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Ethnopharmacological relevance: G. uralensis Fisch. (Glycyrrhiza uralensis) is an ancient and widely used traditional Chinese medicine with good efficacy in clearing heat and detoxifying action. Studies suggest that Glycyrrhiza Uralensis Polysaccharides (GUP), one of the major components of G. uralensis, has anti-inflammatory, anti-cancer and hepatoprotective effects., but its exact molecular mechanism has not been explored in depth. Aim of the study: Objectives of our research are about exploring the anti-inflammatory role of GUP and the mechanisms of its action. Materials and methods: ELISA kits, Western blotting, immunofluorescence, quantitative real-time PCR, immunoprecipitation and DMXAA-mediated STING activation mice models were performed to investigate the role of GUP on the cGAS-STING pathway. To determine the anti-inflammatory effects of GUP, cecal ligation and puncture (CLP) sepsis models were employed. Results: GUP could effectively inhibit the activation of the cGAS-STING signaling pathway accompany by a decrease the expression of type I interferon-related genes and inflammatory factors in BMDMs, THP-1, and human PBMCs. Mechanistically, GUP does not affect the oligomerization of STING, but affects the interaction of STING with TBK1 and TBK1 with IRF3. Significantly, GUP had great therapeutic effects on DMXAA-induced agonist experiments in vivo as well as CLP sepsis in mice. Conclusion: Our studies suggest that GUP is an effective inhibitor of the cGAS-STING pathway, which may be a potential medicine for the treatment of inflammatory diseases mediated by the cGAS-STING pathway.
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BACKGROUND: The cGAS-STING signaling pathway is an essential section of the natural immune system. In recent years, an increasing number of studies have shown a strong link between abnormal activation of the cGAS-STING signaling pathway, a natural immune pathway mediated by the nucleic acid receptor cGAS, and the development and progression of autoimmune diseases. Therefore, it is important to identify an effective compound to specifically downregulate this pathway for disease. METHODS: The effect of Glabridin (Glab) was investigated in BMDMs and Peripheral blood mononuclear cell (PBMC) by establishing an in vitro model of cGAS-STING signaling pathway activation. An activation model stimulated by DMXAA was also established in mice to study the effect of Glab. On the other hand, we investigated the possible mechanism of action of Glab and the effect of Glab on Trex1-deficient mice. RESULTS: In this research, we report that Glab, a major component of licorice, specifically inhibits the cGAS-STING signaling pathway by inhibiting the level of type I interferon and inflammatory cytokines (IL-6 and TNF-α). In addition, Glab has a therapeutic effect on innate immune diseases caused by abnormal cytoplasmic DNA in Trex1-deficient mice. Mechanistically, Glab can specifically inhibit the interaction of STING with IRF3. CONCLUSION: Glab is a specific inhibitor of the cGAS-STING signaling pathway and may be used in the clinical therapy of cGAS-STING pathway-mediated autoimmune diseases.
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Doenças Autoimunes , Interferon Tipo I , Isoflavonas , Fenóis , Animais , Camundongos , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/genética , Leucócitos Mononucleares/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Isoflavonas/uso terapêutico , Fenóis/uso terapêuticoRESUMO
OBJECTIVES: Breast cancer is a common malignancy in women. More than 90% of breast cancer deaths are caused by metastasis. Epimedii Folium (EF) is a commonly used herb with anti-tumor benefits, but its underlying mechanisms and active components for breast cancer prevention are little understood. This study assessed the therapeutic role of Icariside I (ICS I) in Epimedium flavonoids (EF) on lung metastasis of breast cancer, including the underlying mechanism. METHODS: Western blot, RT-qPCR, wound healing assay, colony formation assay, and flow cytometry were used to investigate the inhibition of breast cancer cells growth and migration by EF and ICS I through disrupting the IL-6/STAT3 pathway. Combined with 4T1 breast cancer model in mice, Western blot, RT-qPCR, Hematoxylin and Eosin staining, immunohistochemistry were used to evaluate the therapeutic role of ICS I in proliferation, apoptosis, invasion, and metastasis of breast cancer. KEY FINDINGS: EF can inhibit STAT3 phosphorylation and reduce the colony formation and migration of breast cancer cells. Detecting the active ingredients in EF, we found ICS I can reduce the activation of STAT3 in 4T1 breast cancer cells, impair colony formation and migration. Moreover, ICS I induced cells G1 phase arrest and modulated Cyclin D1, CDK4, bcl-2, and bax to inhibit proliferation and survival of breast cancer cells. Similarly, the in vivo studies demonstrated that ICS I significantly suppressed tumor development and lung metastasis in the 4T1 mouse model. Tumor cells in vehicle group were arranged in a spoke-like pattern with obvious heterogeneity, and multinucleated tumor giant cells were seen. But, the tumor cells in the ICS I group were disorganized and necrotic lysis was seen in some areas. In ICS I-treated group, tumors' STAT3 phosphorylation level, IL-6, Cyclin D1, CDK4, bcl-2, and vimentin expression were downregulated, bax and cleaved caspase 3 expression were upregulated. In the lung tissue, we could find less metastasis of breast cancer cells and less lung injury in the ICS I group. Besides, the expression of metastasis-related genes MMP9 and vimentin was decreased in the lung tissue of ICS I group. CONCLUSIONS: These findings suggest that ICS I can inhibit breast cancer proliferation, apoptosis, invasion and metastasis probably via targeting IL-6/STAT3 pathway. Therefore, ICS I has the potential to become an innovative therapeutic candidate to breast cancer prevention and treatment.
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OBJECTIVE: To investigate the effect of moxibustion on the proteins related with apoptosis and nuclear transcription factor kappa B (NF-κB) in hippocampus of diabetic rats with cognitive impairment (CI), so as to explore its mechanism underlying improvement of learning-memory ability. METHODS: Thirty SD rats were randomly divided into normal, model and moxibustion groups (n=10 rats/group). The diabetic model was established by i.p. injection of streptozotocin solution (25 mg·kg-1·d-1), followed by high-fat diet raising for 4 weeks, and the CI model was confirmed by Morris water maze test. The rats in the moxibustion group were given moxibustion at "Shenting" (GV24), "Baihui" (GV20) and "Dazhui" (GV14) for 20 min each time, the treatment was conducted 6 times a week for 4 weeks. The learning-memory ability was detected by Morris water maze test, the random blood glucose of rats was measured by glucometer and test strips. The protein and mRNA expression levels of Bcl-2, Bax, Caspase-3 and NF-κB p65 in hippocampus were detected by Western blot and quantitative real-time PCR, separately. RESULTS: After modeling, the random blood glucose, escape latency, and the expression levels of Bax, Caspase-3 and NF-κB p65 proteins and mRNAs in the model group were significantly increased, while the expression levels of Bcl-2 protein and mRNA were decreased (P<0.001ï¼P<0.01, P<0.05) in comparison with the normal group. Following the treatment, the modeling induced increase of blood glucose, escape latency, and the expression levels of Bax, Caspase-3 and NF-κB p65 proteins and mRNAs, as well as decrease of Bcl-2 protein and mRNA expression levels were reversed (P<0.05, P<0.01, P<0.001). CONCLUSION: Moxibustion can improve learning-memory ability in diabetic rats with cognitive impairment, which may be related to its function in regulating the expression levels of hippocampal Bcl-2, Bax, Caspase-3 and NF-κB.
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Disfunção Cognitiva , Diabetes Mellitus Experimental , Moxibustão , Animais , Ratos , Ratos Sprague-Dawley , Caspase 3 , NF-kappa B , Glicemia , Proteína X Associada a bcl-2 , Proteínas Proto-Oncogênicas c-bcl-2 , Apoptose , HipocampoRESUMO
In recent years, we have found that the dysregulation of the cyclic-GMP-AMP synthase (cGAS)âstimulator of interferon genes (STING) pathway leads to the development of immune and inflammatory diseases, therefore, finding compounds that can specifically regulate this pathway is essential for effective regulation of the immune pathway for addressing inflammatory diseases. Licorice flavonoids (LFs), are active ingredients extracted from the Chinese herb licorice, which has been reported to have strong anti-inflammatory activity in previous studies. Here, we report that LFs inhibit the activation of the cGAS-STING pathway evidenced by the inhibition of the expression of type I interferons and related downstream genes such as interferon-stimulated gene 15 (ISG15) and C-X-C motif chemokine ligand 10 (CXCL10), as well as inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Notably, LFs markedly improve the LPS-induced acute lung injury by inhibiting the excessive activation of cGAS-STING signaling pathway. Mechanistically, LFs treatment leads to the blocking of 2'3'-cyclic GMP-AMP (cGAMP) synthesis resulting in an inhibition of the activation of the cGAS-STING pathway. Our results indicate that LFs is a specific inhibitor of the cGAS-STING pathway, which is suggested to be a potential candidate for the treatment of cGAS-STING pathway-mediated inflammatory diseases.
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Lesão Pulmonar Aguda , Glycyrrhiza , Interferon Tipo I , Camundongos , Animais , Lipopolissacarídeos/toxicidade , Transdução de Sinais , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Interferon Tipo I/metabolismoRESUMO
AIM: A two-body wear test experiment was performed on human enamel, in simulated chewing motion, against non-veneered zirconia ceramic. Aim-1 was to ascertain the effect of zirconia roughness on enamel wear. Aim-2 was to ascertain the relative enamel wear between enamel-zirconia wear pair and enamel-enamel control pair. MATERIALS: Six molar and premolar human enamel cusps per group were used for a dental wear test against laboratory polished (LP) zirconia and laboratory polished and clinically adjusted (LP + CA) zirconia. Enamel antagonists were tested against incisor teeth as a control group to demonstrate laboratory enamel wear. METHODOLOGY: Two-body wear tests were conducted in a dual-axis biomimetic dental wear simulator. 49N loading force was used for 120,000 cycles with 1 mm lateral movement of the test specimen at 1.6Hz frequency, under constant ambient temperature water flow. Surface roughness before testing was determined using 3D profilometry. Loss of enamel height and volume i.e. vertical wear and volumetric wear respectively, were measured by superimposition of before and after testing scans by 3D laser scanning. Scanning electron microscopy was used for surface morphology assessment. One-way ANOVA and Post Hoc Multiple Comparisons with Bonferroni corrections were used at the 5% significance level to determine whether surface finish affected volumetric and vertical enamel loss. The relationship between volumetric and vertical loss of enamel was assessed using Pearson's correlation test. RESULTS: No significant difference was found between LP and LP + CA zirconia in vertical and volumetric enamel wear results. Control enamel had significantly higher vertical and volumetric enamel wear than LP and LP + CA zirconia. Pearson correlation revealed a strong relationship between vertical wear and volumetric wear of enamel. CONCLUSION: Within the constraints of the test method in this experiment, zirconia irrespective of surface preparation, was found to cause less vertical and volumetric enamel wear compared to control enamel. No statistically significant difference was seen between LP zirconia and LP + CA zirconia.
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Laboratórios , Zircônio , Esmalte Dentário , Porcelana Dentária , Desgaste de Restauração Dentária , Humanos , Teste de Materiais , Propriedades de SuperfícieRESUMO
PURPOSE: The aim of this project is to investigate the usefulness of the absolute liver lesion ADC value and ratio of Apparent diffusion coefficient (ADC) values of a liver lesion and liver parenchyma to discriminate between a benign and malignant lesion. METHODS: Liver MRI scans performed between January 2009 and June 2015 were retrospectively analysed. Scans were performed on either a 1.5 T or 3 T MRI unit. The type of liver lesion (benign or malignant) was determined by its radiological appearance, histology result and clinical management. Lesions with undetermined diagnosis or MRI studies degraded by artifacts were excluded. Liver cysts were also excluded from the analysis. ADC value of a lesion and liver parenchyma was measured and ADCratio was calculated. The values were analysed using independent samples t-test Results:Data set contained 39 benign lesions and 36 malignant lesions. Mean ADC value for benign lesions was 1678, and the mean value for malignant lesions was 1097 with a statistically significant difference of p < 0.001. All lesions with ADC value below 955 were malignant, while all lesions with ADC value above 1880 were benign. ADC value of 1260 was identified as the best available cut-off value for differentiating benign and malignant lesions, achieving sensitivity of 92%, specificity of 80% and an overall accuracy of 89%. The mean lesion to liver ADCratio for benign lesions was 1.3467 and for malignant lesions was 0.9038 with a statistically significant difference of p < 0.001. All lesions with ADCratio measuring <0.9 were malignant while lesions with ADCratio>1.5 were benign. ADCratio of 1.1 was identified statistically as the best available cut-off value for differentiating benign from malignant lesions, with sensitivity of 82%, specificity of 86% and an overall accuracy of 92%. CONCLUSION: Our dataset indicates that lesion to background liver ADCratio is superior in discriminating between benign and malignant focal lesions compared to absolute ADC values of the hepatic lesions.
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Digital image correlation (DIC) is an optical technique commonly used for measuring displacement fields by tracking artificially applied random speckle patterns, which can sometimes be a problem for tracking small-scale displacements. DIC is particularly useful for tracking the crack mouth opening displacement (CMOD) of a notched metallic specimen subjected to three-point bending for fracture toughness determination because the edges of the notch provide the required textural features for DIC without the need for speckle patterns. This simplifies the set-up process as the specimen and stage geometries do not need to account for the placement of a strain gauge. To enhance the accuracy of DIC, this study then successfully downscaled a photogrammetry technique commonly used to track crack propagation in large scale concrete tests so that the pixel coordinates of the captured images can be automatically related to their real-world coordinates, allowing for small scale displacements to be accurately tracked.
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OBJECTIVE: To study the chemical constituents of Psammosilene tunicoides. METHOD: The two chemical constituents were separated by various chromatographic methods, and their structures were identified on the basis of spectroscopic analysis. RESULT: Two beta-carboline alkaloids were separated from normal butanol fraction of P. tunicoides, and identified as 1-acetyl-3-methoxycarbonyl-beta-carboline (1) and 1-acetyl-3-methoxycarbonyl-4-hydroxyl-beta-carboline (2). CONCLUSION: Compound 1 is separated from this plant as natural products for the first time, and compound 2 was a new compound.
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Alcaloides/análise , Carbolinas/análise , Caryophyllaceae/químicaRESUMO
OBJECTIVES: Because administration of 17beta-estradiol following trauma-hemorrhage improves cardiovascular responses, we investigated whether the salutary effects of 17beta-estradiol on cardiac function are mediated via Akt-dependent heme oxygenase-1 up-regulation under those conditions. DESIGN: Experimental animal study. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats underwent trauma-hemorrhage (mean blood pressure approximately 40 mm Hg for 90 mins) followed by fluid resuscitation. Before resuscitation, rats received either vehicle, 17beta-estradiol (1 mg/kg), or 17beta-estradiol plus the phosphoinositide 3-kinase inhibitor wortmannin (1 mg/kg). At 2 hrs after trauma-hemorrhage or sham operation, the rats were killed. MEASUREMENTS AND MAIN RESULTS: Cardiac function, heart tissue myeloperoxidase activity, cardiac and circulatory cytokine levels, cardiac intercellular adhesion molecule-1, and chemokine levels were measured. Cardiac Akt and heme oxygenase-1 were also determined. We found that 17beta-estradiol prevented the trauma-hemorrhage-induced impairment in cardiac function and increase in cardiac myeloperoxidase activity. Cardiac and systemic interleukin-6 and tumor necrosis factor-alpha levels as well as cardiac intercellular adhesion molecule-1, cytokine-induced neutrophil chemoattractant-1, and macrophage inflammatory protein-2 contents were increased following trauma-hemorrhage, which were normalized by 17beta-estradiol. Administration of 17beta-estradiol following trauma-hemorrhage restored cardiac Akt phosphorylation and further increased heme oxygenase-1 expression. Coadministration of wortmannin following trauma-hemorrhage abolished the previous effects by 17beta-estradiol. CONCLUSIONS: These results suggest that the 17beta-estradiol-meditated improvement in cardiac function following trauma-hemorrhage occurs via Akt-dependent heme oxygenase-1 up-regulation.
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Estradiol/farmacologia , Estrogênios/farmacologia , Coração/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Choque Hemorrágico/tratamento farmacológico , Regulação para Cima , Androstadienos/farmacologia , Animais , Quimiocinas/metabolismo , Citocinas/metabolismo , Masculino , Peroxidase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , WortmaninaRESUMO
BACKGROUND: Extracellular signal-regulated protein kinase (ERK) is known to be involved in pro-inflammatory and chemotactic events in response to injury. The aim of this study is to elucidate whether ERK plays any role in 17beta-estradiol (E2)-mediated attenuation of lung injury and pro-inflammatory mediators after trauma-hemorrhage. METHODS: Male Sprague-Dawley rats underwent trauma-hemorrhage (mean blood pressure approximately 40 mm Hg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle (cyclodextrin), E2 (1 mg/kg body weight [BW]), or the ERK inhibitor PD98059 (2 mg/kg BW). At 2 h after sham operation or trauma-hemorrhage, various parameters were measured. RESULTS: Trauma-hemorrhage led to a significant increase in lung ERK phosphorylation, which was associated with increased lung myeloperoxidase activity, wet-to-dry weight ratio, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and macrophage inflammatory protein-2 levels. Circulatory IL-6, TNF-alpha, and lactate levels were also increased after trauma-hemorrhage compared with shams. Administration of E2 or ERK inhibitor PD98059 after trauma-hemorrhage attenuated the trauma-hemorrhage-induced increase in lung injury markers, ERK phosphorylation and cytokines/chemokines, ICAM-1 production, as well as circulatory cytokines and lactate levels. CONCLUSION: These results collectively suggest that the salutary effects of E2 on the lung after trauma-hemorrhage are mediated via an ERK pathway and subsequent downregulation of pro-inflammatory mediator production.
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Lesão Pulmonar Aguda/prevenção & controle , Estradiol/uso terapêutico , Estrogênios/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Choque Hemorrágico/tratamento farmacológico , Lesão Pulmonar Aguda/enzimologia , Lesão Pulmonar Aguda/etiologia , Animais , Biomarcadores/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/sangue , Ácido Láctico/sangue , Fígado/enzimologia , Pulmão/enzimologia , Pulmão/patologia , Masculino , Peroxidase/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Choque Hemorrágico/complicações , Choque Hemorrágico/enzimologia , Fator de Necrose Tumoral alfa/sangueRESUMO
Although MIP-1alpha is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1alpha plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1alpha-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 +/- 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum alpha-glutathione transferase, TNF-alpha, IL-6, IL-10, MCP-1, and MIP-1alpha and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1alpha KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1alpha plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1alpha, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1alpha levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.
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Quimiocina CCL3/fisiologia , Mediadores da Inflamação/fisiologia , Insuficiência de Múltiplos Órgãos/imunologia , Traumatismo Múltiplo/imunologia , Choque Hemorrágico/imunologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Animais , Quimiocina CCL3/deficiência , Quimiocina CCL3/genética , Quimiocinas/biossíntese , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Citocinas/biossíntese , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/patologia , Traumatismo Múltiplo/genética , Traumatismo Múltiplo/patologia , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Choque Hemorrágico/genética , Choque Hemorrágico/patologia , Síndrome de Resposta Inflamatória Sistêmica/genética , Síndrome de Resposta Inflamatória Sistêmica/patologiaRESUMO
Although trauma-hemorrhage produces tissue hypoxia, systemic inflammatory response and organ dysfunction, the mechanisms responsible for these alterations are not clear. Using a potent selective inducible nitric oxide (NO) synthase inhibitor, N-[3-(aminomethyl) benzyl]acetamidine (1400W), and a nonselective NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), we investigated whether inducible NO synthase plays any role in producing hepatic injury, inflammation, and changes of protein expression following trauma-hemorrhage. To investigate this, male Sprague-Dawley rats were subjected to midline laparotomy and hemorrhagic shock (mean blood pressure 35-40 mmHg for approximately 90 min) followed by fluid resuscitation. Animals were treated with either vehicle (DMSO) or 1400W (10 mg/kg body wt ip), or L-NAME (30 mg/kg iv), 30 min before resuscitation and killed 2 h after resuscitation. Trauma-hemorrhage/resuscitation induced a marked hypotension and increase in markers of hepatic injury (i.e., plasma alpha-glutathione S-transferase, tissue myeloperoxidase activity, and nitrotyrosine formation). Hepatic expression of iNOS, hypoxia-inducible factor-1alpha, ICAM-1, IL-6, TNF-alpha, and neutrophil chemoattractant (cytokine-induced neutrophil chemoattractant-1 and macrophage inflammatory protein-2) protein levels were also markedly increased following trauma-hemorrhage/resuscitation. Administration of the iNOS inhibitor 1400W significantly attenuated hypotension and expression of these mediators of hepatic injury induced by trauma-hemorrhage/resuscitation. However, administration of L-NAME could not attenuate hepatic dysfunction and tissue injury mediated by trauma-hemorrhage, although it improved mean blood pressure as did 1400W. These results indicate that increased expression of iNOS following trauma-hemorrhage plays an important role in the induction of hepatic damage under such conditions.
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Amidinas/farmacologia , Benzilaminas/farmacologia , Inibidores Enzimáticos/farmacologia , Hepatopatias/prevenção & controle , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Ressuscitação/efeitos adversos , Choque Hemorrágico/terapia , Ferimentos e Lesões/terapia , Animais , Pressão Sanguínea/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Hidratação/efeitos adversos , Glutationa Transferase/sangue , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Isoenzimas/sangue , Fígado/efeitos dos fármacos , Fígado/enzimologia , Hepatopatias/enzimologia , Hepatopatias/etiologia , Hepatopatias/fisiopatologia , Masculino , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III , Nitritos/metabolismo , Peroxidase/metabolismo , Ratos , Ratos Sprague-Dawley , Choque Hemorrágico/enzimologia , Choque Hemorrágico/fisiopatologia , Tirosina/análogos & derivados , Tirosina/metabolismo , Ferimentos e Lesões/enzimologia , Ferimentos e Lesões/fisiopatologiaRESUMO
OBJECTIVE: In this study, we tested the hypothesis that 17beta-estradiol (E2) administration after trauma-hemorrhage reduces lung injury through a mechanism involving estrogen receptor (ER)-dependent activation of the endothelial nitric oxide (NO) synthase (eNOS)/protein kinase G (PKG)/vasodilator-stimulated phosphoprotein (VASP) pathway. BACKGROUND: Estrogen provides protection after injury via activation of multiple signaling cascades, including the cyclic GMP-dependent PKG pathway. Phosphorylation of VASP at Ser239 (p-VASP) can be used to assess PKG signaling activity. METHODS: Male Sprague-Dawley rats (275-325 g) underwent soft tissue trauma (midline laparotomy) and hemorrhagic shock (mean blood pressure 35-40 mm Hg for 90 minutes) followed by fluid resuscitation. Animals were pretreated with a nonselective NOS inhibitor (N(omega)-nitro-L-arginine methyl ester; 30 mg/kg), a soluble guanylyl cyclase (sGC) inhibitor [1H-(1, 2, 4) oxadiazolo (3, 4-alpha) quinoxalin-1-one; 10 mg/kg] or an ER antagonist (ICI 182,780; 3 mg/kg) 30 minutes before E2 (100 microg/kg) or vehicle administration. Animals were killed at 2 hours after resuscitation. RESULTS: Lung injury induced by trauma-hemorrhage is evidenced by edema (wet/dry ratio), neutrophil infiltration (myeloperoxidase activity), and with an increased expression of cytokines, chemokines, and adhesion molecules. E2 treatment after trauma-hemorrhage resulted in an increase in eNOS expression/phosphorylation, PKG-I activation, and VASP/p-VASP expression, which paralleled a decrease in lung injury. Inhibition of NOS and sGC abolished the E2-induced increase in PKG-I activity, VASP/p-VASP expression. Blockade of eNOS, PKG-I, and ER exacerbated lung inflammation and injury. CONCLUSIONS: These results collectively suggest that activation of the eNOS-PKG/VASP pathway by E2 protects against trauma-hemorrhage-induced lung injury.
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Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citocinas/metabolismo , Antagonistas de Estrogênios/farmacologia , Pneumopatias/prevenção & controle , Lesão Pulmonar , Análise de Variância , Animais , Western Blotting , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Quimiocinas/análise , Quimiocinas/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Citocinas/análise , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Masculino , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Probabilidade , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Choque Hemorrágico/complicações , Transdução de Sinais , Ferimentos e Lesões/complicaçõesRESUMO
Although studies have shown that administration of testosterone receptor antagonist, flutamide, following trauma-hemorrhage, improves hepatic, cardiovascular, and immune functions, the precise cellular/molecular mechanisms responsible for producing these salutary effects remain largely unknown. To study this, male C3H/HeN mice were subjected to a midline laparotomy and hemorrhagic shock (35+/-5 mmHg for approximately 90 min), followed by resuscitation with Ringer lactate. Flutamide (25 mg/kg) or vehicle was administered subcutaneously at the onset of resuscitation, and animals were killed 2 h thereafter. Hepatic injury was assessed by plasma alpha-glutathione S-transferase concentration, liver myeloperoxidase activity, and nitrotyrosine formation. Hepatic malondialdehyde and 4-hydroxyalkenals (lipid peroxidation indicators), cellular DNA fragmentation, and the expression of inducible nitric oxide synthase and hypoxia-inducible factor-1alpha were also evaluated. Cytokines (TNF-alpha, IL-6) and chemokines (keratinocyte-derived chemokine and monocyte chemoattractant protein-1) levels were determined by cytometric bead array. The results indicate that flutamide administration after trauma-hemorrhage reduced liver injury, which was associated with decreased levels of alpha-glutathione S-transferase, myeloperoxidase activity, nitrotyrosine formation, lipid peroxidation, and cytokines/chemokines (systemic, liver tissue, and intracellular cytokines/chemokines). Cellular apoptosis, hepatocyte hypoxia-inducible factor-1alpha, and inducible nitric oxide synthase expression were also decreased under such conditions. Thus administration of flutamide following trauma-hemorrhage protects against liver injury via reduced inflammation, cellular oxidative stress, and apoptosis.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Apoptose/efeitos dos fármacos , Flutamida/uso terapêutico , Hemorragia/prevenção & controle , Hepatite/prevenção & controle , Fígado/lesões , Estresse Oxidativo/efeitos dos fármacos , Animais , Western Blotting , Separação Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Glutationa Transferase/metabolismo , Hepatite/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células de Kupffer/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Óxido Nítrico Sintase Tipo II/biossíntese , Peroxidase/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
p38 MAPK has been reported to regulate the inflammatory response in various cell types via extracellular stimuli. p38 MAPK activation also results in the induction of heme oxygenase (HO)-1, which exerts potent anti-inflammatory effects. Although studies have shown that 17beta-estradiol (E(2)) prevented organ dysfunction following trauma-hemorrhage, it remains unknown whether p38 MAPK/HO-1 plays any role in E(2)-mediated attenuation of intestinal injury under those conditions. To study this, male rats underwent trauma-hemorrhage (mean blood pressure approximately 40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E(2) (1 mg/kg body wt), the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt) or E(2) plus SB-203580. Two hours thereafter, intestinal myeloperoxidase (MPO) activity and lactate, TNF-alpha, IL-6, ICAM-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and macrophage inflammatory protein (MIP)-2 levels were measured. Intestinal p38 MAPK and HO-1 protein levels were also determined. Trauma-hemorrhage led to an increase in intestinal MPO activity and lactate, TNF-alpha, IL-6, ICAM-1, CINC-1, and MIP-2 levels. This was accompanied with a decrease in intestinal p38 MAPK activity and increase in HO-1 expression. Administration of E(2) normalized all the above parameters except HO-1, which was further increased following trauma-hemorrhage. Administration of SB-203580 with E(2) abolished the E(2)-mediated restoration of the above parameters as well as the increase in intestinal HO-1 expression following trauma-hemorrhage. These results suggest that the p38 MAPK/HO-1 pathway plays a critical role in mediating the salutary effects of E(2) on shock-induced intestinal injury.
Assuntos
Estrogênios/farmacologia , Heme Oxigenase-1/fisiologia , Hemorragia/metabolismo , Mucosa Intestinal/metabolismo , Ferimentos e Lesões/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Intestinos/efeitos dos fármacos , Ácido Láctico/metabolismo , Masculino , Modelos Animais , Peroxidase/metabolismo , Ratos , Ratos Sprague-Dawley , Choque Hemorrágico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Although 17beta-estradiol (estrogen) and estrogen receptor (ER) agonist administration after trauma-hemorrhage improves cardiac function, it remains unknown what the optimal estrogen or ER agonist dosage is to elicit this beneficial effect. To study this, the dose-dependent effects of estrogen, propylpyrazole triol (ER-alpha agonist), and diarylpropionitrile (DPN; ER-beta agonist) on heart performance (+dP/dt) were determined in sham rats and in experimental animals at the time of maximal bleedout (MBO) or at 2 h after trauma-hemorrhage. The results showed that estrogen and DPN induced dose-dependent increases in the maximal rate of left ventricular pressure increase (+dP/dt) in all groups, whereas propylpyrazole triol was ineffective at all doses. The maximal dose and the 50% effective dose of DPN were approximately 100-fold lower than those of estrogen. The half-life of estrogen in plasma was approximately 25 min in sham and MBO groups. A positive correlation between the estrogen-induced increase in +dP/dt and survival in MBO rats were observed. These results collectively suggest that the salutary effects of estrogen on cardiac performance are dose-dependent and mediated via ER-beta.
Assuntos
Estrogênios/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Hemorragia/fisiopatologia , Animais , Estradiol/farmacologia , Receptor beta de Estrogênio/agonistas , Masculino , Nitrilas/farmacologia , Fenóis , Propionatos/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Studies have shown that p38 MAPK and nitric oxide (NO), generated by endothelial NO synthase (eNOS), play key roles under physiological and pathophysiological conditions. Although administration of 17beta-estradiol (E2) protects cardiovascular injury from trauma-hemorrhage, the mechanism by which E2 produces those effects remains unknown. Our objective was to determine whether the E2-mediated activation of myocardial p38 MAPK and subsequent eNOS expression/phosphorylation would protect the heart following trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent soft-tissue trauma (midline laparatomy) and hemorrhagic shock (mean blood pressure 35-40 mmHg for 90 min), followed by fluid resuscitation. Animals were pretreated with specific p38 MAPK inhibitor SB-203580 (SB; 2 mg/kg), and nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 30 mg/kg) 30 min before vehicle (cyclodextrin) or E2 (100 microg/kg) treatment, followed by resuscitation, and were killed 2 h thereafter. Cardiovascular performance and other parameters were measured. E2 administration following trauma-hemorrhage increased cardiac p38 MAPK activity, eNOS expression and phosphorylation at Ser(1177), and nitrate/nitrite levels in plasma and heart tissues; these were associated with normalized cardiac performance, which was reversed by SB administration. In addition, E2 also prevented trauma-hemorrhage-induced increase in cytokines (IL-6 and TNF-alpha), chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant-1), and ICAM-1, which was reversed by l-NAME administration. Administration of E2 following trauma-hemorrhage attenuated cardiac tissue injury markers, myeloperoxidase activity, and nitrotyrosine level, which were reversed by treatment with SB and l-NAME. The salutary effects of E2 on cardiac functions and tissue protection following trauma-hemorrhage are mediated, in part, through activation of p38 MAPK and subsequent eNOS expression and phosphorylation.