Role and mechanism of SIRT1 in regulating Nrf2/HO-1 signaling pathway in septic liver injury / 中华危重病急救医学

OBJECTIVE@#To investigate the role and mechanism of silent information regulator 1 (SIRT1) in regulating nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in oxidative stress and inflammatory response to sepsis-induced liver injury.@*METHODS@#A total of 24 male Sprague-Dawley (SD) rats were randomly divided into sham operation (Sham) group, cecal ligation and puncture (CLP) group, SIRT1 agonist SRT1720 pretreatment (CLP+SRT1720) group and SIRT1 inhibitor EX527 pretreatment (CLP+EX527) group, with 6 rats in each group. Two hours before operation, SRT1720 (10 mg/kg) or EX527 (10 mg/kg) were intraperitoneally injected into the CLP+SRT1720 group and CLP+EX527 group, respectively. Blood was collected from the abdominal aorta at 24 hours after modeling and the rats were sacrificed for liver tissue. The serum levels of interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by microplate method. Hematoxylin-eosin (HE) staining was used to observe the pathological injury of rats in each group. The levels of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), glutathione (GSH) and superoxide dismutase (SOD) in liver tissue were detected by corresponding kits. The mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting.@*RESULTS@#Compared with the Sham group, the serum levels of IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were significantly increased; histopathological results showed that liver cords were disordered, hepatocytes were swollen and necrotic, and a large number of inflammatory cells infiltrated; the contents of MDA and 8-OHdG in liver tissue increased, while the contents of GSH and SOD decreased; and the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were significantly decreased. These results suggest that sepsis rats have liver dysfunction, and the levels of SIRT1, Nrf2, HO-1 and antioxidant protein in liver tissues were decreased, while the levels of oxidative stress and inflammation were increased. Compared with the CLP group, the levels of inflammatory factors and oxidative stress were significantly decreased in the CLP+SRT1720 group, the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 were significantly increased [IL-6 (ng/L): 34.59±4.21 vs. 61.84±3.78, IL-1β (ng/L): 41.37±2.70 vs. 72.06±3.14, TNF-α (ng/L): 76.43±5.23 vs. 130.85±5.30, ALT (U/L): 30.71±3.63 vs. 64.23±4.59, AST (U/L): 94.57±6.08 vs. 145.15±6.86, MDA (μmol/g): 6.11±0.28 vs. 9.23±0.29, 8-OHdG (ng/L): 117.43±10.38 vs. 242.37±11.71, GSH (μmol/g): 11.93±0.88 vs. 7.66±0.47, SOD (kU/g): 121.58±5.05 vs. 83.57±4.84, SIRT1 mRNA (2-ΔΔCt): 1.20±0.13 vs. 0.46±0.02, Nrf2 mRNA (2-ΔΔCt): 1.21±0.12 vs. 0.58±0.03, HO-1 mRNA (2-ΔΔCt): 1.71±0.06 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.89±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.87±0.08 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.93±0.14 vs. 0.54±0.12, all P < 0.05], these results indicated that SIRT1 agonist SRT1720 pretreatment could improve liver injury in sepsis rats. However, pretreatment with SIRT1 inhibitor EX527 showed the opposite effect [IL-6 (ng/L): 81.05±6.47 vs. 61.84±3.78, IL-1β (ng/L): 93.89±5.83 vs. 72.06±3.14, TNF-α (ng/L): 177.67±5.12 vs. 130.85±5.30, ALT (U/L): 89.33±9.52 vs. 64.23±4.59, AST (U/L): 179.59±6.44 vs. 145.15±6.86, MDA (μmol/g): 11.39±0.51 vs. 9.23±0.29, 8-OHdG (ng/L): 328.83±11.26 vs. 242.37±11.71, GSH (μmol/g): 5.07±0.34 vs. 7.66±0.47, SOD (kU/g): 59.37±4.28 vs. 83.57±4.84, SIRT1 mRNA (2-ΔΔCt): 0.34±0.03 vs. 0.46±0.02, Nrf2 mRNA (2-ΔΔCt): 0.46±0.04 vs. 0.58±0.03, HO-1 mRNA (2-ΔΔCt): 0.21±0.03 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.47±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.32±0.07 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.19±0.09 vs. 0.54±0.12, all P < 0.05].@*CONCLUSIONS@#SIRT1 can inhibit the release of proinflammatory factors and alleviate the oxidative damage of hepatocytes by activating Nrf2/HO-1 signaling pathway, thus playing a protective role against CLP-induced liver injury.

Echinococcus granulosus cyst fluid(EgCF) inhibits the migration and phagocytic function of mouse macrophages induced by LPS via inducing cytoskeletal rearrangement / 细胞与分子免疫学杂志

Objective To investigate the effect of Echinococcus granulosus cyst fluid(EgCF) on the cytoskeletal rearrangement and phagocytosis and the migration of macrophages induced by lipopolysaccharide(LPS). Methods Peritoneal macrophages of C57BL/6 mice were isolated and cultured in vitro, and divided into control group and LPS group and LPS combined with EgCF group. After 48 hours of treatment, filamentous actin (F-actin) changes were observed with rhodamine-labelled phalloidin staining and fluorescence microscopy; TranswellTM chamber was used to test cell migration ability and flow cytometry to test cell phagocytosis. After 1 hour of treatment, PI3K and AKT, phosphorylated AKT (p-AKT), Rac1, guanosine triphospho-Rac1 (GTP-Rac1), WASP and Arp2 protein expressions were detected with Western blot analysis. Results Compared with the control group, after LPS stimulation, macrophages were deformed significantly; pseudopodia increased; actin cytoskeleton increased and was more distributed in pseudopodia; the ability of migration and phagocytosis were significantly improved, and the expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 proteins significantly increased. EgCF treatment caused cell shrinkage and disappearance of pseudopodia protrusions of LPS-activated cells, and led to the reduced phagocytic and migratory of cells; the protein expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 decreased significantly compared with the LPS group. Conclusion LPS induces the migration and enhances phagocytosis of macrophages while EgCF inhibits these effects, which is related to actin cytoskeleton rearrangement.

Effect of nanoparticles of different stiffness combined with menthol/curcumol on mechanical properties of bEnd.3 cells / 中国中药杂志

This study aimed to investigate the effects of nanoparticles PLGA-NPs and mesoporous silicon nanoparticles(MSNs) of different stiffness before and after combination with menthol or curcumol on the mechanical properties of bEnd.3 cells. The particle size distributions of PLGA-NPs and MSNs were measured by Malvern particle size analyzer, and the stiffness of the two nanoparticles was quantified by atomic force microscopy(AFM). The bEnd.3 cells were cultured in vitro, and the cell surface morphology, roughness, and Young's modulus were examined to characterize the roughness and stiffness of the cell surface. The changes in the mechanical properties of the cells were observed by AFM, and the structure and expression of cytoskeletal F-actin were observed by a laser-scanning confocal microscope. The results showed that both nanoparticles had good dispersion. The particle size of PLGA-NPs was(98.77±2.04) nm, the PDI was(0.140±0.030), and Young's modulus value was(104.717±8.475) MPa. The particle size of MSNs was(97.47±3.92) nm, the PDI was(0.380±0.016), and Young's modulus value was(306.019±8.822) MPa. The stiffness of PLGA-NPs was significantly lower than that of MSNs. After bEnd.3 cells were treated by PLGA-NPs and MSNs separately, the cells showed fine pores on the cell surface, increased roughness, decreased Young's modulus, blurred and broken F-actin bands, and reduced mean gray value. Compared with PLGA-NPs alone, PLGA-NPs combined with menthol or curcumol could allow deepened and densely distributed surface pores of bEnd.3 cells, increase roughness, reduce Young's modulus, aggravate F-actin band breakage, and diminish mean gray value. Compared with MSNs alone, MSNs combined with menthol could allow deepened and densely distributed surface pores of bEnd.3 cells, increase roughness, reduce Young's modulus, aggravate F-actin band breakage, and diminish mean gray value, while no significant difference was observed in combination with curcumol. Therefore, it is inferred that the aromatic components can increase the intracellular uptake and transport of nanoparticles by altering the biomechanical properties of bEnd.3 cells.

Study on mechanism of curcumol against liver fibrosis based on autophagy and apoptosis of hepatic stellate cells / 中国中药杂志

The present study clarified the molecular mechanism of curcumol against liver fibrosis based on its effects on the autopha-gy and apoptosis of hepatic stellate cells. The hepatic stellate cells were divided into a blank control group, a transforming growth factor-β1(TGF-β1)(10 ng·mL~(-1)) group, and low-(12.5 mg·L~(-1)), medium-(25 mg·L~(-1)), and high-dose(50 mg·L~(-1)) curcumol groups. The effect of curcumol on the viability of hepatic stellate cells induced by TGF-β1 was detected by the MTT assay kit. The apo-ptosis in each group was determined by flow cytometry. Real-time fluorescence-based quantitative PCR(RT-PCR) was employed for the detection of mRNA expression of α-smooth muscle actin(α-SMA), type Ⅰ collagen(collagen Ⅰ), and type Ⅲ collagen(collagen Ⅲ). Western blot was used to detect the protein expression of p62, microtubule-associated protein 1 light chain 3(LC3), beclin1, B cell lymphoma 2(Bcl-2), and Bcl-2-associated X protein(Bax). Transmission electron microscopy(TEM) was used to observe cell morphology and autophagosome formation in each group. The autophagic flux was observed after cell infection with adenovirus under double fluorescence labeling. The cell viability assay revealed that compared with the TGF-β1 group, the curcumol groups showed significantly decreased cell viability. The apoptosis assay showed that the apoptosis rates of the curcumol groups were significantly higher than that of the TGF-β1 group. RT-PCR indicated that the mRNA expression of α-SMA, collagenⅠ, and collagen Ⅲ in the curcumol groups was significantly lower than that of the TGF-β1 group. Western blot showed that the expression of p62, LC3, beclin1, Bcl-2, and Bax in the curcumol groups was significantly different from that in the TGF-β1 group. As demonstrated by TEM, compared with the TGF-β1 group, the curcumol groups showed significantly increased autophagosomes. The detection of autophagic flow by the adenovirus under double fluorescence labeling showed that autolysosomes in the curcumol groups were significantly increased compared with those in the TGF-β1 group. Curcumol can induce the autophagy and apoptosis of hepatic stellate cells, which may be one of its anti-liver fibrosis mechanisms.

Notum attenuates HBV-related liver fibrosis through inhibiting Wnt 5a mediated non-canonical pathways

BACKGROUND: Non-canonical Wnt pathways play important roles in liver fibrosis. Notum is a newly discovered inhibitor to Wnt proteins. This study was to investigate anti-fibrotic effects of Notum. METHODS: 53 patients with hepatitis B virus (HBV) infection as well as a cell co-culture system of LX-2 and Hep AD38 cells were engaged in this study. Clinical, biological and virological data of each patient were analyzed. Cell viability was detected at different time points. mRNA and protein levels of NFATc1 (Nuclear factor of activated T-cells), Jnk, α-SMA, Col1A1 and TIMP-1 were detected both in LX-2 and liver tissue. Protein levels of NFATc1 and Jnk in liver tissue and their correlations with fibrosis score were analyzed. RESULTS: Hepatitis B virus replication up-regulated Wnt5a induced NFATc1 and Jnk activity in Hep AD38. Notum suppressed NFATc1, Jnk and fibrosis genes expression, reduced cell viability in co-cultured LX-2 cells induced by HBV. Interestingly, Patients with HBV DNA > 5log copies/ml had higher mRNA levels of NFATc1 and fibrosis genes than patients with HBV DNA < 5log copies/ml. Most importantly, protein expressions of NFATc1 and pJnk have positive correlations with liver fibrosis scores in HBV-infected patients. CONCLUSIONS: Our data showed that Notum inhibited HBV-induced liver fibrosis through down-regulating Wnt 5a mediated non-canonical pathways. This study shed light on anti-fibrotic treatment.

Localization and changes of tyrosine phosphorylated proteins and ß actin in epididymis of rats treated with valproic acid / Localización y cambios de las proteínas tirosina fosforiladas y la ß actina en epidídimos de ratas tratadas con ácido valproico

Tyrosine phosphorylated proteins have been localized and identified in male reproductive tissues such as testis and capacitated/ acrosome reacted sperm except epididymis. The changes of such proteins are associated with decreased sperm quality of valproic acid treatment. This study aimed to investigate the presence and alterations of protein phosphorylation in epididymal epithelium and fluid of rats treated VPA. Sixteen adult male rats were divided into control and VPA-treated groups (n=8/ each). Treated rats were injected with VPA (500 mg/ kgBW, intraperitoneally) for 10 consecutive days. At the end of experiment, the monoclonal antiphosphotyrosine (clone 4G10) was used for immunohistochemistry to probe tyrosine phosphorylated proteins and also to examine the expression of such proteins using immuno-Western blotting in epididymal tissue and fluid. The result showed that positive reactivity of phosphorylated proteins was clearly observed in cytoplasmic principle cells, nuclei of apical & basal cells and sperm mass surrounded with epididymal fluids. The profiles of phosphorylated proteins in epididymal fluid were 182, 127, 80, 70, 57, 45, 34, and 31 kDas, respectively. Interestingly, VPA affected the changes of phosphorylated proteins and β actin in head, body, and tail epididymal fluids. We conclude that tyrosine phosphorylated proteins were detected in epididymal epithelium and fluid. The expressions of those proteins and actin were altered under VPA treating.

Las proteínas tirosina fosforiladas han sido localizadas e identificadas en tejidos reproductores masculinos tales como testículos y espermatozoides, capacitados a nivel acrosómico, excepto en el epidídimo. Los cambios de estas proteínas están asociadas con una disminución de la calidad del esperma en el tratamiento con ácido valproico (AVP). Este estudio tuvo como objetivo investigar la presencia y las alteraciones de la fosforilación de proteínas en el epitelio epididimal y en el fluido espermático de ratas tratadas con AVP. Dieciséis ratas macho adultas se dividieron en dos grupos: control y tratadas con AVP (n = 8 / cada uno). A las ratas tratadas se les inyectó AVP por vía intraperitoneal (500 mg / kg de peso corporal) durante 10 días consecutivos. Al final del experimento, se realizó inmunohistoquímica con la anti-fosfotirosina monoclonal (clon 4G10) para sondear las proteínas tirosina fosforiladas y también para examinar la expresión de tales proteínas usando inmunotransferencia Western, en tejido y fluido epididimarios. El resultado mostró reactividad positiva de proteínas fosforiladas en células citoplásmicas principales, en los núcleos de las células apicales y basales y en la masa de esperma rodeada por fluidos epididimarios. Los perfiles de proteínas fosforiladas en el fluido epididimal fueron 182, 127, 80, 70, 57, 45, 34 y 31 kDas, respectivamente. El AVP provocó cambios en las proteínas fosforiladas y en la β actina de los fluidos epididimarios de cabeza, cuerpo y cola del epidídimo. Concluimos que las proteínas tirosina fosforiladas se detectaron en el epitelio y el fluido epididimarios. Las expresiones de esas proteínas y de la β actina se alteraron bajo tratamiento con AVP.

Correlation between RNA Degradation Patterns of Rat's Brain and Early PMI at Different Temperatures / 法医学杂志

OBJECTIVES@#To explore the correlation between early postmortem interval （PMI） and eight RNA markers of rat's brain at different temperatures.@*METHODS@#Total 222 SD rats were randomly divided into control group （PMI=0 h） and four experimental groups. And the rats in the experimental groups were sacrificed by cervical dislocation and respectively kept at 5 ℃, 15 ℃, 25 ℃ and 35 ℃ in a controlled environment chamber. The RNA was extracted from brain tissues, which was taken at 9 time points from 1 h to 24 h postmortem. The expression levels of eight markers, β-actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, were detected using real-time fluorescent quantitative PCR, respectively. Proper internal reference was selected by geNorm software. Regression analysis of normalized RNA markers was performed by SPSS software. Mathematical model for PMI estimation was established using R software. Another 6 SD rats with known PMI were used to verify the mathematical model.@*RESULTS@#5S rRNA, miR-9 and miR-125b were suitable as internal reference markers for their stable expression. Both β-actin and GAPDH had well time-dependent degradation patterns and degraded continually with prolongation of PMI in 24 h postmortem. The mathematical model of the variation of ΔCt values with PMI and temperature was set up by R software and the model could be used for PMI estimation. The average error rates of model validation using β-actin and GAPDH were 14.1% and 22.2%, respectively.@*CONCLUSIONS@#The expression levels of β-actin and GAPDH are well correlated with PMI and environmental temperature. The mathematical model established in present study can provide references for estimating early PMI under various temperature conditions.

Inactivated Pseudomonas aeruginosa inhibits hypoxia-induced pulmonary hypertension by preventing TGF-beta1/Smad signaling

Pseudomonas aeruginosa is one of the common colonizing bacteria of the human body and is an opportunistic pathogen frequently associated with respiratory infections. Inactivated P. aeruginosa (IPA) have a variety of biological effects against inflammation and allergy. Transforming growth factor-β (TGF-β) signaling plays a critical role in the regulation of cell growth, differentiation, and development in a wide range of biological systems. The present study was designed to investigate the effects of IPA on TGF-β/Smad signaling in vivo, using a hypoxia-induced pulmonary hypertension (PH) rat model. Sprague Dawley rats (n=40) were exposed to 10% oxygen for 21 days to induce PH. At the same time, IPA was administered intravenously from day 1 to day 14. Mean pulmonary artery pressure (mPAP) and the right ventricle (RV) to left ventricle plus the interventricular septum (LV+S) mass ratio were used to evaluate the development of PH. Vessel thickness and density were measured using immunohistochemistry. Primary arterial smooth muscle cells (PASMCs) were isolated and the proliferation of PASMCs was assayed by flow cytometry. The production of TGF-β1 in cultured supernatant of PASMCs was assayed by ELISA. The expression levels of α-smooth muscle actin (α-SMA), TGF-β1 and phospho-Smad 2/3 in PASMCs were assayed by western blot. Our data indicated that IPA attenuated PH, RV hypertrophy and pulmonary vascular remodeling in rats, which was probably mediated by restraining the hypoxia-induced overactive TGF-β1/Smad signaling. In conclusion, IPA is a promising protective treatment in PH due to the inhibiting effects on TGF-β1/Smad 2/3 signaling.

Effect of intranasal rosiglitazone on airway inflammation and remodeling in a murine model of chronic asthma

BACKGROUND/AIMS: Asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Peroxisome proliferator-activated receptors have been reported to regulate inflammatory responses in many cells. In this study, we examined the effects of intranasal rosiglitazone on airway remodeling in a chronic asthma model. METHODS: We developed a mouse model of airway remodeling, including smooth muscle thickening, in which ovalbumin (OVA)-sensitized mice were repeatedly exposed to intranasal OVA administration twice per week for 3 months. Mice were treated intranasally with rosiglitazone with or without an antagonist during OVA challenge. We determined airway inflammation and the degree of airway remodeling by smooth muscle actin area and collagen deposition. RESULTS: Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation, compared with control mice. Additionally, the mice developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer. Administration of rosiglitazone intranasally inhibited the eosinophilic inflammation significantly, and, importantly, airway smooth muscle remodeling in mice chronically exposed to OVA. Expression of Toll-like receptor (TLR)-4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) was increased in the OVA group and decreased in the rosiglitazone group. Co-treatment with GW9660 (a rosiglitazone antagonist) and rosiglitazone increased the expression of TLR-4 and NF-kappaB. CONCLUSIONS: These results suggest that intranasal administration of rosiglitazone can prevent not only air way inf lammation but also air way remodeling associated with chronic allergen challenge. This beneficial effect is mediated by inhibition of TLR-4 and NF-kappaB pathways.

α-Smooth Muscle Actin and ACTA2 Gene Expressions in Vasculopathies

ABSTRACT α-smooth muscle actin, encoded by ACTA2 gene, is an isoform of the vascular smooth muscle actins, typically expressed in the vascular smooth muscle cells contributing to vascular motility and contraction. ACTA2 gene mutations cause a diversity of diffuse vasculopathies such as thoracic aortic aneurysms and dissections as well as occlusive vascular diseases, including premature coronary artery disease and ischemic stroke. Dynamics of differentiation-specific α-smooth muscle actin in arterial smooth muscle cells and proliferation of the proteins have been well described. Although a variety of research works have been undertaken in terms of modifications of α-smooth muscle actin and mutations of ACTA2 gene and myosin, the underlying mechanisms towards the pathological processes by way of gene mutations are yet to be clarified. The purpose of the present article is to describe the phenotypes of α-smooth muscle actin and implications of ACTA2 mutations in vasculopathies in order to enhance the understanding of potential mechanisms of aortic and coronary disorders.

Manual de monitoramento da qualidade dos registros de enfermagem na assistência domiciliar / Manual de monitoreo de la calidad de los registros de enfermería en la atención domiciliaria / Manual for monitoring the quality of nursing home care records

RESUMO Objetivo: construir e validar um instrumento para monitorar a qualidade dos registros de enfermagem no Programa de Assistência Domiciliar (PAD) em um hospital universitário. Método: estudo metodológico envolvendo a elaboração de um manual e submetido à validação de conteúdo por seis juízes sob consenso ≥ 80%. A coleta ocorreu em 2012 por meio de questionário contendo: evolução de enfermagem, diagnóstico e prescrição de enfermagem e normas para os registros da equipe de enfermagem preconizadas pelo Conselho Regional de Enfermagem-SP e pela instituição. Os itens do manual foram julgados de acordo com as variáveis - relevância, pertinência, clareza e simplicidade. Resultados: das 39 proposições 100% atingiram consenso ≥ 80% em relevância, pertinência e clareza; 92,3% em simplicidade. Os itens sono/repouso, mobilidade e checagem nas atividades prescritas não atingiram consenso mínimo favorável, sendo aprimorados pelas sugestões dos juízes. Conclusão: acreditamos que o instrumento possibilitará a melhoria dos processos de trabalho no PAD. .

RESUMEN Objetivo: construir y validar un instrumento para monitorear la calidad del registros de enfermería en Programa de Atención Domiciliaria (PAD) de un Hospital Universitario. Metodo: estudio metodológico. Fue construido un manual y sometió a validación de contenido por seis jueces bajo el consenso ≥80%. La recogida currió en 2012, con un cuestionario que contiene: evolución de enfermería, diagnóstico y prescripción de enfermería y normas para los registros del personal de enfermaria estabelecidas por Consejo Regional de Enfermería-SP y por la institución. Los artículos del manual fueran juzgadso conforme las variables relevancia, pertinencia, claridad y sencillez. Resultados: de las 39 proposiciones 100% alcanzó consenso ≥ 80% en la relevancia, pertinencia y claridad; 92,3% en la simplicidad. Los itens sueño/resto, movilidad y verificar las actividades prescritas no alcanzó consenso favorable, siendo mejoradas por las sugerencias de los jueces. Conclusión: creemos que el instrumento permitirá la mejora de los procesos de trabajo en PAD. .

ABSTRACT Objective: to build and validate an instrument aimed at monitoring the quality of nursing records in the Home Care Program (HCP) of a university hospital. Method: methodological study involving the elaboration of a manual, whose content was later submitted to six experts for validation, reaching a ≥ 80% consensus. The data collection process was carried out in 2012 by means of a questionnaire comprised of the following issues: nursing evolution, nursing diagnosis, and nursing prescription, and standards for the nursing team recommended by the Regional Nursing Council of São Paulo and by the assessed institution. Manual items were judged according to the following variables: relevance, pertinence, clarity and simplicity. Results: of the 39 propositions, 100% achieved ≥ 80% agreement in the relevance, pertinence and clarity variables; 92.3% in the simplicity variable. Sleep/rest, Mobility and Check-out variables did not reach a favorable minimum consensus in the prescribed activities and were improved following suggestions from the experts. Conclusion: we believe that the instrument will enable the improvement of the HCP’s work process. .

Fatores psicossociais e socioeconômicos relacionados à insônia e menopausa: Estudo Pró-Saúde / Psychosocial and socioeconomic factors related to insomnia and menopause: Pró-Saúde Study / Factores psicosociales y socioeconómicos relacionados con el insomnio y la menopausia: Estudio Pro-Salud

Foi avaliada a associação entre menopausa e insônia e a influência de variáveis socioeconômicas e psicossociais, em estudo transversal com 2.190 funcionárias de uma universidade (Estudo Pró-Saúde), a partir de um questionário autopreenchível com variáveis sobre menopausa, insônia, transtorno mental comum, eventos de vida estressantes, apoio social e variáveis socioeconômicas. Odds ratios foram calculados por meio de regressão logística multivariada, com desfecho politômico. Após ajuste para potenciais confundidoras sociodemográficas, mulheres na menopausa há mais de 60 meses apresentaram maior chance de reportar queixas de sono frequentes (OR entre 1,53 e 1,86) do que as que estavam na menopausa há menos de 60 meses. Após os ajustes, no primeiro grupo, para as variáveis psicossociais, a magnitude dos ORs reduziu para 1,53 (IC95%: 0,92-2,52) para dificuldade em iniciar o sono, 1,81 (IC95%: 1,09-2,98) para dificuldade em manter o sono e 1,71 (IC95%: 1,08-2,73) para queixa geral de insônia. Fatores psicossociais podem mediar a manifestação da insônia em mulheres na menopausa.

This study evaluated the association between insomnia and menopausal status and the influence of socioeconomic and psychosocial variables on this association in a cross-sectional analysis of 2,190 university employees (the Pró-Saúde Study). A self-administered questionnaire was used, covering menopausal status, complaints of insomnia, common mental disorders, stressful life events, social support, and socioeconomic variables. Odds ratios were calculated using logistic regression with a polytomous outcome. After adjusting for potential socio-demographic confounders, women who had entered menopause more than 60 months previously were more likely to report complaints with sleep (OR 1.53-1.86) as compared to women in menopause for less than 60 months. After adjusting for psychosocial variables, in the first group the ORs decreased to 1.53 (95%CI: 0.92-2.52) for difficulty initiating sleep, 1.81 (95%CI: 1.09-2.98) for difficulty maintaining sleep, and 1.71 (95%CI: 1.08-2.73) for general complaints of insomnia. Psychosocial factors can mediate the manifestation of insomnia among menopausal women.

En este estudio se evaluó la asociación entre insomnio y menopausia y la influencia de las variables socioeconómicas y psicosociales, en un estudio transversal con 2.190 mujeres de una universidad (Estudio Pro-Salud), a partir de un cuestionario autoadministrado con variables de la menopausia, insomnio, trastornos mentales, situaciones de estrés vital, apoyo social y variables socioeconómicas. Se calcularon los odds ratio mediante regresión logística multivariante con desenlace politómico. Después de ajustar por factores de confusión sociodemográficos potenciales, las mujeres menopáusicas desde hace más de 60 meses fueron más propensas a reportar quejas frecuentes de sueño (OR entre 1,53 y 1,86) que las menopáusicas hace menos de 60 meses. Después de los ajustes, en el primer grupo, para las variables psicosociales la magnitud de los OR se redujo a 1,53 (IC95%: 0,92-2,52) para la dificultad para iniciar el sueño, un 1,81 (IC95%: 1,09-2,98) para mantener el sueño y un 1,71 (IC95%: 1,08-2,73) para las quejas de insomnio en general. Los factores psicosociales pueden mediar en la manifestación del insomnio en las mujeres menopáusicas.

Quantification of C4d deposition and hepatitis C virus RNA in tissue in cases of graft rejection and hepatitis C recurrence after liver transplantation

Histology is the gold standard for diagnosing acute rejection and hepatitis C recurrence after liver transplantation. However, differential diagnosis between the two can be difficult. We evaluated the role of C4d staining and quantification of hepatitis C virus (HCV) RNA levels in liver tissue. This was a retrospective study of 98 liver biopsy samples divided into four groups by histological diagnosis: acute rejection in patients undergoing liver transplant for hepatitis C (RejHCV+), HCV recurrence in patients undergoing liver transplant for hepatitis C (HCVTx+), acute rejection in patients undergoing liver transplant for reasons other than hepatitis C and chronic hepatitis C not transplanted (HCVTx-). All samples were submitted for immunohistochemical staining for C4d and HCV RNA quantification. Immunoexpression of C4d was observed in the portal vessels and was highest in the HCVTx- group. There was no difference in C4d expression between the RejHCV+ and HCVTx+ groups. However, tissue HCV RNA levels were higher in the HCVTx+ group samples than in the RejHCV+ group samples. Additionally, there was a significant correlation between tissue and serum levels of HCV RNA. The quantification of HCV RNA in liver tissue might prove to be an efficient diagnostic test for the recurrence of HCV infection.

A low-protein diet during pregnancy prevents modifications in intercellular communication proteins in rat islets

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Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic beta-cells

AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic beta-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic beta-cells.

Toward understanding the role of the neuron-specific BAF chromatin remodeling complex in memory formation

The long-term storage of memory requires the finely tuned coordination of intracellular signaling with the transcriptional, translational and epigenetic regulations of gene expression. Among the epigenetic mechanisms, however, we know relatively little about the involvement of chromatin remodeling-dependent control of gene expression in cognitive brain functions, compared with our knowledge of other such mechanisms (for example, histone modifications and DNA methylation). A few recent studies have implicated the Brm/Brg-associated factor (BAF) chromatin-remodeling complex, a mammalian homolog of the yeast Swi/Snf complex, in neuronal structural/functional plasticity and memory formation. The BAF complex was previously known to have a critical role in neurodevelopment, but these recent findings indicate that it also contributes to both cognitive functions in the adult brain and human mental disorders characterized by intellectual disability. In this review, we provide a brief overview of the BAF complexes, introduce recent research findings that link their functions to memory formation, and speculate on the yet-unknown molecular mechanisms that may be relevant to these processes.

Dynamic expression of desmin, α-SMA and TGF-β1 during hepatic fibrogenesis induced by selective bile duct ligation in young rats

We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-β1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT‐PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-β1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT‐PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-β1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT‐PCR technique was more sensitive to expression changes than the semiquantitative method.

Immunohistochemical profile of stromal constituents and lymphoid cells over the course of wound healing in murine model

PURPOSE: To assess the evolution profile of the immunohistochemical expression of stromal constituents over the time-course of wound healing in a murine model. METHODS: Surgical wounds were performed in the back of 24 Wistar rats. After three, seven, 14 and 21 days, six rats were euthanized and the wounded histologically processed to assess the immunohistochemical expression of CD3, CD20, CD31, α-SMA and type-I collagen. Non-injured skin samples (NSS) were used as control. Data were subjected to statistical analysis using ANOVA and Tukey test. RESULTS: The mean of CD3 and CD20 positive cells in the wounds was significantly higher than in NSS at seven and 14 days (p<0.001). The blood vessels content was significantly lower than in NSS (p<0.05) at three days, but increased at seven and 14 days (p<0.01). The mean of α-SMA positive cells at seven, 14 and 21 days was higher than in NSS (p<0.05). The relative content of type I collagen increased from three to 21 days, but remained lower than in NSS (p<0.05). CONCLUSIONS: Lymphoid cells, myofibroblasts and microvessels contents varied over the time-course of wound healing, with peak at seven days and progressive reduction until 21 days. The type I collagen content increased over time. .

Histone demethylase retinoblastoma binding protein 2 regulates the expression of -smooth muscle actin and vimentin in cirrhotic livers

Liver cirrhosis is one of the most common diseases of Chinese patients. Herein, we report the high expression of a newly identified histone 3 lysine 4 demethylase, retinoblastoma binding protein 2 (RBP2), and its role in liver cirrhosis in humans. The siRNA knockdown of RBP2 expression in hepatic stellate cells (HSCs) reduced levels of α-smooth muscle actin (α-SMA) and vimentin and decreased the proliferation of HSCs; and overexpression of RBP2 increased α-SMA and vimentin levels. Treatment with transforming growth factor β (TGF-β) upregulated the expression of RBP2, α-SMA, and vimentin, and the siRNA knockdown of RBP2 expression attenuated TGF-β-mediated upregulation of α-SMA and vimentin expression and HSC proliferation. Furthermore, RBP2 was highly expressed in cirrhotic rat livers. Therefore, RBP2 may participate in the pathogenesis of liver cirrhosis by regulating the expression of α-SMA and vimentin. RBP2 may be a useful marker for the diagnosis and treatment of liver cirrhosis.