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BACKGROUND: Chronic remote ischemic conditioning (CRIC) has been shown to improve myocardial ischemia in experimental animal studies; however, its effectiveness in patients with chronic stable angina (CSA) has not been investigated. We conducted a proof-of-concept study to investigate the efficacy and safety of a six-month CRIC treatment in patients with CSA. METHODS: The EARLY-MYO-CSA trial was a prospective, randomized, controlled trial evaluating the CRIC treatment in patients with CSA with persistent angina pectoris despite receiving ≥ 3-month guideline-recommended optimal medical therapy. The CRIC and control groups received CRIC (at 200 mmHg) or sham CRIC (at 60 mmHg) intervention for 6 months, respectively. The primary endpoint was the 6-month change of myocardial flow reserve (MFR) on single-photon emission computed tomography. The secondary endpoints were changes in rest and stress myocardial blood flow (MBF), angina severity according to the Canadian Cardiovascular Society (CCS) classification, the Seattle Angina Questionnaire (SAQ), and a 6-min walk test (6-MWT). RESULTS: Among 220 randomized CSA patients, 208 (105 in the CRIC group, and 103 in the control group) completed the treatment and endpoint assessments. The mean change in MFR was significantly greater in the CRIC group than in the control group (0.27 ± 0.38 vs. - 0.04 ± 0.25; P < 0.001). MFR increased from 1.33 ± 0.48 at baseline to 1.61 ± 0.53 (P < 0.001) in the CRIC group; however, a similar increase was not seen in the control group (1.35 ± 0.45 at baseline and 1.31 ± 0.44 at follow-up, P = 0.757). CRIC treatment, when compared with controls, demonstrated improvements in angina symptoms assessed by CCS classification (60.0% vs. 14.6%, P < 0.001), all SAQ dimensions scores (P < 0.001), and 6-MWT distances (440 [400-523] vs. 420 [330-475] m, P = 0.016). The incidence of major adverse cardiovascular events was similar between the groups. CONCLUSIONS: CSA patients benefit from 6-month CRIC treatment with improvements in MFR, angina symptoms, and exercise performance. This treatment is well-tolerated and can be recommended for symptom relief in this clinical population. TRIAL REGISTRATION: [chictr.org.cn], identifier [ChiCTR2000038649].
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Angina Estável , Isquemia Miocárdica , Animais , Angina Estável/terapia , Estudos Prospectivos , Canadá , Doença CrônicaRESUMO
Objectives: The aims of this study was to investigate the use of cytologic samples for subclassification of lung adenocarcinoma and the cytologic-histologic correlation in lung adenocarcinoma subtypes using small samples. Methods and Methods: Cytological characteristics of lung adenocarcinoma subtypes were summarized by a literature review. Cytology samples from 115 patients with lung adenocarcinoma confirmed by small biopsies were classified by subtype. The diagnostic concordance of subtypes between biopsy and cytology samples was assessed. Results: Among the 115 cases, 62 (53.9%) had acinar predominant pattern, 16 (13.9%) were papillary predominant pattern, 29 (25.2%) had solid predominant pattern, 3 (2.6%) had lepidic predominant pattern, and 5 (4.3%) had micropapillary predominant pattern. All corresponding cytologic samples were classified into five subtypes based on cytomorphology features, with concordance rates of 74.2% (46 patients) in c-acinar subtype, 56.3% (nine patients) in c-papillary subtype, 24.1% (seven patients) in c-solid subtype, 66.7% (two patients) in c-lepidic subtype, and 40% (two patients) in c-micropapillary subtype. Collectively, the cytology and small biopsy concordance rate was approximately 57.4%. Conclusion: Subtyping of lung adenocarcinoma using cytologic specimens is challenging and the consistency rate varies with the subtype. Acinar predominant tumors have an excellent cytologic-histologic correlation compared to tumors with predominant solid or micropapillary pattern. Evaluating cytomorphologic features of different lung adenocarcinoma subtypes can reduce the false-negative rate of lung adenocarcinoma, particularly for the mild, atypical micropapillary subtype, and improve diagnostic accuracy.
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Background: This study explored the efficacy of the "L-sandwich" strategy, which involves the implantation of stents in the main vessel (MV) and shaft of the side branch (SB) with a drug-coated balloon (DCB) applied to the SB ostium, for coronary true bifurcation lesions. Methods and Results: Of 99 patients with true bifurcation lesions, 38 patients underwent the "L-sandwich" strategy (group A), 32 patients underwent a two-stent strategy (group B), and 29 patients underwent a single-stent + DCB strategy (group C). Angiography outcomes (late lumen loss [LLL], minimum lumen diameter [MLD]), and clinical outcomes (major adverse cardiac events [MACEs]) were analyzed. At 6 months, the MLD of the SB ostium in groups A and B were similar (P > 0.05) and group A larger than group C (P < 0.05). The LLL of group B was the largest among the three groups (P < 0.05). The MLD of the SB shaft in groups A and B were larger than in group C (P < 0.05). The LLL of the SB shaft in group C was the lowest (P < 0.05). Two patients in group B received target vessel revascularization at the 6-month followup (P > 0.05), and patients in the other groups had no MACEs. Conclusions: The "L-sandwich" strategy was feasible for the treatment of true coronary bifurcation lesions. It is a simpler procedure with similar acute lumen gain than the two-stent strategy, results in a larger SB lumen than the single-stent + DCB strategy, and it can also be used as a remedy for dissection following the single-stent + DCB strategy.
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Angioplastia Coronária com Balão , Doença da Artéria Coronariana , Stents Farmacológicos , Humanos , Angioplastia Coronária com Balão/métodos , Angiografia Coronária/métodos , Vasos Coronários/diagnóstico por imagem , Vasos Coronários/cirurgia , Vasos Coronários/patologia , Resultado do Tratamento , Stents , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/cirurgiaRESUMO
OBJECTIVE: MicroRNA (miR) therapeutics is a promising approach to manage coronary artery disease (CAD). Herein, this research was aimed to explore miR-181c-5p-related mechanisms in CAD through regulating SIRT1. METHODS: A CAD mouse model was established by feeding a high-fat diet in 8-week-old ApoE-/- mice. miR-181c-5p, SIRT1, and acetylated p65 levels in mouse myocardial tissues were evaluated by RT-qPCR and Western blot. Hemodynamic parameters included the maximum rising rate of the left ventricular pressure (lv + dp/dtmax) and the time values from the onset of contraction to dp/dtmax (t-dp/dtmax), while hemorheological indices included whole blood viscosity (low shear, middle shear, or high shear), plasma viscosity, hematocrit, and platelet adhesion were measured. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 were detected. Mouse pathological changes, degree of fibrosis, and cardiomyocyte apoptosis in myocardial tissues were assessed by HE, Masson, and TUNEL staining, respectively. The targeting relationship between miR-181c-5p and SIRT1 was verified by bioinformatics tools, dual luciferase reporter gene assay, and RNA pull-down assays. RESULTS: In myocardial tissue of CAD mice, miR-181c-5p and acetylated p65 were upregulated while SIRT1 was downregulated. Downregulating miR-181c-5p or upregulating SIRT1 effectively ameliorated CAD by improving hemodynamics and hemorheology and reducing inflammation, pathological changes, degree of fibrosis, and cardiomyocyte apoptosis in myocardial tissues of mice. miR-181c-5p targeted SIRT1, and overexpression of SIRT1 relieved upregulated miR-181c-5p-induced injuries in CAD mice. Regulating miR-181c-5p and SIRT1 affected the acetylation of p65. CONCLUSION: Downregulation of miR-181c-5p may ameliorate myocardial pathological changes and cardiomyocyte apoptosis in CAD by upregulating SIRT1 expression and decreasing acetylated p65 levels.
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Doença da Artéria Coronariana , MicroRNAs , Animais , Camundongos , Apoptose , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Fibrose , MicroRNAs/genética , MicroRNAs/metabolismo , Miocárdio/patologia , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
Myocardial infarction (MI), which is due to cardiac dysfunction, results in morbidity and mortality. Moreover, the cellular activity of transplanted mesenchymal stem cells (MSCs) generally limits their therapeutic efficacy in the treatment of MI. Here, inject able hyaluronic acid-chitosan/ß-glycerophosphate (HA-CS/ß-GP) hydrogel-loaded MSCs are prepared, after which their effects on the treatment of MI are investigated. The synthesized HA-CS/ß-GP hydrogels exhibit swelling ratio, an in vitro degradation value, and a gelatin time of 82.19 ± 4.1, 88.18% ± 2.4%, and 9 s, respectively. Further, rheological studies revealed that the elastic modulus of the HA-CS/ß-GP hydrogels is ≥230 Pa, exhibiting large elastic to viscous modulus ratio, which indicates their mechanical strength. Furthermore, the in vitro 3T3 cell and MSC culture studies confirm the good biocompatibility of the HA-CS and HA-CS/ß-GP hydrogels. The implantation of the synthesized hydrogels in the mouse MI model considerably improves the therapeutic effect of the MSCs (enhanced cardiac function, reduced cardiomyocyte apoptosis, and increased vascularization) for the first time. The innovative synergistic strategy of combining injectable HA-CS and HA-CS/ß-GP hydro gels with MSCs may be suitable for the effective treatment of cardiac morbidity due to MIs.
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Quitosana , Células-Tronco Mesenquimais , Infarto do Miocárdio , Animais , Quitosana/farmacologia , Glicerofosfatos , Ácido Hialurônico/farmacologia , Hidrogéis/farmacologia , Camundongos , Infarto do Miocárdio/terapiaRESUMO
BACKGROUND: Expansion-mediated replicative senescence and age-related natural senescence have adverse effects on mesenchymal stem cell (MSC) regenerative capability and functionality, thus severely impairing the extensive applications of MSC-based therapies. Emerging evidences suggest that microRNA-34a (miR-34a) has been implicated in the process of MSC senescence; however, the molecular mechanisms with regard to how miR-34a influencing MSC senescence remain largely undetermined. METHODS: MiR-34a expression in MSCs was evaluated utilizing RT-qPCR. The functional effects of miR-34a exerting on MSC senescence were investigated via gene manipulation. Relevant gene and protein expression levels were analyzed by RT-qPCR and western blot. Luciferase reporter assays were applied to confirm that Nampt is a direct target of miR-34a. The underlying regulatory mechanism of miR-34a targeting Nampt in MSC senescence was further explored by measuring intracellular NAD+ content, NAD+/NADH ratio and Sirt1 activity. RESULTS: In contrast to Nampt expression, miR-34a expression incremented in senescent MSCs. MiR-34a overexpression in young MSCs resulted in senescence-associated characteristics as displayed by senescence-like morphology, prolonged cell proliferation, declined osteogenic differentiation potency, heightened senescence-associated-ß-galactosidase activity, and upregulated expression levels of the senescence-associated factors. Conversely, miR-34a suppression in replicative senescent and natural senescent MSCs contributed to diminished senescence-related phenotypic features. We identified Nampt as a direct target gene of miR-34a. In addition, miR-34a repletion resulted in prominent reductions in Nampt expression levels, NAD+ content, NAD+/NADH ratio, and Sirt1 activity, whereas anti-miR-34a treatment exerted the opposite effects. Furthermore, miR-34a-mediated MSC senescence was evidently rescued following the co-treatment with Nampt overexpression. CONCLUSION: This study identifies a significant role of miR-34a playing in MSC replicative senescence and natural senescence via targeting Nampt and further mediating by NAD+-Sirt1 pathway, carrying great implications for optimal strategies for MSC therapeutic applications.
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Células-Tronco Mesenquimais , MicroRNAs , Senescência Celular , MicroRNAs/genética , NAD , Osteogênese , Sirtuína 1/genéticaRESUMO
Oxidative stress is one of the main causes of aging. The process of physiological aging is always accompanied by increased levels of endogenous oxidative stress. Exogenous oxidants have contributed to premature cellular senescence. As a deacetylase located in mitochondrial matrix, Sirt3 plays critical roles in mitochondrial energy metabolism, oxidative stress regulation, and cellular senescence. However, it remains unknown whether Sirt3 exerts the analogous role in cellular senescence caused by two different oxidation pathways. In this study, the function of Sirt3 was investigated in age-related natural senescence and H2O2-induced premature senescence of rat bone marrow mesenchymal stem cells (MSCs). Our results showed that Sirt3 expression was significantly decreased in both senescent MSCs, which was concerned with reduced cellular reactive oxygen species (ROS) and aggravated DNA injury. Sirt3 repletion could partly reverse the senescence-associated phenotypic features in natural and premature senescent MSCs. Moreover, Sirt3 replenishment led to the reduction in the levels of cellular ROS by enhancing the expression and activity of superoxide dismutase 2 (SOD2), thus maintaining the balance of intracellular oxidation and antioxidation and ameliorating oxidative stress damage. Altogether, Sirt3 inhibits MSC natural senescence and H2O2-induced premature senescence through alleviating ROS-induced injury and upregulating SOD2 expression and activity. Our research indicates that Sirt3 might contribute to uncovering the novel mechanisms underlying MSC senescence and provide new insights to aging and oxidative stress-related diseases.
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OBJECTIVE: In 2013, The Paris System for Reporting Urinary Cytology (TPS) was developed as a uniform practical urine cytology system that could be applied worldwide. Here, we investigated the effectiveness of TPS diagnostic approach compared with that of the traditional urine cytological diagnosis method used in China. METHODS: Based on the diagnostic criteria of TPS, 412 urine samples from 143 patients with histological follow-up data were retrospectively analysed, and the diagnoses were compared with the original cytological diagnoses. RESULTS: In total, 110 patients were histologically diagnosed with high-grade urothelial carcinoma (HGUC), and 33 patients were diagnosed with low-grade urothelial neoplasia. Based on the traditional urine cytological analysis method, 50 patients (34.9%) were diagnosed as negative, 48 patients (33.6%) were diagnosed as having atypical urothelial cells, and 45 patients (31.5%) were diagnosed as positive. After reclassification using TPS, urine samples from 11 cases (7.7%) were categorised as unsatisfactory, 34 cases (23.8%) were negative, 21 cases (14.7%) were categorised as having atypical urothelial cells, 12 cases (8.4%) were diagnosed as suspicious for HGUC, 59 cases (41.2%) were diagnosed with HGUC, and six cases (4.2%) were reclassified as having low-grade urothelial neoplasia. Thus, after implementing TPS criteria, the sensitivity for positive malignancy diagnoses (HGUC alone) increased from 38.2% to 50.9%, while the specificity of the diagnosis was barely changed. CONCLUSIONS: The Paris System for Reporting Urinary Cytology greatly contributes to the standardisation of urine cytology reports and significantly improves the diagnostic sensitivity for HGUC.
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Carcinoma de Células de Transição/urina , Citodiagnóstico , Diagnóstico Diferencial , Neoplasias Urológicas/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células de Transição/classificação , Carcinoma de Células de Transição/patologia , China/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sistema Urinário/diagnóstico por imagem , Sistema Urinário/patologia , Neoplasias Urológicas/classificação , Neoplasias Urológicas/patologia , Urotélio/diagnóstico por imagem , Urotélio/patologiaRESUMO
The present study aimed to investigate the involvement of long intergenic non-coding RNA for kinase activation (LINK-A) long non-coding RNA (lncRNA) in osteosarcoma. Plasma levels of LINK-A lncRNA and transforming growth factor ß1 (TGF-ß1) were measured by reverse transcription-quantitative polymerase chain reaction and ELISA, respectively. Correlation between LINK-A lncRNA and TGF-ß1 was analyzed by Pearson correlation coefficient. LINK-A lncRNA and TGF-ß1 were upregulated in patients with osteosarcoma compared with healthy controls. LINK-A lncRNA and TGF-ß1 were positively correlated in the two groups. LINK-A lncRNA short hairpin RNAs (shRNAs) were transfected into osteosarcoma cell lines and Transwell migration assay, Matrigel invasion assay and flow cytometry were used to evaluate cell migration, invasion and stemness, respectively. Effects of LINK-A lncRNA silencing and overexpression on TGF-ß1 expression were analyzed by western blotting. LINK-A lncRNA shRNA silencing inhibited, whereas TGF-ß1 treatment promoted cell migration, invasion and stemness. LINK-A lncRNA silencing inhibited TGF-ß1 expression, whereas TGF-ß1 treatment had no effects on LINK-A lncRNA expression. TGF-ß1 reduced the inhibitory effects of LINK-A lncRNA knockdown on cancer cell migration, invasion and stemness. These data indicated that LINK-A lncRNA is upregulated in osteosarcoma and may regulate migration, invasion and stemness of osteosarcoma cells through TGF-ß1.
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The aim of our study was to explore the roles of miR-671-5p in mediating biological processes of osteosarcoma (OS) cells and clinical implications. On the basis of the OS samples acquired from the GEO database, the expression difference and overall survival analyses of miR-671-5p and TUFT1 were determined. The expression of MiR-671-5p was verified using OS cell lines. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound-healing, and Transwell assays were respectively carried out to probe whether miR-671-5p regulated OS cell vitality, migration, and invasion. The expression of miR-671-5p was downregulated in OS tissues and cell lines. High expression of MiR-671-5p blocked OS cell growth, migration, and invasion. TUFT1 was predicted and validated as the target of miR-671-5p in OS cells using in silico analysis and luciferase reporter assays. Forced expression of TUFT1 reversed the suppressive influence of miR-671-5p on cell viability, migration, and invasion of OS cells. Moreover, the low expression of miR-671-5p and the high expression of TUFT1 led to poor prognosis. Taken together, targeting miR-671-5p/TUFT1 may be a promising strategy for treating OS.
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Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/mortalidade , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Proteínas do Esmalte Dentário/metabolismo , MicroRNAs/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/mortalidade , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proteínas do Esmalte Dentário/genética , Progressão da Doença , Humanos , MicroRNAs/genética , Osteossarcoma/patologia , Prognóstico , Taxa de Sobrevida , TransfecçãoRESUMO
As a chronic metabolic disease, diabetes mellitus (DM) is broadly characterized by elevated levels of blood glucose. Novel epidemiological studies demonstrate that some diabetic patients have an increased risk of developing dementia compared with healthy individuals. Alzheimer's disease (AD) is the most frequent cause of dementia and leads to major progressive deficits in memory and cognitive function. Multiple studies have identified an increased risk for AD in some diabetic populations, but it is still unclear which diabetic patients will develop dementia and which biological characteristics can predict cognitive decline. Although few mechanistic metabolic studies have shown clear pathophysiological links between DM and AD, there are several plausible ways this may occur. Since AD has many characteristics in common with impaired insulin signaling pathways, AD can be regarded as a metabolic disease. We conclude from the published literature that the body's diabetic status under certain circumstances such as metabolic abnormalities can increase the incidence of AD by affecting glucose transport to the brain and reducing glucose metabolism. Furthermore, due to its plentiful lipid content and high energy requirement, the brain's metabolism places great demands on mitochondria. Thus, the brain may be more susceptible to oxidative damage than the rest of the body. Emerging evidence suggests that both oxidative stress and mitochondrial dysfunction are related to amyloid-ß (Aß) pathology. Protein changes in the unfolded protein response or endoplasmic reticulum stress can regulate Aß production and are closely associated with tau protein pathology. Altogether, metabolic disorders including glucose/lipid metabolism, oxidative stress, mitochondrial dysfunction, and protein changes caused by DM are associated with an impaired insulin signal pathway. These metabolic factors could increase the prevalence of AD in diabetic patients via the promotion of Aß pathology.
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Doença de Alzheimer/epidemiologia , Glicemia , Diabetes Mellitus/epidemiologia , Estresse do Retículo Endoplasmático , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Metabolismo dos Lipídeos , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Resposta a Proteínas não DobradasRESUMO
Vascular smooth muscle cell (VSMC) accumulation and endothelial cell dysfunction are associated with pathogenesis of atherosclerosis. Long noncoding RNA taurine up-regulated gene 1 (TUG1) has been reported to play an important role in cardiovascular diseases, including atherosclerosis. However, the regulatory mechanism underlying TUG1 in atherosclerosis is far from understood. VSMC and human umbilical vein endothelial cells (HUVEC) stimulated by oxidized low-density lipoprotein (ox-LDL) were used as cellular model of atherosclerosis. Cell proliferation and apoptosis were detected by CCK-8, flow cytometry and Western blot. The expression levels of TUG1, microRNA (miR)-148b and insulin-like growth factor 2 (IGF2) were measured by quantitative real-time polymerase chain reaction or Western blot. The target association among TUG1, miR-148b and IGF2 was determined by luciferase reporter assay and RNA immunoprecipitation. The expression of TUG1 was increased in ox-LDL-treated VSMC and HUVEC. Silence of TUG1 inhibited proliferation and promoted apoptosis in ox-LDL-treated VSMC but induced proliferation promotion and apoptosis inhibition in HUVEC stimulated by ox-LDL. miR-148b was a target of TUG1 and its knockdown reversed the effect of TUG1 silence on proliferation and apoptosis of VSMC and HUVEC challenged by ox-LDL. IGF2 was a target of miR-148b and miR-148b regulated proliferation and apoptosis in ox-LDL-treated VSMC and HUVEC by targeting IGF2. TUG1 promoted IGF2 protein expression by sponging miR-148b. TUG1 knockdown attenuated ox-LDL-induced injury through regulating proliferation and apoptosis of VSMC and HUVEC by miR-148b/IGF2 axis, providing a novel mechanism for pathogenesis of atherosclerosis.
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Apoptose/fisiologia , Proliferação de Células/fisiologia , Fator de Crescimento Insulin-Like II/fisiologia , Lipoproteínas LDL/farmacologia , MicroRNAs/fisiologia , RNA Longo não Codificante/fisiologia , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , HumanosRESUMO
Diabetes mellitus adversely affects human bones and increases the risk of developing osteoporosis. In the present study, treatment with 30 mmol/l glucose was used to establish a high glucose (HG) cell model in vitro. Plasmids were used to overexpress the P2X purinoceptor 7 (P2X7) gene. Brilliant blue G and (4benzoylbenzoyl)ATP were used as a P2X7 antagonist and agonist, respectively. Proliferation of osteogenic MC3T3E1 cells and alkaline phosphatase (ALP) activity were determined using MTT and colorimetric assays, respectively. Alizarin Red S was used to assess calcification of MC3T3E1 cells. Western blotting and reverse transcriptionquantitative PCR were performed to determine protein and mRNA expression levels. The results demonstrated that HG inhibited MC3T3E1 cell proliferation and P2X7 expression, reduced calcification, and downregulated the expression levels of ALP and osteocalcin (Ocn) in MC3T3E1 cells. Overexpression of P2X7 in HG conditions increased calcification and proliferation, and upregulated the levels of ALP and Ocn in MC3T3E1 cells. Inhibition of P2X7 downregulated the expressions of ALP and Ocn in MC3T3E1 cells under HG conditions. Therefore, the present results indicated that HG caused damage to osteogenic MC3T3E1 cells. Thus, P2X7 may be a regulatory factor that may be used to counteract the effects of HG on osteogenesis.
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Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glucose/antagonistas & inibidores , Osteogênese/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2X/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Células 3T3 , Trifosfato de Adenosina/agonistas , Trifosfato de Adenosina/análogos & derivados , Animais , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Regulação para Baixo , Humanos , Camundongos , Osteocalcina/metabolismo , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2X7/genética , Regulação para CimaRESUMO
Decreased bone volume and strength with aging and enhanced risk of fractures are in part due to reduced number of bone-forming mesenchymal stem cells (MSCs) and cellular dysfunction. In a previous study, we found that osteogenic differentiation of the multipotent and omnipotent preosteoblasts are accompanied by the alterations of intracellular NAD metabolism in which nicotinamide phosphoribosyltransferase (Nampt) plays a regulatory role. The increased Nampt during osteoblast differentiation, the enzyme catalyzing NAD resynthesis from nicotinamide was noted. However, whether Nampt will also be able to affect osteogenic differentiation of primary bone marrow-derived mesenchymal stem cells (BM-MSCs), it is still uncertain. Here we report the role of Nampt in regulating osteoblast differentiation in primary mouse BM-MSCs. We found that Nampt expression was progressively elevated during BM-MSCs osteogenic differentiation. The Nampt inhibitor FK866 or knock-down of Nampt in BM-MSCs led to declined osteoblastogenesis, including attenuated ALP activity, diminished matrix mineralization and down-regulated osteoblast specific marker genes. In addition, declined osteoblastogenesis by Nampt deficiency or addition of FK866 was related to lower intracellular NAD concentration and decreased Sirt1 activity. The present findings demonstrate that osteogenic differentiation in MSCs can be modulated by intracellular NAD metabolism, in which Nampt may serve as an applicable marker for the osteoblast determination.
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Medula Óssea/metabolismo , Diferenciação Celular , Citocinas/metabolismo , Células-Tronco Mesenquimais/citologia , Nicotinamida Fosforribosiltransferase/metabolismo , Osteoblastos/citologia , Animais , Apoptose , Western Blotting , Proliferação de Células , Células Cultivadas , Citocinas/genética , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Osteoblastos/metabolismo , Osteogênese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
Senescence restricts the development of applications involving mesenchymal stem cells (MSCs) in research fields, such as tissue engineering, and stem cell therapeutic strategies. Understanding the mechanisms underlying natural aging processes may contribute to the development of novel approaches to preventing age-related diseases or slowing individual aging processes. Nampt is a rate-limiting NAD biosynthetic enzyme that plays critical roles in energy metabolism, cell senescence and maintaining life spans. However, it remains unknown whether Nampt influences stem cell senescence. In this study, the function of Nampt was investigated using a rat model of natural aging. Our data show that Nampt expression was significantly lower in MSCs obtained from aged rats than in those obtained from young rats during physiological aging. Reducing the level of Nampt in aged MSCs resulted in lower intracellular concentrations of NAD+ and downregulated Sirt1 expression and activity. After the Nampt inhibitor FK866 was added, young MSCs were induced to become aged cells. The enhanced senescence was correlated with NAD+ depletion and Sirt1 activity attenuation. In addition, Nampt overexpression attenuated cell senescence in aged MSCs. Our findings provide a new explanation for the mechanisms underlying stem cell senescence and a novel target for delaying stem cell senescence and preventing and treating age-related diseases.
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Células da Medula Óssea/metabolismo , Senescência Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Sirtuína 1/metabolismo , Acrilamidas/farmacologia , Fatores Etários , Envelhecimento/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/genética , Piperidinas/farmacologia , Ratos , Sirtuína 1/genéticaRESUMO
Wheat (Triticum aestivum), one of the most important cereal crops, is often threatened by drought. In this study, water deficit significantly reduced the height of plants and yield of grains. To explore further the effect of drought stress on the development and yield of grains, we first performed a large scale phosphoproteome analysis of developing grains in wheat. A total of 590 unique phosphopeptides, representing 471 phosphoproteins, were identified under well-watered conditions. Motif-X analysis showed that four motifs were enriched, including [sP], [Rxxs], [sDxE], and [sxD]. Through comparative phosphoproteome analysis between well-watered and water-deficit conditions, we found that 63 unique phosphopeptides, corresponding to 61 phosphoproteins, showed significant changes in phosphorylation level (≥2-fold intensities). Functional analysis suggested that some of these proteins may be involved in signal transduction, embryo and endosperm development of grains, and drought response and defense under water-deficit conditions. Moreover, we also found that some chaperones may play important roles in protein refolding or degradation when the plant is subjected to water stress. These results provide a detailed insight into the stress response and defense mechanisms of developmental grains at the phosphoproteome level. They also suggested some potential candidates for further study of transgenosis and drought stress as well as incorporation into molecular breeding for drought resistance.
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Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Proteoma , Triticum/metabolismo , Água , Irrigação Agrícola , Triticum/crescimento & desenvolvimentoRESUMO
This study explored the expression of connexin 43 (Cx43) in the testes of prepubertal Sprague-Dawley (SD) rats following in utero flutamide (Flu) exposure. Connexins constitute the major protein type in gap junctions. Connexin 43, the most prominent connexin family member expressed by testes, is localized at the base of seminiferous tubules in humans and rodents, and may be involved in fertility. Flutamide was injected subcutaneously into pregnant SD rats on gestational days 12-21 (25 mg/kg/day). Immunohistochemistry, Western blotting, and real-time PCR was used to investigate the distribution and the expression of Cx43 protein and mRNA in the testis on postnatal day 20 (PD20). Following Flu-exposure, Cx43 was observed between Sertoli cells in the seminiferous tubules. On PD20, no Cx43 protein was expressed by the spermatogonial cell layer of the seminiferous tubules in the controls, but was observed in the Flu-exposed group. Western blotting showed that Cx43 was expressed at significantly lower levels in Flu-exposed testes than controls on PD20 (p < 0.001). On PD20, levels of Cx43 mRNA in undescended Flu-exposed testes were significantly lower than in controls (p < 0.05) and descended Flu-exposed testes (p < 0.01). After Flu-exposure in the rat embryonic period, Cx43 mRNA and protein expression were downregulated, and its distribution in the seminiferous tubules was abnormal.
Assuntos
Antagonistas de Androgênios/farmacologia , Conexina 43/metabolismo , Flutamida/farmacologia , Exposição Materna , Túbulos Seminíferos/efeitos dos fármacos , Animais , Conexina 43/genética , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Masculino , Gravidez , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos/metabolismoRESUMO
<p><b>OBJECTIVE</b>In oder to treat periodontitis by using tissue engineering and gene engineering technology, the article established an transient expression system of bone marrow stromal cells (BMSC) modified by osteoprotegerin (OPG) gene and detected its expression using eukaryotic secreted expression pSecTag2/B-OPG plasmid.</p><p><b>METHODS</b>By solation and culture of BMSC in vitro, the identified recombined plasmid was transiently transfected into BMSC by Lipofectamine 2000 and OPG expression in BMSC was determined by RT-PCR and Western blot in 6 weeks.</p><p><b>RESULTS</b>The fragments of the recombinant plasmid digested with Hind III, EcoR I and BamH I and examined by 10 g/L agarose electrophoresis, were consistent with predicted size. The sequence of OPG was identical to the sequence provided by GeneBank [gi:33878056]. OPG transcribing in BMSC was confirmed by RT-PCR and OPG sustainable expressing in BMSC was detected by Western blot in 39 days.</p><p><b>CONCLUSION</b>The transiently expression system of BMSC modified by OPG gene was successfully established.</p>