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1.
FASEB J ; 38(15): e23859, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-39082187

RESUMO

Metabolic dysfunction-associated steatotic liver disease (MASLD) is strongly associated with insulin resistance development. Hepatic lipid accumulation and inflammation are considered the main drivers of hepatic insulin resistance in MASLD. Cysteine-rich 61 (Cyr61 also called CCN1), a novel secretory matricellular protein, is implicated in liver inflammation, and its role in MASLD is not clearly understood. Therefore, we investigated the role of Cyr61 in hepatic insulin resistance and lipid metabolism as major factors in MASLD pathogenesis. In high-fat diet (HFD)-fed C57BL/6J mice, Cyr61 was downregulated or upregulated via viral transduction. Measurements of glucose homeostasis, histological assessment of liver tissues, and gene expression and signaling pathways of lipogenesis, fatty acid oxidation, and inflammation were performed using liver samples from these mice. Cyr61 levels in HepG2 cells were reduced using RNAi-mediated gene knockdown. Inflammation and insulin resistance were evaluated using real-time polymerase chain reaction and western blotting. HFD/AAV-shCyr61 mice exhibited enhanced glucose tolerance via the protein kinase B pathway, reduced hepatic inflammation, decreased lipogenesis, and increased fatty acid oxidation. Notably, HFD/AAV-shCyr61 mice showed elevated protein expression of sirtuin 6 and phosphorylated-AMP-activated protein kinase. In vitro experiments demonstrated that inhibition of Cyr61 downregulated pro-inflammatory cytokines such as interleukin-1 beta, IL-6, and tumor necrosis factor-alpha via the nuclear factor kappa B/c-Jun N-terminal kinase pathway, and alleviated insulin resistance. Cyr61 affected hepatic inflammation, lipid metabolism, and insulin resistance. Inhibition of Cyr61 reduced inflammation, recovered insulin resistance, and altered lipid metabolism in vivo and in vitro. Therefore, Cyr61 is a potential therapeutic target in MASLD.


Assuntos
Proteína Rica em Cisteína 61 , Dieta Hiperlipídica , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado , Camundongos Endogâmicos C57BL , Animais , Proteína Rica em Cisteína 61/metabolismo , Proteína Rica em Cisteína 61/genética , Células Hep G2 , Humanos , Camundongos , Dieta Hiperlipídica/efeitos adversos , Masculino , Fígado/metabolismo , Lipogênese
2.
Mediators Inflamm ; 2023: 2364121, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37868614

RESUMO

Background: Inflammation is a major cause of hepatic tissue damage and accelerates the progression of nonalcoholic fatty liver disease (NAFLD). Amphiregulin (AREG), an epidermal growth factor receptor ligand, is associated with human liver cirrhosis and hepatocellular carcinoma. We aimed to investigate the effects of AREG on hepatic inflammation during NAFLD progression, in vivo and in vitro. Methods: AREG gene expression was measured in the liver of mice fed a methionine choline-deficient (MCD) diet for 2 weeks. We evaluated inflammatory mediators and signaling pathways in HepG2 cells after stimulation with AREG. Nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were analyzed using an enzyme-linked immunosorbent assay and western blotting. Nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, were analyzed using western blotting. Results: Proinflammatory cytokines (interleukin (IL)-6, IL-1ß, and IL-8) and immune cell recruitment (as indicated by L3T4, F4/80, and ly6G mRNA expression) increased, and expression of AREG increased in the liver of mice fed the MCD diet. AREG significantly increased the expression of IL-6 and IL-1ß and the production of NO, PGE2, and IL-8 in HepG2 cells. It also activated the protein expression of iNOS and COX-2. AREG-activated NF-κB and MAPKs signaling, and together with NF-κB and MAPKs inhibitors, AREG significantly reduced the protein expression of iNOS and COX-2. Conclusion: AREG plays a role in hepatic inflammation by increasing iNOS and COX-2 expression via NF-κB and MAPKs signaling.


Assuntos
NF-kappa B , Hepatopatia Gordurosa não Alcoólica , Camundongos , Humanos , Animais , NF-kappa B/metabolismo , Ciclo-Oxigenase 2/metabolismo , Anfirregulina/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Dinoprostona , Interleucina-8/metabolismo , Inflamação/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Óxido Nítrico/metabolismo
3.
Biochem Biophys Res Commun ; 588: 154-160, 2022 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34971904

RESUMO

Non-alcoholic fatty liver disease (NAFLD) includes a broad spectrum of liver diseases characterized by steatosis, inflammation, and fibrosis. This study aimed to investigate the potential of dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter 2 inhibitors in alleviating the progression of NAFLD. The NAFLD model was generated by feeding male C57BL/6J mice a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 7 weeks. After 2 weeks of CDAHFD feeding, the NAFLD model mice were assigned to four groups, namely (ⅰ) VEHICLE, (ⅱ) gemigliptin (GEMI), (ⅲ) empagliflozin (EMPA), and (ⅳ) GEMI + EMPA. For the next 5 weeks, mice received the vehicle or the drug based upon the group to which they belonged. Thereafter, the triglyceride concentration, extent of fibrosis, and the expression of genes encoding inflammatory cytokines, chemokines, and antioxidant enzymes were analyzed in the livers of mice. The NAFLD activity score and hepatic fibrosis grade were assessed via hematoxylin and eosin and Sirius Red staining of the liver tissue samples. All mice belonging to the GEMI, EMPA, and GEMI + EMPA groups showed improvements in the accumulation of liver triglycerides and the expression of inflammatory cytokines and chemokines. Additionally, the oxidative stress was reduced due to inhibition of the c-Jun N-terminal kinase pathway and upregulation of the antioxidant enzymes. Furthermore, in these three groups, the galectin-3 and interleukin 33-induced activity of tumor necrosis factor-α was inhibited, thereby preventing the progression of liver fibrosis. These findings suggest that the GEMI, EMPA, and GEMI + EMPA treatments ameliorate hepatic steatosis, inflammation, oxidative stress, and fibrosis in CDAHFD-induced NAFLD mouse models.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Dieta Hiperlipídica , Glucosídeos/uso terapêutico , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Piperidonas/farmacologia , Piperidonas/uso terapêutico , Substâncias Protetoras/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Aminoácidos , Animais , Compostos Benzidrílicos/farmacologia , Colina , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Glucosídeos/farmacologia , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Substâncias Protetoras/farmacologia
4.
J Immunol Res ; 2021: 9944880, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34124273

RESUMO

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT2) and dipeptidyl peptidase-4 (DPP-4) inhibitors are glucose-lowering drugs whose anti-inflammatory properties have recently become useful in tackling metabolic syndromes in chronic inflammatory diseases, including diabetes and obesity. We investigated whether empagliflozin (SGLT2 inhibitor) and gemigliptin (DPP-4 inhibitor) improve inflammatory responses in macrophages, identified signalling pathways responsible for these effects, and studied whether the effects can be augmented with dual empagliflozin and gemigliptin therapy. METHODS: RAW 264.7 macrophages were first stimulated with lipopolysaccharide (LPS), then cotreated with empagliflozin, gemigliptin, or empagliflozin plus gemigliptin. We conducted quantitative RT-PCR (qRT-PCR) to determine the most effective anti-inflammatory doses without cytotoxicity. We performed ELISA and qRT-PCR for inflammatory cytokines and chemokines and flow cytometry for CD80, the M1 macrophage surface marker, to evaluate the anti-inflammatory effects of empagliflozin and gemigliptin. NF-κB, MAPK, and JAK2/STAT signalling pathways were examined via Western blotting to elucidate the molecular mechanisms of anti-inflammation. RESULTS: LPS-stimulated CD80+ M1 macrophages were suppressed by coincubation with empagliflozin, gemigliptin, and empagliflozin plus gemigliptin, respectively. Empagliflozin and gemigliptin (individually and combined) inhibited prostaglandin E2 (PGE2) release and COX-2, iNOS gene expression in LPS-stimulated RAW 264.7 macrophages. These three treatments also attenuated the secretion and mRNA expression of proinflammatory cytokines, such as TNF-α, IL-1ß, IL-6, and IFN-γ, and proinflammatory chemokines, such as CCL3, CCL4, CCL5, and CXCL10. All of them blocked NF-κB, JNK, and STAT1/3 phosphorylation through IKKα/ß, MKK4/7, and JAK2 signalling. CONCLUSIONS: Our study demonstrated the anti-inflammatory effects of empagliflozin and gemigliptin via IKK/NF-κB, MKK7/JNK, and JAK2/STAT1 pathway downregulation in macrophages. In all cases, combined empagliflozin and gemigliptin treatment showed greater anti-inflammatory properties.


Assuntos
Anti-Inflamatórios/farmacologia , Compostos Benzidrílicos/farmacologia , Inibidores da Dipeptidil Peptidase IV/farmacologia , Glucosídeos/farmacologia , Macrófagos/imunologia , Piperidonas/farmacologia , Pirimidinas/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Animais , Quinase I-kappa B/metabolismo , Janus Quinase 2/metabolismo , Lipopolissacarídeos/imunologia , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase 7/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Células RAW 264.7 , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais
5.
J Gastroenterol Hepatol ; 36(9): 2592-2600, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33600604

RESUMO

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to non-alcoholic steatohepatitis, which is characterized by hepatic inflammation that can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Visfatin, an adipocytokine, was reported to induce pro-inflammatory cytokines and can be associated with liver fibrosis. We investigated the role of visfatin on hepatic inflammation and fibrosis in a methionine-choline-deficient (MCD)-diet-induced steatohepatitis mouse model. METHODS: Eight-week-old male C57BL/6 J mice were randomly assigned into one of three groups: (1) saline-injected control diet group; (2) saline-injected MCD diet group; and (3) visfatin-injected MCD diet group (n = 8 per group). Mice were administered intravenous saline or 10 µg/kg of recombinant murine visfatin for 2 weeks. Histologic assessment of liver and biochemical and molecular measurements of endoplasmic reticulum (ER) stress, reactive oxidative stress (ROS), inflammation, and fibrosis were performed in livers from these animals. RESULTS: Visfatin injection aggravated hepatic steatosis and increased plasma alanine aminotransferase and aspartate aminotransferase concentrations. Visfatin increased inflammatory cell infiltration (as indicated by F4/80, CD68, ly6G, and CD3 mRNA expression) and expression of chemokines in the liver. Visfatin also increased the expression of pro-inflammatory cytokines (IL-1ß, TNF-α, and IL-6) and activated fibrosis markers (CTGF, TIMP1, collagen 1α2, collagen 3α2, αSMA, fibronectin, and vimentin) in liver. Livers of visfatin-injected mice showed upregulation of ER stress and ROS and activation of JNK signaling. CONCLUSIONS: These results suggest that visfatin aggravates hepatic inflammation together with induction of ER and oxidative stress and exacerbates fibrosis in an MCD-diet-fed mouse model of NAFLD.


Assuntos
Adipocinas , Doença Hepática Induzida por Substâncias e Drogas , Dieta , Nicotinamida Fosforribosiltransferase , Hepatopatia Gordurosa não Alcoólica , Adipocinas/efeitos adversos , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Deficiência de Colina/complicações , Dieta/efeitos adversos , Modelos Animais de Doenças , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Masculino , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Nicotinamida Fosforribosiltransferase/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia
6.
Mol Biol Rep ; 47(6): 4285-4293, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32418112

RESUMO

Chemokines interact with hepatic resident cells during inflammation and fibrosis. CC chemokine ligand (CCL) 20 has been reported to be important in inflammation and fibrosis in the liver. We hypothesized that visfatin, an adipocytokine, could play a role in hepatic fibrosis via CCL20. We investigated the effect of visfatin on CCL20 in THP-1 human promonocytic cells and examined the molecular mechanisms involved. Following treatment of THP-1 cells with visfatin, CCL20 expression and secretion were assessed. We assessed the intracellular signaling molecules IKK/NF-κB, JAK2/STAT3, MAPKs, and MKK3/6 by western blotting. We treated THP-1 cells with visfatin and signaling inhibitors, and examined CCL20 mRNA and protein levels. To investigate the effect of visfatin-induced CCL20 expression in hepatic stellate cells (HSCs), LX-2 cells were co-cultured with the culture supernatant of THP-1 cells with or without anti-CCL20 neutralizing antibodies, and fibrosis markers were examined by RT-PCR and immunoblotting. In THP-1 cells, visfatin increased the CCL20 mRNA and protein levels. visfatin increased the activities of the NF-κB, p38, and MLK3/6 signaling pathways but not those of the JAK2/STAT3 and ERK pathways. Visfatin treatment together with an NF-κB, p38, or MLK3 inhibitor reduced the mRNA and protein levels of CCL20. The visfatin-induced CCL20 increased the expression of fibrosis markers and CCR6 in HSCs. Following neutralization of CCL20, the levels of fibrosis markers and CCR6 were decreased. Visfatin increases the expression of CCL20 via the NF-κB and MKK3/6-p38 signaling pathways in macrophages, and visfatin-induced CCL20 expression promotes the fibrosis markers in HSCs.


Assuntos
Quimiocina CCL20/metabolismo , Células Estreladas do Fígado/metabolismo , Nicotinamida Fosforribosiltransferase/farmacologia , Quimiocina CCL20/fisiologia , Quimiocinas/metabolismo , Hepatócitos/metabolismo , Humanos , Janus Quinase 2/metabolismo , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 6/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1 , Fator de Transcrição RelA/metabolismo
7.
Sci Rep ; 9(1): 17839, 2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31780766

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is excessive fat build-up in the liver without alcohol consumption and includes hepatic inflammation and damage. Excessive influx of fatty acids to liver from circulation is thought to be a pathogenic cause for the development of NAFLD. Thus, inhibition of fatty acid intake into hepatocyte would be a maneuver for protection from high fat diet (HFD)-induced NAFLD. This study was initiated to determine whether sodium fluorocitrate (SFC) as a fatty acid uptake inhibitor could prevent palmitate-induced lipotoxicity in hepatocytes and protect the mice from HFD-induced NAFLD. SFC significantly inhibited the cellular uptake of palmitate in HepG2 hepatocytes, and thus prevented palmitate-induced fat accumulation and death in these cells. Single treatment with SFC reduced fasting-induced hepatic steatosis in C57BL/6J mice. Concurrent treatment with SFC for 15 weeks in HFD-fed C57BL/6J mice prevented HFD-induced fat accumulation and stress/inflammatory signal activation in the liver. SFC restored HFD-induced increased levels of serum alanine aminotransferase and aspartate aminotransferases as hepatic injury markers in these mice. SFC treatment also improved HFD-induced hepatic insulin resistance, and thus ameliorated HFD-induced hyperglycemia. In conclusion, inhibition of fatty acid mobilization into liver through SFC treatment can be a strategy to protect from HFD-induced NAFLD.


Assuntos
Citratos/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Ácido Palmítico/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Citratos/farmacologia , Dieta Hiperlipídica/efeitos adversos , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia
8.
J Diabetes Res ; 2019: 4021623, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396538

RESUMO

BACKGROUND: It has been suggested that visfatin, which is an adipocytokine, exhibits proinflammatory properties and is associated with insulin resistance. Insulin resistance and inflammation are the principal pathogeneses of nonalcoholic fatty liver disease (NAFLD), but the relationship, if any, between visfatin and NAFLD remains unclear. Here, we evaluated the effects of visfatin on hepatic inflammation and insulin resistance in HepG2 cells and examined the molecular mechanisms involved. METHODS: After treatment with visfatin, the inflammatory cytokines IL-6, TNF-α, and IL-1ß were assessed by real-time polymerase chain reaction (RT-PCR) and immunocytochemical staining in HepG2 cells. To investigate the effects of visfatin on insulin resistance, we evaluated insulin-signaling pathways, such as IR, IRS-1, GSK, and AKT using immunoblotting. We assessed the intracellular signaling molecules including STAT3, NF-κB, IKK, p38, JNK, and ERK by western blotting. We treated HepG2 cells with both visfatin and either AG490 (a JAK2 inhibitor) or Bay 7082 (an NF-κB inhibitor); we examined proinflammatory cytokine mRNA levels using RT-PCR and insulin signaling using western blotting. RESULTS: In HepG2 cells, visfatin significantly increased the levels of proinflammatory cytokines, reduced the levels of proteins (e.g., phospho-IR, phospho-IRS-1 (Tyr612), phospho-AKT, and phospho-GSK-3α/ß) involved in insulin signaling, and increased IRS-1 S307 phosphorylation compared to controls. Interestingly, visfatin increased the activities of the JAK2/STAT3 and IKK/NF-κB signaling pathways but not those of the JNK, p38, and ERK pathways. Visfatin-induced inflammation and insulin resistance were regulated by JAK2/STAT3 and IKK/NF-κB signaling; together with AG490 or Bay 7082, visfatin significantly reduced mRNA levels of IL-6, TNF-α and IL-1ß and rescued insulin signaling. CONCLUSION: Visfatin induced proinflammatory cytokine production and inhibited insulin signaling via the STAT3 and NF-κB pathways in HepG2 cells.


Assuntos
Hepatócitos/efeitos dos fármacos , Inflamação/induzido quimicamente , Resistência à Insulina , NF-kappa B/metabolismo , Nicotinamida Fosforribosiltransferase/farmacologia , Fator de Transcrição STAT3/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina/fisiologia , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
9.
J Transl Med ; 14(1): 191, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27350539

RESUMO

BACKGROUND: Foxp3 is a key regulator of the development and function of regulatory T cells (Tregs), and its expression is thought to be T cell-restricted. We found that B cells in mice can express Foxp3 and B cells expressing Foxp3 may play a role in preventing the development of collagen-induced arthritis (CIA) in DBA/1J mice. METHODS: Foxp3 expression was modulated in CD19(+) B cells by transfection with shRNA or using an over-expression construct. In addition, Foxp3-transfected B cells were adoptively transferred to CIA mice. We found that LPS or anti-IgM stimulation induced Foxp3 expression in B cells. Foxp3-expressing B cells were found in the spleens of mice. RESULTS: Over-expression of Foxp3 conferred a contact-dependent suppressive ability on proliferation of responder T cells. Down-regulation of Foxp3 by shRNA caused a profound induction in proliferation of responder T cells. Adoptive transfer of Foxp3(+)CD19(+) B cells attenuated the clinical symptoms of CIA significantly with concomitant suppression of IL-17 production and enhancement of Foxp3 expression in CD4(+) T cells from splenocytes. CONCLUSION: Our data indicate that Foxp3 expression is not restricted to T cells. The expression of Foxp3 in B cells is critical for the immunoregulation of T cells and limits autoimmunity in a mouse model.


Assuntos
Transferência Adotiva , Artrite Experimental/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B Reguladores/imunologia , Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Artrite Experimental/patologia , Comunicação Celular , Linhagem Celular Tumoral , Proliferação de Células , Imunoglobulina M/metabolismo , Terapia de Imunossupressão , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos DBA , Baço/patologia , Transfecção
10.
J Immunol ; 195(7): 3001-10, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26324771

RESUMO

IL-23 is the key cytokine that induces the expansion of Th17 cells. It is composed of p19 and p40 subunits of IL-12. The p40 subunit binds competitively to the receptor of IL-23 and blocks its activity. Our aim was to assess the preventive and therapeutic effect of the IL-12p40 homodimer (p40)2 subunit in autoimmune arthritis animal models. In the current study, using IL-1R antagonist-knockout mice and a collagen-induced arthritis model, we investigated the suppressive effect of (p40)2 on inflammatory arthritis. We demonstrated that the recombinant adenovirus-expressing mouse (p40)2 model prevented the development of arthritis when given before the onset of arthritis. It also decreased the arthritis index and joint erosions in the mouse model if transferred after arthritis was established. (p40)2 inhibited the production of inflammatory cytokines and Ag-specific T cell proliferation. It also induced CD4(+)CD25(+)Foxp3 regulatory T (Treg) cells in vitro and in vivo, whereas the generation of retinoic acid receptor-related organ receptor γt and Th17 cells was suppressed. The induction of Treg cells and the suppression of Th17 cells were mediated via activated STAT5 and suppressed STAT3. Our data suggest that (p40)2 suppressed inflammatory arthritis successfully. This could be a useful therapeutic approach in autoimmune arthritis to regulate the Th17/Treg balance and IL-23 signaling.


Assuntos
Artrite Experimental/prevenção & controle , Subunidade p40 da Interleucina-12/farmacologia , Subunidade p19 da Interleucina-23/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/imunologia , Citocinas/biossíntese , Subunidade p40 da Interleucina-12/imunologia , Interleucina-17/biossíntese , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/biossíntese , Multimerização Proteica , Receptores de Interleucina/imunologia , Receptores Tipo I de Interleucina-1/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/imunologia
11.
Exp Mol Med ; 45: e46, 2013 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-24091748

RESUMO

Interleukin (IL)-27 is a novel cytokine of the IL-6/IL-12 family that has been reported to be involved in the pathogenesis of autoimmune diseases and has a pivotal role as both a pro- and anti-inflammatory cytokine. We investigated the in vivo effects of IL-27 on arthritis severity in a murine collagen-induced arthritis (CIA) model and its mechanism of action regarding control of regulatory T (Tregs) and IL-17-producing T helper 17 (Th17) cells. IL-27-Fc-treated CIA mice showed a lower severity of arthritis. IL-17 expression in the spleens was significantly decreased in IL-27-Fc-treated CIA mice compared with that in the CIA model. The Th17 population was decreased in the spleens of IL-27-Fc-treated CIA mice, whereas the CD4(+)CD25(+)Foxp3(+) Treg population increased. In vitro studies revealed that IL-27 inhibited IL-17 production in murine CD4(+) T cells, and the effect was associated with retinoic acid-related orphan receptor γT and signal transducer and activator of transcription 3 inhibition. In contrast, fluorescein isothiocyanate-labeled forkhead box P3 (Foxp3) and IL-10 were profoundly augmented by IL-27 treatment. Regarding the suppressive capacity of Treg cells, the proportions of CTLA-4(+) (cytotoxic T-lymphocyte antigen 4), PD-1(+) (programmed cell death protein 1) and GITR(+) (glucocorticoid-induced tumor necrosis factor receptor) Tregs increased in the spleens of IL-27-Fc-treated CIA mice. Furthermore, in vitro differentiated Treg cells with IL-27 exerted a more suppressive capacity on T-cell proliferation. We found that IL-27 acts as a reciprocal regulator of the Th17 and Treg populations in CD4(+) cells isolated from healthy human peripheral blood mononuclear cells (PBMCs), as well as from humans with rheumatoid arthritis (RA) PBMCs. Our study suggests that IL-27 has the potential to ameliorate overwhelming inflammation in patients with RA through a reciprocal regulation of Th17 and Treg cells.


Assuntos
Artrite Experimental/tratamento farmacológico , Interleucinas/uso terapêutico , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Artrite Experimental/imunologia , Células Cultivadas , Humanos , Interleucinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA
12.
Arthritis Rheum ; 64(11): 3543-52, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22736176

RESUMO

OBJECTIVE: To investigate the impact of STAT-3-mediated regulation on Th17 differentiation in patients with rheumatoid arthritis (RA). METHODS: CD4+ T cells isolated from peripheral blood (PB) and synovial fluid (SF) were stimulated to differentiate into Th17 cells or Treg cells. The activity of STAT-3 was knocked down by transfecting CD4+ T cells with small interfering RNA (siRNA). After 3 days in culture, the proportions of Th17 cells and Treg cells were measured by flow cytometry, and the production of interleukin-17 (IL-17) was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: The levels of IL-17, IL-6, IL-23, IL-1, and tumor necrosis factor α were significantly higher in RA SF and synovial tissue than in SF and synovial tissue from osteoarthritis patients. In RA synovial tissue, the expression of STAT-3 increased in proportion to the severity of synovitis, as shown by stromal cellularity, intimal hyperplasia, and inflammatory infiltration. The degree of Th17 differentiation was highest in RA SF, followed by RA PB, and lowest in normal subjects. In CD4+ T cells, transfection with STAT-3 siRNA prevented Th17 differentiation of mononuclear cells from RA PB and SF but increased the proportion of Treg cells. In contrast, inhibition of STAT-5, the transcription factor for Treg cells, increased the proportion of Th17 cells and reduced that of Treg cells. CONCLUSION: Our findings indicate that modulation of STAT-3 in CD4+ T cells affects the differentiation of Th17 cells and Treg cells in patients with RA. This role of STAT-3 in RA synovial T cells may provide a new therapeutic target for the management of RA.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Fator de Transcrição STAT3/genética , Linfócitos T Reguladores/citologia , Células Th17/citologia , Artrite Reumatoide/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismo
13.
J Korean Med Sci ; 26(10): 1333-8, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22022187

RESUMO

The aim of this study was to evaluate and compare the Child-Turcotte-Pugh (CTP) classification system and the model for end-stage liver disease (MELD) score in predicting the severity of the systemic inflammatory response in living-donor liver transplantation patients. Recipients of liver graft were allocated to a recipient group (n = 39) and healthy donors to a donor group (n = 42). The association between the CTP classification, the MELD scores and perioperative cytokine concentrations in the recipient group was evaluated. The pro-inflammatory cytokines measured included interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α; the anti-inflammatory cytokines measured included IL-10 and IL-4. Cytokine concentrations were quantified using sandwich enzyme-linked immunoassays. The IL-6, TNF-α, and IL-10 concentrations in the recipient group were significantly higher than those in healthy donor group patients. All preoperative cytokine levels, except IL-6, increased in relation to the severity of liver disease, as measured by the CTP classification. Additionally, all cytokine levels, except IL-6, were significantly correlated preoperatively with MELD scores. However, the correlations diminished during the intraoperative period. The CTP classification and the MELD score are equally reliable in predicting the severity of the systemic inflammatory response, but only during the preoperative period.


Assuntos
Citocinas/sangue , Falência Renal Crônica/classificação , Falência Renal Crônica/cirurgia , Transplante de Fígado , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico , Adulto , Feminino , Humanos , Interleucina-10/sangue , Interleucina-1beta/sangue , Interleucina-4/sangue , Interleucina-6/sangue , Fígado/patologia , Doadores Vivos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Índice de Gravidade de Doença , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangue
14.
J Korean Med Sci ; 26(9): 1132-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21935266

RESUMO

The interleukin-33 (IL-33)/ST2 pathway has emerged as an intercellular signaling system that participates in antigen-allergen response, autoimmunity and fibrosis. It has been suggested that IL-33/ST2 signaling has been involved in the pathogenesis of rheumatoid arthritis (RA), because IL-33 and its receptor have been specifically mapped to RA synovium. The aim of this study was to determine the levels of IL-33 and sST2 in sera and synovial fluids in patients with RA. The serum level of IL-33 was significantly higher in patients with RA (294.9 ± 464.0 pg/mL) than in healthy controls (96.0 ± 236.9 pg/mL, P = 0.002). The synovial fluid level of IL-33 was significantly higher in RA patients than in osteoarthritis patients. The level of serum sST2 was higher in RA patients than in healthy controls (P = 0.042). A significant relationship was found between the levels of IL-33 and IL-1ß (r = 0.311, P = 0.005), and IL-33 and IL-6 (r = 0.264, P = 0.017) in 81 RA patients. The levels of IL-33, sST2 and C-reactive protein decreased after conventional disease-modifying antirheumatic drugs treatment in 10 patients with treatment-naïve RA. Conclusively, IL-33 is involved in the pathogenesis of RA and may reflect the degree of inflammation in patients with RA.


Assuntos
Artrite Reumatoide/patologia , Interleucinas/análise , Receptores de Superfície Celular/análise , Adulto , Idoso , Antirreumáticos/uso terapêutico , Artrite Reumatoide/sangue , Artrite Reumatoide/tratamento farmacológico , Proteína C-Reativa/análise , Feminino , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-1beta/análise , Interleucina-1beta/sangue , Interleucina-33 , Interleucina-6/análise , Interleucina-6/sangue , Interleucinas/sangue , Masculino , Pessoa de Meia-Idade , Osteoartrite/sangue , Osteoartrite/patologia , Receptores de Superfície Celular/sangue , Líquido Sinovial/metabolismo
15.
Arthritis Res Ther ; 13(4): R113, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21749686

RESUMO

INTRODUCTION: The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of arthritis. We conducted this study to determine the effect of interleukin (IL)-17 on the expression and production of RAGE in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). The role of nuclear factor-κB (NF-κB) activator 1 (Act1) in IL-17-induced RAGE expression in RA-FLS was also evaluated. METHODS: RAGE expression in synovial tissues was assessed by immunohistochemical staining. RAGE mRNA production was determined by real-time polymerase chain reaction. Act-1 short hairpin RNA (shRNA) was produced and treated to evaluate the role of Act-1 on RAGE production. RESULTS: RAGE, IL-17, and Act-1 expression increased in RA synovium compared to osteoarthritis synovium. RAGE expression and production increased by IL-17 and IL-1ß (*P <0.05 vs. untreated cells) treatment but not by tumor necrosis factor (TNF)-α in RA-FLS. The combined stimuli of both IL-17 and IL-1ß significantly increased RAGE production compared to a single stimulus with IL-17 or IL-1ß alone (P <0.05 vs. 10 ng/ml IL-17). Act-1 shRNA added to the RA-FLS culture supernatant completely suppressed the enhanced production of RAGE induced by IL-17. CONCLUSIONS: RAGE was overexpressed in RA synovial tissues, and RAGE production was stimulated by IL-17 and IL-1ß. Act-1 contributed to the stimulatory effect of IL-17 on RAGE production, suggesting a possible inhibitory target for RA treatment.


Assuntos
Artrite Reumatoide/metabolismo , Regulação da Expressão Gênica/fisiologia , Interleucina-17/metabolismo , Receptores Imunológicos/biossíntese , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , RNA Mensageiro , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Receptor para Produtos Finais de Glicação Avançada , Membrana Sinovial/citologia , Transfecção
16.
Immunol Lett ; 124(2): 102-10, 2009 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-19446580

RESUMO

To examine whether grape seed proanthocyanidin extract (GSPE) which is known to act as an antioxidant has therapeutic effect on collagen-induced arthritis (CIA) in mice, an animal model of rheumatoid arthritis. Mice were treated with an intraperitoneal injection of GSPE (10, 50, or 100 mg/kg) or saline. Clinical, histological, and biochemical parameters were assessed. The effects of GSPE on osteoclastogenesis were determined by tartrate-resistant acid phosphatase (TRAP) staining of the inflamed joints and bone-marrow cells cultured with the receptor activator of nuclear factor B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Intracellular levels of hydrogen peroxide were determined using carboxy-dichlorodihydrofluorescein diacetate. GSPE treatment significantly attenuated the severity of CIA in a dose-dependent manner and reduced the histology scores for synovial inflammation, cartilage erosion, bone erosion, and the number of TRAP+ osteoclasts. GSPE treatment significantly reduced the numbers of tumor necrosis factor alpha (TNF-alpha)- or interleukin 17 (IL-17)-producing cells in the synovial tissue and the spontaneous production of TNF-alpha and IL-17 by splenocytes compared with those in the control mice. The serum levels of type-II-collagen-specific IgG2a and plasma levels of 8-isoprostane in the GSPE-treated mice were significantly lower than those in the control mice. GSPE dose-dependently suppressed osteoclastogenesis in vitro. GSPE significantly reduced hydrogen peroxide production by anti-CD3-monoclonal-antibody-stimulated CD4+ splenocytes. These results indicate that intraperitoneal injection of GSPE attenuated CIA in mice. GSPE may be useful in the treatment of rheumatoid arthritis.


Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Proantocianidinas/uso terapêutico , Fosfatase Ácida/imunologia , Fosfatase Ácida/metabolismo , Animais , Articulação do Tornozelo/efeitos dos fármacos , Articulação do Tornozelo/imunologia , Articulação do Tornozelo/metabolismo , Articulação do Tornozelo/patologia , Anticorpos/sangue , Anticorpos/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo II/farmacologia , Modelos Animais de Doenças , Extrato de Sementes de Uva , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/imunologia , Peróxido de Hidrogênio/metabolismo , Interleucina-17/antagonistas & inibidores , Interleucina-17/biossíntese , Interleucina-17/imunologia , Isoenzimas/imunologia , Isoenzimas/metabolismo , Isoprostanos/antagonistas & inibidores , Isoprostanos/sangue , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos DBA , Osteoclastos/efeitos dos fármacos , Osteoclastos/imunologia , Osteoclastos/metabolismo , Extratos Vegetais/administração & dosagem , Proantocianidinas/administração & dosagem , Ligante RANK/farmacologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Fosfatase Ácida Resistente a Tartarato , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia
17.
Immunol Lett ; 123(1): 21-30, 2009 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-19428548

RESUMO

To examine the effects of nuclear factor kappa B (NF-kappaB) inhibition on the secretion of macrophage migration inhibitory factor (MIF) in human CD4(+) T cells. Isolated human CD4(+) T cells were cultured for 24h with pharmacological inhibitors of NF-kappaB including parthenolide, pyrrolidine dithiocarbamate, BAY 11-7082, gliotoxin, oridonin, andrographolide, and NF-kappaB shRNA. MIF concentration was measured by intracellular flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. The intracellular concentrations O(2)(-), H(2)O(2), and glutathione were measured using the oxidation-sensitive fluorescent dyes dihydroethidium, dichlorodihydrofluorescein diacetate, and monochlorobimane, respectively. The amount of phosphorylated c-Jun was measured by Western blotting. Treatment of CD4(+) T cells with NF-kappaB inhibitors significantly increased MIF concentration in culture supernatants, MIF gene expression, and O(2)(-) production, and decreased the intracellular concentrations of MIF, H(2)O(2), and glutathione. Treatment with LY294002 (PI3K inhibitor) and SP600125 (JNK inhibitor) suppressed NF-kappaB inhibitor induced MIF mRNA expression and MIF secretion. LY294002 and SP600125 inhibited the parthenolide-induced phosphorylation of c-Jun. Treatment with H(2)O(2) decreased the amount of intracellular MIF protein and increased MIF concentration in the culture supernatant. N-acetylcysteine, an antioxidant precursor of glutathione, inhibited the parthenolide-induced and H(2)O(2)-induced secretion of MIF. These results indicate that pharmacological inhibition of NF-kappaB causes the release of MIF through de novo synthesis of MIF and the secretion of preformed MIF in CD4(+) T cells through the production of reactive oxygen species.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , NF-kappa B/antagonistas & inibidores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Glutationa/imunologia , Glutationa/metabolismo , Humanos , Oxirredutases Intramoleculares/biossíntese , Fatores Inibidores da Migração de Macrófagos/biossíntese , NF-kappa B/genética , NF-kappa B/metabolismo , Peroxidase/imunologia , Peroxidase/metabolismo , Peroxidase/farmacologia , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo
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