inflammatory diseases.

6.
J Cell Biochem ; 2018 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-30315711

RESUMO

OBJECTIVE: We aim to explore the effect of adipose derived mesenchymal stem cells (ADMSCs) on a knee osteoarthritis rat model and analyze how ADMSCs affect chondrocyte apoptosis. MATERIALS AND METHODS: A surgically induced rat knee osteoarthritis (OA) model was constructed. ADMSCs were engrafted into the right knee cavity. Hematoxylin and eosin (H&E), Masson, and Safranin O were used to compare the histopathology of synovial membrane and cartilage. Immunohistochemical (IHC) was used to measure MMP-13, Collagen 2 (Col-2), Caspase-3 (Cas-3), PARP, p62, LC3b, DDR-2, FGFR-1, Wnt, P-AKT/AKT, p-CAMKII/CAMKII, and p-Smad1/Smad1 expression in the articular cartilage. qPCR and Western blot analysis were used to detect mRNA and protein levels of markers in chondrocytes. TUNEL and Annexin-V were used to assess apoptosis. RESULTS: Histological analysis showed that ADMSCs alleviated the deterioration of cartilage and osteoarthritis. ADMSCs coculture increase the expression of Col2 and Sox-9, while down regulated MMP-13 in IL-1ß stimulated chondrocytes. ADMSCs decreased proinflammatory cytokines IL-1ß, IL-6, and TNF-α. ADMSCs enhanced the viability of IL-1ß stimulated chondrocytes. ADMSC attenuated chondrocyte apoptosis. The pretreatment of 3-methyladenine (3-MA) reversed the reduction of Caspase-3 caused by ADMSCs, showing that the antiapoptotic effect was associated with autophagy inducing. ADMSCs significantly reduced the expression of FGFR-1, DDR-2, and Wnt in IL-1ß stimulated chondrocytes. ADMSCs reduced the ratio of p-Smad1/Smad1 and p-CAMK II/CAMKII, and increased the ratio of p-AKT/AKT. CONCLUSIONS: ADMSCs treatment alleviate osteoarthritis in rat OA models. AMDSCs reduced the secretion of proinflammatory cytokines and protected against apoptosis through autophagy inducing. ADMSCs' function could be related to multiple signaling pathway.

7.
J Clin Invest ; 128(10): 4510-4524, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30198906

RESUMO

Fibroblast-like synoviocytes (FLSs) are critical to synovial aggression and joint destruction in rheumatoid arthritis (RA). The role of long noncoding RNAs (lncRNAs) in RA is largely unknown. Here, we identified a lncRNA, LERFS (lowly expressed in rheumatoid fibroblast-like synoviocytes), that negatively regulates the migration, invasion, and proliferation of FLSs through interaction with heterogeneous nuclear ribonucleoprotein Q (hnRNP Q). Under healthy conditions, by binding to the mRNA of RhoA, Rac1, and CDC42 - the small GTPase proteins that control the motility and proliferation of FLSs - the LERFS-hnRNP Q complex decreased the stability or translation of target mRNAs and downregulated their protein levels. But in RA FLSs, decreased LERFS levels induced a reduction of the LERFS-hnRNP Q complex, which reduced the binding of hnRNP Q to target mRNA and therefore increased the stability or translation of target mRNA. These findings suggest that a decrease in synovial LERFS may contribute to synovial aggression and joint destruction in RA and that targeting the lncRNA LERFS may have therapeutic potential in patients with RA.

8.
Int Immunopharmacol ; 63: 261-269, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30121047

RESUMO

OBJECTIVE: Hydroxychloroquine (HCQ) is an antimalarial drug that is widely used in the treatment of some autoimmune diseases. In the present study, we explore the role of HCQ in regulating endothelial inflammation and its underlying mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords. Protein expression was measured by Western blot or immunofluorescence staining. Endothelial adhesion ability was determined by leukocyte-endothelial monolayer adhesion assay. Transwell assay was used to measure the transendothelial-migration of PBMCs. RESULTS: TNF-α-induced endothelial-leukocyte adhesion and the leukocyte transmigration were profoundly reduced by HCQ treatment. HCQ treatment dramatically inhibited the expression of TNF-α-induced endothelial ICAM-1 and VCAM-1. Furthermore, treatment with HCQ prevented the TNF-α-induced translocation of NF-κB p65 into the nucleus and the phosphorylation of the p65 subunit in HUVECs. HCQ inhibited the expression of phosphorylated p38 and JNK protein but not ERK. Treatment with NF-κB, p38 and JNK inhibitor could also reduce TNF-α-induced endothelial-leukocyte adhesion and the endothelial expression of ICAM-1 and VCAM-1. HCQ administration also suppressed TNF-α induced lung injury in mice by reducing neutrophil infiltration in pulmonary interstitial tissue. CONCLUSIONS: This work shows the inhibitory effect of HCQ on endothelial inflammatory response through, at least in part, blocking NF-κB, p38 and JNK pathways. Our findings suggest that HCQ may be a promising approach for the treatment of inflammatory vascular disease beyond its immunomodulatory actions.

9.
Front Immunol ; 9: 1714, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30100905

RESUMO

Objective: To investigate the role of glycogen metabolism in regulating rheumatoid fibroblast-like synoviocyte (FLS)-mediated synovial inflammation and its underlying mechanism. Methods: FLSs were separated from synovial tissues (STs) obtained from rheumatoid arthritis (RA) patients. Glycogen content was determined by periodic acid Schiff staining. Protein expression was analyzed by Western blot or immunohistochemistry. Gene expression of cytokines and matrix metalloproteinases (MMPs) was evaluated by quantitative real-time PCR. FLS proliferation was detected by EdU incorporation. Migration and invasion were measured by Boyden chamber assay. Results: Glycogen levels and glycogen synthase 1 (GYS1) expression were significantly increased in the ST and FLSs of RA patients. TNF-α or hypoxia induced GYS1 expression and glycogen synthesis in RA FLSs. GYS1 knockdown by shRNA decreased the expression of IL-1ß, IL-6, CCL-2, MMP-1, and MMP-9 and proliferation and migration by increasing AMP-activated protein kinase (AMPK) activity in RA FLS. AMPK inhibitor or knockdown AMPK could reverse the inhibitory effect of GYS1 knockdown on the inflammatory response in RA FLSs; however, an AMPK agonist blocked RA FLS activity. We further determined that hypoxia-inducible factor-1α mediates TNF-α- or hypoxia-induced GYS1 expression and glycogen levels. Local joint depletion of GYS1 or intraperitoneal administration with an AMPK agonist ameliorated the severity of arthritis in rats with collagen-induced arthritis. Conclusion: Our data demonstrate that GYS1-mediated glycogen accumulation contributes to FLS-mediated synovial inflammation in RA by blocking AMPK activation. In our knowledge, this is a first study linking glycogen metabolism to chronic inflammation. Inhibition of GYS1 might be a novel therapeutic strategy for chronic inflammatory arthritis, including RA.

10.
Clin Exp Rheumatol ; 36(5): 884-890, 2018 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29652659

RESUMO

OBJECTIVES: To evaluate the efficacy of different tapering or discontinuation strategies of etanercept in a cohort of axial spondyloarthritis from South China. METHODS: We performed a retrospective cohort study. Axial SpA patients who achieved clinical remission for at least 6 months after receiving a standard dose of etanercept therapy were enrolled. Different tapering or discontinuation strategies were compared. RESULTS: Altogether, 258 cases were enrolled. No differences were found in baseline characteristics among the three groups. Significantly more patients on discontinuation group (19%) than tapering group (5.4%, p<0.001) relapsed as early as 6 months. Almost all of the patients (103/107, 96.3%) in taper 25% group and more than 80% (71/88, 80.7%) of the patients in taper 50% group maintained low disease activity (LDA) or clinical remission during the first year. At the end of the 2-year follow-up, the percentage of patients maintaining LDA or remission were 28.6% (discontinuation), 55.7% (taper 50%), 84.1% (taper 25%), respectively. Activity indexes were significantly lower in taper 25% group compared to the other two groups. Patients in discontinuation group and tapering 50% group, with longer SpA duration were more likely to relapse, and remission>12 months before discontinuation/tapering helped to reduce relapse. CONCLUSIONS: It is feasible to slowly increase the dosing interval and transit to the lowest effective dosing interval for some patients in remission/LDA. Prolonging the time under remission before tapering help to improve the outcome. Tapering 25% of the etanercept dose every 3 months may be a pragmatic approach for more cost-effective use of the drug.

11.
Inflamm Res ; 67(3): 233-243, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29119225

RESUMO

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/metabolismo
12.
Mod Rheumatol ; 28(2): 339-344, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28485997

RESUMO

OBJECTIVE: Flare prophylaxis is recommended during urate-lowering therapy (ULT) despite lack of proven benefit especially when initiating febuxostat. We investigated if colchicine or steroids administration during initiation of febuxostat for chronic gouty arthritis reduces the frequency and/or severity of acute gout flares. METHODS: Patients with confirmed diagnosis of gout starting febuxostat were retrospectively studied. Frequency, severity, and length of flares were analyzed. Assessment of severity based on a visual analog scale (VAS). RESULTS: Two hundred and seventy-three patients were studied. The mean dose of colchicine and steroids was 0.53 ± 0.15 mg PO QD and 7.55 ± 1.30 mg prednisone equivalent PO QD; while the duration was 6.13 ± 1.14 and 6.20 ± 1.36 months, respectively. Subjects treated with colchicine and steroids suffered fewer total flares (0.30, 0.96 vs 2.47, p = .000), fewer flares from 0 to 3 months (0.26, 0.71 vs 1.72, p = .000), less severe flares assessed by VAS than those without prophylactic therapy (3.65, 3.49 vs 5.54, p = .000). Both total flares (p = .003) and flares from 0 to 3 months (p = .008) of the colchicine group were fewer than the steroids group. There were no significant differences in length of flares among groups (p = .815). Both colchicine and steroids were well tolerated. CONCLUSION: The use of colchicine or steroids prophylaxis reduces the frequency and severity of acute gout flares during initiation of febuxostat for chronic gouty arthritis. Colchicine is superior to steroids in flares prophylaxis. Prophylactic therapy with colchicine 0.5 mg PO QD or steroids 7.5 mg prednisone equivalent PO QD for 6 months is suggested.


Assuntos
Artrite Gotosa/tratamento farmacológico , Colchicina/uso terapêutico , Febuxostat/uso terapêutico , Supressores da Gota/uso terapêutico , Adulto , Artrite Gotosa/prevenção & controle , Colchicina/administração & dosagem , Esquema de Medicação , Quimioterapia Combinada , Febuxostat/administração & dosagem , Feminino , Supressores da Gota/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
13.
Oncotarget ; 8(55): 94893-94899, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29212275

RESUMO

Objective: to assess the impact of longitudinal change of the overweight and physical activity on hyperuricemia. Methods: We performed a retrospective cohort study. Demographic information, clinical features, laboratory findings, body weight and physical exercises pattern were documented. Results: Altogether 4678 cases of hyperuricemia were enrolled. The median aged males were most affected. Individuals in the middle age had the highest prevalence of being overweight (2501/3382, 74.0%). Middle aged with BMI≥25 kg/m2 were more likely to lose weight (963/2807, 34.3%). BMI and waist circumference control helped to reduce serum uric acid. Overweight population was more likely to use urate-lowering or uricosuric medication (3025/3382, 89.4%). Intermediate and heavy activity were associated with bigger SUA improvement. Patients in the age of 35-60 were more likely to do physical exercises than the others. Conclusion: Being overweight is strongly associated with hyperuricemia. Successful weight control was correlated with significant uric acid reduction. Intermediate to heavy physical activity helps to reduce waist circumference and SUA. In the hyperuricemia population, obese, middle aged men were the most affected, and also the most likely to do more exercises and get their bodyweight back to normal.

14.
Rheumatology (Oxford) ; 56(8): 1417-1427, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28431044

RESUMO

Objective: To explore the role of leonurine in the regulation of synovial inflammation and joint destruction inRA. Methods: Fibroblast-like synoviocytes were isolated from synovial tissue from RA patients. Pro-inflammatory cytokine and MMP expression was evaluated using real-time PCR and a cytometric bead array. Cell migration and invasion in vitro were measured using the Boyden chamber method and the scratch assay, respectively. Protein expression was measured by western blotting. Nuclear factor kappa B (NF-κB) nuclear translocation was detected by immunofluorescence. The in vivo effect of leonurine was evaluated in mice with CIA. Results: Leonurine treatment significantly decreased the production of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNFα) and MMPs (MMP-1 and MMP-3) and suppressed the migration and invasion of RA fibroblast-like synoviocytes. The molecular analysis revealed that leonurine impaired TNFα-induced NF-κB signalling by inhibiting the phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) and subsequently preventing the nuclear translocation of the NF-κB p65 subunit. Leonurine also inhibited the p38 and Jun N-terminal kinase mitogen-activated protein kinases signalling pathways without affecting ERK signalling. Intraperitoneal injection of leonurine reduced synovial inflammation, joint destruction and the serum IL-1ß, IL-6 and TNFα levels in mice with CIA. Conclusion: Our findings show that leonurine reduces synovial inflammation and joint destruction in RA through the NF-κB and mitogen-activated protein kinases pathways. Leonurine has potential as a therapeutic agent for RA.


Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Ácido Gálico/análogos & derivados , Adulto , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Citocinas/efeitos dos fármacos , Feminino , Fibroblastos/metabolismo , Ácido Gálico/farmacocinética , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Camundongos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/patologia , Fator de Transcrição RelA/efeitos dos fármacos
15.
J Immunol ; 198(9): 3719-3728, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28348270

RESUMO

The B cells inhabited in mucosa play a vital role in mediating homeostasis with autoantigens and external Ags. Tumor-infiltrating lymphocytes are potential prognostic markers and therapeutic agents for cancer. However, the spatial heterogeneity of the B cell repertoire in intestinal mucosa and the tumor-infiltrating lymphocytes in colorectal cancer (CRC) remain poorly understood. In this study, we developed an unbiased method to amplify the IgH repertoire, as well as a bioinformatic pipeline to process these high-throughput sequencing data. With biopsies from seven intestinal mucosal segments, we uncovered their strong spatial homogeneity among the large intestine, where the clone overlap rate was up to 62.21%. The heterogeneity between terminal ileum and large intestine was also observed, including discrepant isotype distribution and low clone overlap rate. With tumor and adjacent normal mucosal tissues from CRC and colorectal advanced adenoma (AD) patients, we observed a similar IgH profile between tumor and adjacent normal mucosal tissues in AD, as well as a slight difference in CRC. Interestingly, we found distinct repertoire properties in the CRC tumor from AD and normal mucosa. Finally, we identified 1445 public clones for the normal mucosa, and 22 public clones for the CRC tumor with characteristic features. These data may be of potential use in clinical prognosis, diagnosis, and treatment of CRC.


Assuntos
Adenoma/imunologia , Linfócitos B/fisiologia , Neoplasias do Colo/imunologia , Neoplasias Colorretais/imunologia , Mucosa Intestinal/imunologia , Linfócitos do Interstício Tumoral/fisiologia , Receptores de Antígenos de Linfócitos B/genética , Adenoma/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Biologia Computacional , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias
16.
Inflamm Res ; 66(5): 433-440, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28265680

RESUMO

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/metabolismo
17.
Br J Pharmacol ; 174(9): 893-908, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28239846

RESUMO

BACKGROUND AND PURPOSE: Abnormal glycolytic metabolism contributes to joint inflammation in rheumatoid arthritis (RA). The aims of this study were to investigate the role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a bifunctional enzyme that controls the glycolytic rate, in regulating fibroblast-like synoviocyte (FLS)-mediated synovial inflammation and invasiveness in RA. EXPERIMENTAL APPROACH: A specific inhibitor of PFKFB3, PFK15, and siRNA were used to evaluate the role of PFKFB3. Protein expression was measured by Western blotting or immunofluorescence staining. The expression of cytokines was determined by quantitative real-time PCR. Migration and invasion were measured using a Boyden chamber assay. A mouse model of collagen-induced arthritis (CIA) was used to evaluate the in vivo effect of PFK15. KEY RESULTS: PFKFB3 expression was increased in the synovial tissue and FLSs from RA patients compared with osteoarthritis patients. PFKFB3 inhibition decreased the expression of IL-8, IL-6, CCL-2 and CXCL-10 and the proliferation, migration and invasion of RA FLSs. PFK15 suppressed TNF-α-induced activation of NF-κB and p38, JNK and ERK MAPK signals in RA FLSs. PFK15 treatment also suppressed glucose uptake and lactate secretion. Lactate reversed the inhibitory effect of PFK15 or PFKFB3 siRNA on cytokine expression and migration of RA FLSs. Lactate was also involved in PFKFB3-mediated activation of NF-κB and MAPKs. Intraperitoneal injection of PFK15 in mice with CIA attenuated joint inflammation. CONCLUSION AND IMPLICATIONS: Elevated PFKFB3 expression might contribute to synovial inflammation and aggressive behaviours of RA FLSs, suggesting a novel strategy of targeting PFKFB3 to prevent synovial inflammation and joint destruction in RA.


Assuntos
Artrite Reumatoide/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/metabolismo , Sinoviócitos/metabolismo , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , RNA Interferente Pequeno/farmacologia , Distribuição Aleatória , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinoviócitos/efeitos dos fármacos
18.
J Cell Mol Med ; 21(4): 781-790, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27860244

RESUMO

To probe the role of protein arginine methyltransferase 5 (PRMT5) in regulating inflammation, cell proliferation, migration and invasion of fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). FLSs were separated from synovial tissues (STs) from patients with RA and osteoarthritis (OA). An inhibitor of PRMT5 (EPZ015666) and short interference RNA (siRNA) against PRMT5 were used to inhibit PRMT5 expression. The standard of protein was measured by Western blot or immunofluorescence. The excretion and genetic expression of inflammatory factors were, respectively, estimated by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Migration and invasion in vitro were detected by Boyden chamber assay. FLSs proliferation was detected by BrdU incorporation. Increased PRMT5 was discovered in STs and FLSs from patients with RA. In RA FLSs, the level of PRMT5 was up-regulated by stimulation with IL-1ß and TNF-α. Inhibition of PRMT5 by EPZ015666 and siRNA-mediated knockdown reduced IL-6 and IL-8 production, and proliferation of RA FLSs. In addition, inhibition of PRMT5 decreased in vitro migration and invasion of RA FLSs. Furthermore, EPZ015666 restrained the phosphorylation of IκB kinaseß and IκBα, as well as nucleus transsituation of p65 as well as AKT in FLSs. PRMT5 regulated the production of inflammatory factors, cell proliferation, migration and invasion of RA FLS, which was mediated by the NF-κB and AKT pathways. Our data suggested that targeting PRMT5 to prevent synovial inflammation and destruction might be a promising therapy for RA.


Assuntos
Artrite Reumatoide/enzimologia , Artrite Reumatoide/patologia , Movimento Celular , Fibroblastos/enzimologia , Inflamação/enzimologia , Proteína-Arginina N-Metiltransferases/metabolismo , Sinoviócitos/enzimologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Quinase I-kappa B/metabolismo , Inflamação/complicações , Inflamação/patologia , Mediadores da Inflamação/metabolismo , NF-kappa B/metabolismo , Osteoartrite/enzimologia , Osteoartrite/patologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/patologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
19.
Br J Pharmacol ; 174(1): 101-115, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27774624

RESUMO

BACKGROUND AND PURPOSE: There is increasing evidence indicating that bromodomain and extra-terminal domain (BET) proteins play a critical role in the regulation of immune and inflammatory responses; however, their contribution to vascular inflammation has not yet been elucidated. In this study, we investigated the effect of inhibiting BET bromodomain on vascular inflammation and the underlying mechanisms. EXPERIMENTAL APPROACH: HUVECs were isolated from fresh umbilical cords. JQ1, a specific BET bromodomain inhibitor, and Brd shRNA were used to evaluate the regulation of the BET proteins in vascular inflammation. Leukocyte adhesion to HUVECs was measure by an adhesion assay. Western blot or immunohistochemical analysis was used to detect the protein expression. Real-time PCR was used to evaluate mRNA expression. Leukocyte accumulation in vivo was determined by an acute lung inflammation model. KEY RESULTS: BET bromodomain inhibition suppressed the expression of adhesion molecules induced by TNF-α- or LPS, including ICAM-1, VCAM-1 and E-selectin, and inhibited leukocyte adhesion to activated HUVEC monolayers. Treatment with JQ1 also attenuated the LPS-induced accumulation of leukocytes and expression of endothelial adhesion molecules in the acute lung inflammation model in vivo. Furthermore, BET bromodomain inhibition reduced the activity of p38 and JNK MAPKs and NF-κB in TNF-α-stimulated HUVECs. TNF-α-induced NF-κB activation was also blocked by inhibitors of p38 (SB203580) or JNK (SP600125). CONCLUSIONS AND IMPLICATIONS: BET bromodomain is important for regulating endothelial inflammation. Strategies targeting endothelial BET bromodomain may provide a new therapeutic approach for controlling inflammatory-related diseases.


Assuntos
Azepinas/farmacologia , Inflamação/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Relação Estrutura-Atividade
20.
Int Immunopharmacol ; 43: 187-194, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28013187

RESUMO

Fibroblast-like synoviocytes (FLSs) display an aggressive phenotype that is a critical factor in cartilage destruction in rheumatoid arthritis (RA). Increased FLS migration and proliferation are essential to the pathology of RA. Halofuginone has been found to inhibit cell migration and proliferation in cancer cells. However, whether halofuginone has a role in the treatment of RA FLSs is unclear. In this study, we found that halofuginone reduced migration, invasion, cell proliferation and MMPs expression in RA FLSs. In addition, we demonstrated that halofuginone inhibited reorganization of the actin cytoskeleton during cell migration. To gain insight into the molecular mechanisms, we evaluated the effect of halofuginone on the MAPK and AKT pathways. Our results indicated that halofuginone inhibited the activity of MAPK and AKT. Taken together, these results suggest that halofuginone may protect against joint destruction in RA by regulating synoviocyte migration, invasion and cell proliferation by inhibiting MAPK and AKT activation.


Assuntos
Antirreumáticos/farmacologia , Artrite Reumatoide/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Sinoviócitos/efeitos dos fármacos , Adulto , Idoso , Artrite Reumatoide/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Oncogênica v-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinoviócitos/imunologia , Fator de Necrose Tumoral alfa/imunologia
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