RESUMO
Self-improving dystrophic epidermolysis bullosa (DEB) is a genodermatosis that is inherited autosomal dominantly or recessively, and its clinical symptoms may improve or subside spontaneously. Herein, we report a case of self-improving DEB with COL7A1 p.Gly2025Asp variant. The diagnosis was made through histopathological, electron microscopic examination, and genetic testing. The same variant is also noted on his father, who presents with dystrophic toenails without any blisters. This study highlights that idiopathic nail dystrophy could be linked to congenital or hereditary disease. Furthermore, we conducted a review of the literature on the characteristics of reported cases of self-improving DEB with a personal or family history of nail dystrophy. The results supported our findings that nail dystrophy may be the sole manifestation in some family members. We suggest that individuals suffering from idiopathic nail dystrophy may seek genetic counselling when planning pregnancy to early evaluate the potential risk of hereditary diseases.
Assuntos
Colágeno Tipo VII , Epidermólise Bolhosa Distrófica , Mutação de Sentido Incorreto , Humanos , Epidermólise Bolhosa Distrófica/genética , Colágeno Tipo VII/genética , Masculino , Taiwan , Heterozigoto , Linhagem , Feminino , Adulto , Doenças da Unha/genéticaRESUMO
Vibrio vulnificus, a gram-negative bacterium, causes serious wound infections and septicemia. Once it develops into early phase sepsis, hyperinflammatory immune responses result in poor prognosis in patients. The present study aimed to examine the possible underlying pathogenic mechanism and explore potential agents that could protect against V. vulnificus cytotoxicity. Here, we report that infection of mouse macrophages with V.â¯vulnificus triggers antiphagocytic effects and pyroptotic inflammation via ATP-mediated purinergic P2X7 receptor (P2X7R) signaling. V.â¯vulnificus promoted P2X7-dependent nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 translocation, modulating the expression of the inflammasome sensor NLR family pyrin domain containing 3 (NLRP3), adaptor apoptosis-associated speck-like protein containing a card (ASC), and pyroptotic protein gasdermin D (GSDMD) in mouse macrophages. V.â¯vulnificus induced the NLRP3/caspase-1 inflammasome signaling complex expression that drives GSDMD transmembrane pore formation and secretion of interleukin (IL)-1ß, IL-18, and macrophage inflammatory protein-2 (MIP-2). This effect was blocked by P2X7R antagonists, indicating that the P2X7R mediates GSDMD-related pyroptotic inflammation in macrophages through the NF-κB/NLRP3/caspase-1 signaling pathway. Furthermore, blockade of P2X7R reduced V. vulnificus-colony-forming units in the spleen, immune cell infiltration into the skin and lung tissues, and serum concentrations of IL-1ß, IL-18, and MIP-2 in mice. These results indicate that P2X7R plays a vital role in mediating phagocytosis by macrophages and pyroptotic inflammation during V.â¯vulnificus infection and provides new opportunities for therapeutic intervention in bacterial infections.
RESUMO
The lung is the first and most frequent organ to fail among sepsis patients. The mortality rate of sepsis-related acute lung injury (ALI) is high. Despite appropriate antimicrobial therapy, no treatment strategies are available for sepsis-induced ALI. Stem cell-mediated paracrine signaling is a potential treatment method for various diseases. This study aimed to examine the effects of induced pluripotent stem cell-derived conditioned medium (iPSC-CM) combined with antibiotics on ALI in a rat model of Escherichia coli-induced sepsis. Rats were administered either iPSC-CM or the vehicle (saline) with antibiotics (ceftriaxone). After 72 h, liquid biopsy, bronchoalveolar lavage fluid (BALF), and tissues were harvested for analysis. Survival rates were observed for up to 3 days. Furthermore, we examined the effects of iPSC-CM on cytokine production, metalloproteinase 9 (MMP-9) expression, and NLRP3-ASC interaction in RAW264.7 cells stimulated with lipopolysaccharide/interferon-γ (LPS/IFN-γ). Combined treatment of iPSC-CM with antibiotics significantly improved survival in E. coli-infected rats (p = 0.0006). iPSC-CM ameliorated E. coli-induced infiltration of macrophages, reducing the number of cells in BALF, and suppressing interleukin (IL)-1ß, MIP-2, IL-6, and MMP-9 messenger RNA in lung sections. iPSC-CM treatment attenuated NLRP3 expression and inhibited NLRP3 inflammasome activation by disrupting NLRP3-mediated ASC complex formation in LPS/IFN-γ-primed RAW264.7 cells. This study reveals the mechanisms underlying iPSC-CM-conferred anti-inflammatory activity in ALI through the attenuation of macrophage recruitment to the lung, thus inactivating NLRP3 inflammasomes in macrophages. iPSC-CM therapy may be a useful adjuvant treatment to reduce sepsis-related mortality by ameliorating ALI.
Assuntos
Lesão Pulmonar Aguda , Células-Tronco Pluripotentes Induzidas , Sepse , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Antibacterianos/efeitos adversos , Ceftriaxona/efeitos adversos , Meios de Cultivo Condicionados/farmacologia , Escherichia coli/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos , Sepse/tratamento farmacológicoRESUMO
Glycine N-methyltransferase (GNMT) regulates S-adenosylmethionine (SAMe), a methyl donor in methylation. Over-expressed SAMe may cause neurogenic capacity reduction and memory impairment. GNMT knockout mice (GNMT-KO) was applied as an experimental model to evaluate its effect on neurons. In this study, proteins from brain tissues were studied using proteomic approaches, Haemotoxylin and Eosin staining, immunohistochemistry, Western blotting, and ingenuity pathway analysis. The expression of Receptor-interacting protein 1(RIPK1) and Caspase 3 were up-regulated and activity-dependent neuroprotective protein (ADNP) was down-regulated in GNMT-KO mice regardless of the age. Besides, proteins related to neuropathology, such as excitatory amino acid transporter 2, calcium/calmodulin-dependent protein kinase type II subunit alpha, and Cu-Zn superoxide dismutase were found only in the group of aged wild-type mice; 4-aminobutyrate amino transferase, limbic system-associated membrane protein, sodium- and chloride-dependent GABA transporter 3 and ProSAAS were found only in the group of young GNMT-KO mice and are related to function of neurons; serum albumin and Rho GDP dissociation inhibitor 1 were found only in the group of aged GNMT-KO mice and are connected to neurodegenerative disorders. With proteomic analyses, a pathway involving Gonadotropin-releasing hormone (GnRH) signal was found to be associated with aging. The GnRH pathway could provide additional information on the mechanism of aging and non-aging related neurodegeneration, and these protein markers may be served in developing future therapeutic treatments to ameliorate aging and prevent diseases.
Assuntos
Envelhecimento/metabolismo , Biomarcadores , Doenças Neurodegenerativas/metabolismo , Animais , Biomarcadores/metabolismo , Encéfalo , Senescência Celular , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imuno-Histoquímica , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/etiologia , Neurônios/metabolismo , Prognóstico , Proteoma , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacosRESUMO
Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial proliferation and remodeling, resulting in a specific increase in right ventricle systolic pressure (RVSP) and, ultimately right ventricular failure. Recent studies have demonstrated that caffeic acid phenethyl ester (CAPE) exerts a protective role in NF-κB-mediated inflammatory diseases. However, the effect of CAPE on PAH remains to be elucidated. In this study, monocrotaline (MCT) was used to establish PAH in rats. Two weeks after the induction of PAH by MCT, CAPE was administrated by intraperitoneal injection once a day for two weeks. Pulmonary hemodynamic measurements and pulmonary artery morphological assessments were examined. Our results showed that administration of CAPE significantly suppressed MCT-induced vascular remodeling by decreasing the HIF-1α expression and PDGF-BB production, and improved in vivo RV systolic performance in rats. Furthermore, CAPE inhibits hypoxia- and PDGF-BB-induced HIF-1α expression by decreasing the activation of the AKT/ERK pathway, which results in the inhibition of human pulmonary artery smooth muscle cells (hPASMCs) proliferation and prevention of cells resistant to apoptosis. Overall, our data suggest that HIF-1α is regarded as an alternative target for CAPE in addition to NF-κB, and may represent a promising therapeutic agent for the treatment of PAH diseases.
Assuntos
Ácidos Cafeicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Álcool Feniletílico/análogos & derivados , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Hemodinâmica/efeitos dos fármacos , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/tratamento farmacológico , Hipertrofia Ventricular Direita/tratamento farmacológico , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Imuno-Histoquímica , Álcool Feniletílico/farmacologia , Fator de Crescimento Derivado de Plaquetas/genética , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Remodelação Vascular/efeitos dos fármacosRESUMO
Malignant melanoma is developed from pigment-containing cells, melanocytes, and primarily found on the skin. Malignant melanoma still has a high mortality rate, which may imply a lack of therapeutic agents. Lakoochin A, a compound isolated from Artocarpus lakoocha and Artocarpus xanthocarpus, has an inhibitory function of tyrosinase activity and melanin production, but the anti-cancer effects are still unclear. In the current study, the therapeutic effects of lakoochin A with their apoptosis functions and possible mechanisms were investigated on A375.S2 melanoma cells. Several methods were applied, including 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), flow cytometry, and immunoblotting. Results suggest that lakoochin A attenuated the growth of A375.S2 melanoma cells through an apoptosis mechanism. Lakoochin A first increase the production of cellular and mitochondrial reactive oxygen species (ROSs); mitochondrial ROSs then promote mitogen-activated protein kinases (MAPKs) pathway activation and raise downstream apoptosis-related protein and caspase expression. This is the first study to demonstrate that lakoochin A, through ROS-MAPK, apoptosis-related proteins, caspases cascades, can induce melanoma cell apoptosis and may be a potential candidate compound for treating malignant melanoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Melanoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estilbenos/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
Upregulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via adhesion molecule induction and then causes lung injury. However, the mechanisms underlying LPS-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. We showed that LPS induced ICAM-1 expression in HPAEpiCs, revealed by Western blotting, RT-PCR, real-time PCR, and promoter assay. Pretreatment with the inhibitor of c-Src (protein phosphatase-1, PP1), reactive oxygen species (ROS) (Edaravone), NADPH oxidase (apocynin and diphenyleneiodonium chloride), EGFR (AG1478), PDGFR (AG1296), phosphatidylinositol-3-kinase (PI3K) (LY294002), MEK1/2 (U0126), or NF-κB (Bay11-7082) and transfection with siRNAs of c-Src, EGFR, PDGFR, Akt, p47(phox), Nox2, Nox4, p42, and p65 markedly reduced LPS-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with LPS. In addition, we established that LPS stimulated phosphorylation of c-Src, EGFR, PDGFR, Akt, or p65, which was inhibited by pretreatment with their respective inhibitors. LPS induced Toll-like receptor 4 (TLR4), MyD88, TNF receptor-associated factor 6 (TRAF6), c-Src, p47(phox), and Rac1 complex formation 2, which was attenuated by transfection with c-Src or TRAF6 siRNA. Furthermore, LPS markedly enhanced NADPH oxidase activation and intracellular ROS generation, which were inhibited by PP1. We established that LPS induced p42/p44 MAPK activation via a c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt-dependent pathway in these cells. Finally, we observed that LPS significantly enhanced NF-κB and IκBα phosphorylation, NF-κB translocation, and NF-κB promoter activity, which were inhibited by PP1, Edaravone, apocynin, diphenyleneiodonium chloride, AG1478, AG1296, LY294002, or U0126. These results demonstrated that LPS induces p42/p44 MAPK activation mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase/ROS/EGFR, PDGFR/PI3K/Akt pathway, which in turn initiates the activation of NF-κB and ultimately induces ICAM-1 expression in HPAEpiCs.
Assuntos
Células Epiteliais Alveolares/metabolismo , Molécula 1 de Adesão Intercelular/genética , Lipopolissacarídeos/farmacologia , Quinases da Família src/fisiologia , Células Epiteliais Alveolares/imunologia , Proteína Tirosina Quinase CSK , Células Cultivadas , Receptores ErbB/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/imunologia , Pulmão/imunologia , Pulmão/patologia , NADPH Oxidases/metabolismo , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Ativação Transcricional/imunologiaRESUMO
Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)-1, displays an anti-inflammatory effect in various cellular injuries, but the precise mechanisms of HO-1 expression remain unknown. We used the transition metal carbonyl compound carbon monoxide-releasing molecule-2 (CORM-2) that acts as carbon monoxide donor. The effects of CORM-2 on expression of HO-1 in human tracheal smooth muscle cells (HTSMCs) were determined by Western blot, real-time PCR, and promoter activity assay. In HTSMCs, CORM-2 activated Nrf2 through the activation of a c-Src/EGFR/PI3K/Akt-dependent pathway, resulting in HO-1 expression. We showed that CORM-2-induced HO-1 protein and mRNA levels were inhibited by the inhibitor of c-Src (PP1 or SU6656), EGFR (AG1478), PI3K (LY294002), Akt (SH-5), JNK1/2 (SP600125), or p38 MAPK (SB202190) and transfection with siRNA of c-Src, EGFR, Akt, p38, JNK2, or Nrf2 in HTSMCs. We also showed that CORM-2 stimulated c-Src, EGFR, Akt, p38 MAPK, and JNK1/2 phosphorylation. CORM-2 also enhanced Nrf2 translocation from the cytosol to the nucleus and antioxidant response element (ARE) promoter activity. Moreover, CORM-2 mediated p38 MAPK and JNK1/2 activation via a c-Src/EGFR/PI3K/Akt pathway, which further enhanced Nrf2 activation and translocation. Finally, we observed that CORM-2 induced in vivo binding of Nrf2 to the HO-1 promoter. CORM-2 activates the c-Src/EGFR/PI3K/Akt/JNK1/2 and p38 MAPK pathways, which in turn trigger Nrf2 activation and ultimately induces HO-1 expression in HTSMCs. Thus, the HO-1/CO system might be potential therapeutics in airway diseases.
Assuntos
Monóxido de Carbono/metabolismo , Receptores ErbB/metabolismo , Heme Oxigenase-1/metabolismo , Miócitos de Músculo Liso/metabolismo , Compostos Organometálicos/metabolismo , Traqueia/metabolismo , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologiaRESUMO
Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.
Assuntos
Dinoprostona/metabolismo , Pulmão/patologia , Fosfolipases A2/metabolismo , Pneumonia/patologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular , Cromonas/farmacologia , Proteína p300 Associada a E1A/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Pulmão/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipases A2/biossíntese , Fosfolipases A2/genética , Fosforilação/genética , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/biossíntese , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Tirfostinas/farmacologia , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
BACKGROUND: Matrix metalloproteinase-9 (MMP-9) has been shown to be induced by cytokines including TNF-α and may contribute to bone inflammatory diseases. However, the mechanisms underlying MMP-9 expression induced by TNF-α in MC3T3-E1 cells remain unclear. RESULTS: We applied gelatin zymography, Western blot, RT-PCR, real-time PCR, selective pharmacological inhibitors of transcription (actinomycin D, Act.D), translation (cycloheximide, CHI), c-Src (PP1), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), and NF-κB (Bay11-7082), respective siRNAs transfection, promoter assay, immunofluorescence staining, and ELISA to investigate the MMP-9 expression and soluble ICAM-1 (sICAM-1) release induced by TNF-α in MC3T3-E1 cells. Here we demonstrated that TNF-α-induced MMP-9 expression was attenuated by Act.D, CHI, PP1, U0126, SB202190, SP600125, and Bay11-7082, and by the transfection with siRNAs for ERK2, p38 MAPK, and JNK2. TNF-α-stimulated TNFR1, TRAF2, and c-Src complex formation was revealed by immunoprecipitation and Western blot. Furthermore, TNF-α-stimulated NF-κB phosphorylation and translocation were blocked by Bay11-7082, but not by PP1, U0126, SB202190, or SP600125. TNF-α time-dependently induced MMP-9 promoter activity which was also inhibited by PP1, U0126, SB202190, SP600125, or Bay11-7082. Up-regulation of MMP-9 was associated with the release of sICAM-1 into the cultured medium, which was attenuated by the pretreatment with MMP-2/9i, an MMP-9 inhibitor. CONCLUSIONS: In this study, we demonstrated that TNF-α up-regulates MMP-9 expression via c-Src, MAPKs, and NF-κB pathways. In addition, TNF-α-induced MMP-9 expression may contribute to the production of sICAM-1 by MC3T3-E1 cells. The interplay between MMP-9 expression and sICAM-1 release may exert an important role in the regulation of bone inflammatory diseases.
Assuntos
Molécula 1 de Adesão Intercelular/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Metaloproteinase 9 da Matriz/genética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno , NF-kappa B/metabolismo , Osteíte/metabolismo , Osteíte/patologia , Osteoblastos/metabolismo , Fosforilação , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genética , Fator de Necrose Tumoral alfa/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Rheumatoid arthritis (RA) is characterized by chronic inflammatory infiltration of the synovium and elevation of proinflammatory cytokines. Cytosolic phospholipase A2 (cPLA2) is involved in the development of inflammatory diseases. Heme oxygenase-1 (HO-1) has been shown to possess anti-inflammatory properties. The objective of the study was to investigate the detailed mechanisms of TNF-α-induced cPLA2 expression and to determine whether carbon monoxide releasing molecule-2 (CO-RM2) suppresses TNF-α-induced expression of NF-κB-related proinflammatory genes, including cPLA2, via HO-1 induction in RA synovial fibroblasts (RASFs). Here, we reported that TNF-α-induced cPLA2 expression was mediated through TNFR1/PKCα-dependent signaling pathways, including NADPH oxidase (NOX) activation/ROS production and NF-κB activation. CO-RM2 significantly suppressed TNF-α-induced cPLA2 expression by inhibiting the ROS generation and the phosphorylation of NF-κB p65 and IKK α/ß, but not the phosphorylation of p38 MAPK and JNK1/2. These results were further confirmed by a ChIP assay to detect the NF-κB DNA-binding activity. Our results demonstrated that induction of HO-1 by CO-RM2 exerted anti-inflammatory and antioxidant effects which were required in concert to prevent the activation of NF-κB leading to induction of various inflammatory genes implicated in the pathogenesis of RA.
Assuntos
Heme Oxigenase-1/metabolismo , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Fosfolipases A2/metabolismo , Proteína Quinase C-alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Citosol/enzimologia , Dinoprostona/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Compostos Organometálicos/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Membrana Sinovial/citologiaRESUMO
AIMS: Pulmonary arterial hypertension (PAH) is characterized by extensive pulmonary arterial remodelling. Although mesenchymal stem cell (MSC)-derived exosomes provide protective effects in PAH, MSCs exhibit limited senescence during in vitro expansion compared with the induced pluripotent stem cells (iPSCs). Moreover, the exact mechanism is not known. METHODS AND RESULTS: In this study, we used murine iPSCs generated from mouse embryonic fibroblasts with triple factor (Oct4, Klf4, and Sox2) transduction to determine the efficacy and action mechanism of iPSC-derived exosomes (iPSC-Exo) in attenuating PAH in rats with monocrotaline (MCT)-induced pulmonary hypertension. Both early and late iPSC-Exo treatment effectively prevented the wall thickening and muscularization of pulmonary arterioles, improved the right ventricular systolic pressure, and alleviated the right ventricular hypertrophy in MCT-induced PAH rats. Pulmonary artery smooth muscle cells (PASMC) derived from MCT-treated rats (MCT-PASMC) developed more proliferative and pro-migratory phenotypes, which were attenuated by the iPSC-Exo treatment. Moreover, the proliferation and migration of MCT-PASMC were reduced by iPSC-Exo with suppression of PCNA, cyclin D1, MMP-1, and MMP-10, which are mediated via the HIF-1α and P21-activated kinase 1/AKT/Runx2 pathways. CONCLUSION: IPSC-Exo are effective at reversing pulmonary hypertension by reducing pulmonary vascular remodelling and may provide an iPSC-free therapy for the treatment of PAH.
Assuntos
Exossomos , Hipertensão Pulmonar , Células-Tronco Pluripotentes Induzidas , Hipertensão Arterial Pulmonar , Ratos , Animais , Camundongos , Hipertensão Arterial Pulmonar/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Remodelação Vascular , Exossomos/metabolismo , Fibroblastos/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Artéria Pulmonar , Monocrotalina/efeitos adversos , Monocrotalina/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismoRESUMO
TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-l-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47(phox), p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure.
Assuntos
Quinase 2 de Adesão Focal/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Miocárdio , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Linhagem Celular , Miocárdio/enzimologia , Miocárdio/metabolismo , NF-kappa B/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
BACKGROUND: Endothelin-1 (ET-1) is a proinflammatory mediator and elevated in the regions of several brain injury and inflammatory diseases. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the regulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system in various cell types. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear. Herein we investigated the effects of ET-1 in COX-2 regulation in mouse brain microvascular endothelial (bEnd.3) cells. RESULTS: The data obtained with Western blotting, RT-PCR, and immunofluorescent staining analyses showed that ET-1-induced COX-2 expression was mediated through an ETB-dependent transcriptional activation. Engagement of Gi- and Gq-protein-coupled ETB receptors by ET-1 led to phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 and then activated transcription factor NF-κB. Moreover, the data of chromatin immunoprecipitation (ChIP) and promoter reporter assay demonstrated that the activated NF-κB was translocated into nucleus and bound to its corresponding binding sites in COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, up-regulation of COX-2 by ET-1 promoted PGE2 release in these cells. CONCLUSIONS: These results suggested that in mouse bEnd.3 cells, activation of NF-κB by ETB-dependent MAPK cascades is essential for ET-1-induced up-regulation of COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rationally therapeutic interventions for brain injury or inflammatory diseases.
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OBJECTIVE: Reactive oxygen species (ROS) produced by cytokines induce the expression of inflammatory mediators in rheumatoid arthritis (RA). Heme oxygenase 1 (HO-1) exerts an antiinflammatory effect. The aim of this study was to examine the mechanisms underlying interleukin-1ß (IL-1ß)-induced cytosolic phospholipase A2 (cPLA2) expression through ROS generation as modulated by HO-1 in RA synovial fibroblasts (RASFs). METHODS: IL-1ß-induced ROS generation was determined by flow cytometry. The involvement of MAPKs and NADPH oxidase (NOX)/ROS in IL-1ß-induced cPLA2 expression was investigated using pharmacologic inhibitors and transfection with small interfering RNAs (siRNAs) and was analyzed by Western blotting and promoter assay. Overexpression of HO-1 was performed by transfection of RASFs with a recombinant adenovirus containing human HO-1 plasmid. SCID mice with inflammation caused by IL-1ß were infected with adenovirus containing HO-1. Histologic characterization of joint inflammation and local expression of cPLA2 were evaluated after treatment. RESULTS: IL-1ß-induced cPLA2 expression was mediated through NOX activation/ROS production, which was attenuated by N-acetylcysteine (NAC; a scavenger of ROS), the inhibitors of NOX (diphenyleneiodonium chloride and apocynin), MEK-1/2 (U0126), and JNK-1/2 (SP600125), transfection with the respective siRNAs, and the overexpression of HO-1 in RASFs. IL-1ß-induced cPLA2 expression was mediated through recruitment of activator protein 1 (AP-1) to the cPLA2 promoter region, which was attenuated by NAC and overexpression of HO-1. Furthermore, HO-1 overexpression inhibited IL-1ß-mediated cPLA2 expression in SCID mice. CONCLUSION: In RASFs, IL-1ß induced cPLA2 expression via activation of p42/p44 MAPK and JNK-1/2, leading to p47phox phosphorylation, ROS production, and AP-1 activation. The induction of HO-1 exerted protective effects on the pathogenesis of RA.
Assuntos
Artrite Reumatoide/metabolismo , Heme Oxigenase-1/metabolismo , Interleucina-1beta/farmacologia , NADPH Oxidases/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismoRESUMO
BACKGROUND: Current guideline-recommended multiparameters used to assess the risk levels of pulmonary arterial hypertension (PAH) are invasive hemodynamic measurements or effort-dependent exercise tests. Serum natriuretic peptide is only one kind of effort-free biomarker that has been adopted for risk assessment. This study aimed to investigate the application of homocysteine as a non-invasive and effort-free measurement for the risk assessment of patients with PAH. METHODS: Samples of 50 patients diagnosed with PAH via right heart catheterization were obtained, and the patients were divided into low-, intermediate- and high-risk groups for further analysis. Additionally, serum N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) and homocysteine levels of monocrotaline (MCT)-induced PAH rats were analyzed at each week with progressed severity of PAH, and they were sacrificed on day 28 with pathology being assessed. RESULTS: Hyperhomocysteinemia was an independent predictor (odds ratio [OR]: 1.256; 95% confidence interval [CI]: 1.002-1.574) and showed a linear correlation with NT-proBNP. Hyperhomocysteinemia could discriminate between low/intermediate and high-risk levels in PAH with a cut-off value in 12 µmol/L. Moreover, the elevated homocysteine levels by weeks in MCT rats also demonstrated the association between homocysteine and the severity of PAH. CONCLUSIONS: Homocysteine can be a non-invasive and effort-free risk assessment for patients with pulmonary hypertension. Homocysteine level had a linear correlation with NT-proBNP level, and patients with hyperhomocysteinemia had a higher risk level, higher NT-proBNP level, and decreased lower diffusing capacity for carbon monoxide. The correlation between homocysteine level and PAH severity was also demonstrated in MCT rats.
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Nanofiber meshes (NFMs) loaded with therapeutic agents are very often employed to treat hard-to-heal wounds such as diabetic wounds. However, most of the NFMs have limited capability to load multiple or hydrophilicity distinctive-therapeutic agents. The therapy strategy is therefore significantly hampered. To tackle the innate drawback associated with the drug loading versatility, a chitosan-based nanocapsule-in-nanofiber (NC-in-NF) structural NFM system is developed for simultaneous loading of hydrophobic and hydrophilic drugs. Oleic acid-modified chitosan is first converted into NCs by the developed mini-emulsion interfacial cross-linking procedure, followed by loading a hydrophobic anti-inflammatory agent Curcumin (Cur) into the NCs. Sequentially, the Cur-loaded NCs are successfully introduced into reductant-responsive maleoyl functional chitosan/polyvinyl alcohol NFMs containing a hydrophilic antibiotic Tetracycline hydrochloride. Having a co-loading capability for hydrophilicity distinctive agents, biocompatibility, and a controlled release property, the resulting NFMs have demonstrated the efficacy on promoting wound healing either in normal or diabetic rats.
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BACKGROUND: Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Moreover, bradykinin (BK) induces the expression of several inflammatory proteins in brain astrocytes. Recent studies have suggested that increased oxidative stress is implicated in the brain inflammation and injury. However, whether BK induced MMP-9 expression mediated through oxidative stress remains virtually unknown. Herein we investigated the role of redox signals in BK-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells). RESULTS: In the study, we first demonstrated that reactive oxygen species (ROS) plays a crucial role in BK-induced MMP-9 expression in cultured brain astrocytes (in vitro) and animal brain tissue (in vivo) models. Next, BK-induced MMP-9 expression is mediated through a Ca2+-mediated PKC-α linking to p47phox/NADPH oxidase 2 (Nox2)/ROS signaling pathway. Nox2-dependent ROS generation led to activation and up-regulation of the downstream transcriptional factor AP-1 (i.e. c-Fos and c-Jun), which bound to MMP-9 promoter region, and thereby turned on transcription of MMP-9 gene. Functionally, BK-induced MMP-9 expression enhanced astrocytic migration. CONCLUSIONS: These results demonstrated that in RBA-1 cells, activation of AP-1 (c-Fos/c-Jun) by the PKC-α-mediated Nox2/ROS signals is essential for up-regulation of MMP-9 and cell migration enhanced by BK.
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OBJECTIVE: Cytosolic phospholipase A2 (cPLA2) is a rate-limiting enzyme that plays a critical role in the biosynthesis of eicosanoids. The aim of this study was to investigate the mechanisms underlying interleukin-1ß (IL-1ß)-induced cPLA2 expression in human rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Synovial tissue was obtained from patients with RA who were undergoing joint replacement surgery. In a mouse model of IL-1ß-mediated inflammatory arthritis, neutrophil infiltration, bone erosion, and cPLA2 expression in ankle synovium were analyzed by immunohistochemistry. IL-1ß-induced cPLA2 expression was determined by Western blotting, real-time polymerase chain reaction, and gene promoter assay using pharmacologic inhibitors and transfection with short hairpin RNAs or small interfering RNAs. The recruitment of NF-κB and p300 to the cPLA2 promoter was determined by chromatin immunoprecipitation assay. Prostaglandin E2 (PGE2) biosynthesis was evaluated by enzyme-linked immunosorbent assay. RESULTS: IL-1ß-induced cPLA2 expression and PGE2 release were mediated through a myeloid differentiation factor 88 (MyD88)/c-Src-dependent matrix metalloproteinase (MMP)/heparin-binding epidermal growth factor (HB-EGF) cascade linking to transactivation of the EGF receptor (EGFR)/phosphatidylinositol 3-kinase (PI 3-kinase)/Akt, p300, and NF-κB p65 pathways. IL-1ß also stimulated Akt phosphorylation and nuclear translocation. Activation of Akt eventually led to the acetylation of histone residues by phosphorylation and recruitment of p300 and enhanced its histone acetyltransferase activity on the NF-κB elements of the cPLA2 promoter. IL-1ß-induced NF-κB transcriptional activity was mediated through a PI 3-kinase/Akt-dependent cascade. Up-regulation of cPLA2 by IL-1ß increased PGE(2) biosynthesis in RASFs. CONCLUSION: IL-1ß-induced cPLA2 expression is mediated through activation of the MyD88/c-Src, MMP/HB-EGF, EGFR/PI 3-kinase/Akt, p300, and NF-κB pathways. These results provide insights into the mechanisms underlying IL-1ß-enhanced joint inflammatory responses in RA and may inspire new targeted therapeutic approaches.
Assuntos
Artrite Reumatoide/enzimologia , Dinoprostona/metabolismo , Proteína p300 Associada a E1A/metabolismo , Fibroblastos/enzimologia , Interleucina-1beta/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Membrana Sinovial/enzimologia , Animais , Humanos , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Patients with end-stage renal disease often need dialysis to maintain their lives because of donor organ shortage. The creation of a transplantable graft to permanently replace kidney function would overcome the organ shortage problem and the morbidity associated with immunosuppression. In the present study, we decellularized rat kidneys by the perfusion of detergent, yielding acellular scaffolds with the vascular, uretic, as well as cortical and medullary architecture. To regenerate the functional organ, we seeded tubular epithelial cells and mouse kidney progenitor cells from the ureter together with endothelial cells and mouse kidney progenitor cells from the renal artery. The renal constructs from seeded cells were cultured in a whole-organ bioreactor. After 3 months of organ culture, the seeded cells formed renal tubules, grew in the glomeruli, and some mouse kidney progenitor cells were also scattered in the interstitium. We tested the function of the bioengineered kidney with standardized perfusate in vitro. The bioengineered kidney not only produced urine but also reabsorbed albumin, glucose, and calcium. We conclude that seeded cell-based bioengineering of kidneys with physiological secreting and reabsorbing properties is possible and holds therapeutic promise.