RESUMO
PURPOSE: To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. METHODS: Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. RESULTS: Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. CONCLUSIONS: The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.
Assuntos
Indutores da Angiogênese/farmacologia , Caryophyllaceae/química , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Extratos Vegetais/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Fosfatidilinositol 3-Quinases/análise , Extratos Vegetais/química , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/análise , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/efeitos dos fármacos , Reprodutibilidade dos Testes , Transdução de Sinais , Fatores de TempoRESUMO
Abstract Purpose To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. Methods Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. Results Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. Conclusions The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.
Assuntos
Animais , Masculino , Cicatrização/efeitos dos fármacos , Extratos Vegetais/farmacologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Caryophyllaceae/química , Indutores da Angiogênese/farmacologia , Fatores de Tempo , Imuno-Histoquímica , Extratos Vegetais/química , Transdução de Sinais , Western Blotting , Reprodutibilidade dos Testes , Ratos Sprague-Dawley , Fosfatidilinositol 3-Quinases/análise , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Células Endoteliais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/análise , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacosRESUMO
PURPOSE: This study was conducted to investigate the effect of normal mesenteric lymph (NML) from mice on the spleen injury induced by lipopolysaccharide (LPS) challenge. METHODS: Mice in the LPS and LPS+NML groups received an intraperitoneal injection of LPS (35 mg/kg) and kept for 6 h.. The mice in the LPS+NML group received NML treatment at 1 h after LPS injection. Afterward, the splenic morphology, the levels of lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), phosphorylation mitogen-activated protein kinases (MAPKs), and inflammatory mediators in splenic tissue were investigated. RESULTS: LPS injection induced spleen injury, increased the levels of LBP, CD14, tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interferon γ (IFN-γ), and decreased the IL-4 content in the spleen. By contrast, NML treatment reversed these changes. Meanwhile, the LPS challenge decreased the phosphorylation levels of p38 MAPK, extracellular regulated protein kinases 1/2, and c-Jun N-terminal kinase (JNK). Moreover, the phosphorylation levels of p38 MAPK and JNK were further decreased by the NML administration. CONCLUSION: rRdThe normal mesenteric lymph treatment alleviated lipopolysaccharide induced spleen injury by attenuating LPS sensitization and production of TNF-α, IL-6, and IFN-γ.
Assuntos
Lipopolissacarídeos/administração & dosagem , Linfonodos/transplante , Mesentério , Esplenopatias/terapia , Proteínas de Fase Aguda/análise , Animais , Proteínas de Transporte/análise , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Injeções Intraperitoneais , Receptores de Lipopolissacarídeos/análise , Glicoproteínas de Membrana/análise , Camundongos Endogâmicos BALB C , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Distribuição Aleatória , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
PURPOSE: This study was conducted to investigate the effect of normal mesenteric lymph (NML) from mice on the spleen injury induced by lipopolysaccharide (LPS) challenge.METHODS: Mice in the LPS and LPS+NML groups received an intraperitoneal injection of LPS (35 mg/kg) and kept for 6 h.. The mice in the LPS+NML group received NML treatment at 1 h after LPS injection. Afterward, the splenic morphology, the levels of lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), phosphorylation mitogen-activated protein kinases (MAPKs), and inflammatory mediators in splenic tissue were investigated.RESULTS:LPS injection induced spleen injury, increased the levels of LBP, CD14, tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interferon γ (IFN-γ), and decreased the IL-4 content in the spleen. By contrast, NML treatment reversed these changes. Meanwhile, the LPS challenge decreased the phosphorylation levels of p38 MAPK, extracellular regulated protein kinases 1/2, and c-Jun N-terminal kinase (JNK). Moreover, the phosphorylation levels of p38 MAPK and JNK were further decreased by the NML administration.CONCLUSION:rRdThe normal mesenteric lymph treatment alleviated lipopolysaccharide induced spleen injury by attenuating LPS sensitization and production of TNF-α, IL-6, and IFN-γ.
Assuntos
Animais , Lipopolissacarídeos/administração & dosagem , Linfonodos/transplante , Mesentério , Esplenopatias/terapia , Proteínas de Fase Aguda/análise , /análise , Proteínas de Transporte/análise , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Injeções Intraperitoneais , Camundongos Endogâmicos BALB C , Glicoproteínas de Membrana/análise , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Distribuição Aleatória , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
This study was conducted to investigate the effect of normal mesenteric lymph (NML) from mice on the spleen injury induced by lipopolysaccharide (LPS) challenge. Mice in the LPS and LPS+NML groups received an intraperitoneal injection of LPS (35 mg/kg) and kept for 6 h.. The mice in the LPS+NML group received NML treatment at 1 h after LPS injection. Afterward, the splenic morphology, the levels of lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), phosphorylation mitogen-activated protein kinases (MAPKs), and inflammatory mediators in splenic tissue were investigated.RESULTS:LPS injection induced spleen injury, increased the levels of LBP, CD14, tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interferon γ (IFN-γ), and decreased the IL-4 content in the spleen. By contrast, NML treatment reversed these changes. Meanwhile, the LPS challenge decreased the phosphorylation levels of p38 MAPK, extracellular regulated protein kinases 1/2, and c-Jun N-terminal kinase (JNK). Moreover, the phosphorylation levels of p38 MAPK and JNK were further decreased by the NML administration. rRdThe normal mesenteric lymph treatment alleviated lipopolysaccharide induced spleen injury by attenuating LPS sensitization and production of TNF-α, IL-6, and IFN-γ.(AU)
Assuntos
Animais , Lipopolissacarídeos/administração & dosagem , Linfonodos/transplante , Mesentério , Esplenopatias/terapia , Proteínas de Fase Aguda/análise , Receptores de Lipopolissacarídeos/análise , Proteínas de Transporte/análise , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Injeções Intraperitoneais , Glicoproteínas de Membrana/análise , Camundongos Endogâmicos BALB C , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Distribuição Aleatória , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
In order to achieve the goal of this article, as an example we will describe the strategies followed to analyze the presence of the multi-kinase complex at the mitochondria and the posttranslational modification of two key mitochondrial proteins, which participate in the regulation of cholesterol transport across the mitochondrial membranes and in the regulation of steroid biosynthesis. Hormones, ions or growth factors modulate steroid biosynthesis by the posttranslational phosphorylation of proteins. The question still remains on how phosphorylation events transmit a specific signal to its mitochondrial site of action. Cholesterol transport requires specific interactions in mitochondria between several proteins including a multi-kinase complex. The presence of this multi-kinase complex at the mitochondria reveals the importance of the posttranslational modification of mitochondrial proteins for its activity and functions. The activation of PKA triggers the posttranslational modification of the mitochondrial acyl-CoA thioesterase (Acot2), which releases arachidonic acid (AA) in the mitochondria, and the activation of a kinase cascade that leads to the phoshorylation of the steroidogenic acute regulatory (StAR) protein. The function of StAR is to facilitate the access of cholesterol to the first enzyme of the biosynthesis process and its induction is dependent on Acot2 and intramitochondrial AA release. Truncation of the StAR protein is associated with the steroid deficiency disease, congenital lipoid adrenal hyperplasia.
Assuntos
Mitocôndrias/enzimologia , Proteínas Quinases/análise , Proteínas Quinases/metabolismo , Esteroides/biossíntese , Tioléster Hidrolases/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/análise , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Mitocôndrias/química , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Tioléster Hidrolases/análise , Tioléster Hidrolases/genética , TransfecçãoRESUMO
We studied the proton secretion mechanisms involved with pHi regulation in immortalized rat proximal tubule cells (IRPTC), a SV40-immortalized cell line derived from rat proximal tubule, and characterized the effects of serum deprivation on them. Using pHi measurements with the fluorescent probe BCECF, we demonstrated that the IRPTC express both Na+/H+ exchanger and H+-ATPase, but only NHE1 is modulated by serum deprivation. In these cells, 24 h of serum starvation increased pHi from 7.08+/-0.008 (n=34) to 7.18+/-0.018 (n=33) as well as the pH recovery rate from intracellular acidification with NH4Cl from 0.29+/-0.022 pH U/min (n=14) to 0.50+/-0.024 pH U/min (n=14), without modifying their buffering capacity. These effects were followed by several modifications in morphological features, indicating an increase in differentiation status. The altered activity of NHE1 was consistent with an increase of both transcription and translation of the antiporter, as the utilization of actinomycin D and cycloheximide significantly inhibited the upregulation of NHE1 induced by serum withdrawal. Inhibition of tyrosine phosphorylation by genistein blocked the serum deprivation-dependent activation of NHE. Moreover, the pharmacological inhibition of MEK1/2, the upstream activator of ERK1/2 by UO-126, significantly inhibited the stimulatory effect of serum starvation on Na+/H+ exchanger activity, whereas the putative p38 MAPK inhibitor SB-203580 failed to cause any effect on pHi recovery rates. Our findings indicate that during IRPTC differentiation by serum deprivation, there was a net enhancement of NHE1 activity. This upregulation of NHE by serum removal was consistent with an increase of RNA and protein synthesis of the exchanger, which depends on tyrosine kinase phosphorylation and ERK pathway activation.
Assuntos
Diferenciação Celular/fisiologia , Túbulos Renais Proximais/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Animais , Linhagem Celular , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação da Expressão Gênica , Genisteína/farmacologia , Concentração de Íons de Hidrogênio , MAP Quinase Quinase Quinases/análise , MAP Quinase Quinase Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/análise , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Biossíntese de Proteínas/fisiologia , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/fisiologia , ATPases Translocadoras de Prótons/metabolismo , Ratos , Soro/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Transcrição Gênica/fisiologia , Regulação para CimaRESUMO
In this study we have used the transgenic mouse model Tg2576 to analyze the involvement of anomalous loss of regulation of cdk5 and the stress kinases JNK and p38 in brain neuronal death as related to neurodegenerative disorders such as Alzheimer's disease. Previous studies on hippocampal cells led us to the discovery that the cdk5/p35 complex is activated in neurodegeneration, a finding that was confirmed later in the transgenic mouse model. Here we show a link between the cdk5 system and JNK and p38 phosphoproteins, as an alternative pathway to neuronal death. Brains of the Tg2576 transgenic mice overexpressing amyloid precursor protein exhibited immunoreactivity with the phosphoproteins p-JNK, p-p38 and the GTPase protein Rac1 surrounding neuritic plaques. A significant increase in the immunodetection of JNK and p38 phosphoproteins in the Tg2576 mouse compared with wild type controls confirmed these findings. The significant increase in co-immunoprecipitation of p-JNK, p-p38 and Rac1 proteins with cdk5 in the transgenic mouse provided evidence for these interactions. At the cellular level, p-JNK and cdk5 colocalized in the cytoplasm of the cell bodies and neurites of brain cortical areas of the transgenic mouse. The present evidence suggests a cellular link between the cdk5 system and the stress kinase JNK and p38 pathways in an in vivo model. This study sheds new light on the pathogenesis of neuronal degeneration processes such as those occurring in Alzheimer's disease.