RESUMO
Endothelial dysfunction often accompanies sepsis. Sevoflurane (Sev) is a widely used inhaled anesthetic that has a protective effect on sepsis-associated damage. We aimed to elucidate the role of Sev in endothelial dysfunction by using a model of LPS induced HUVECs. Sev increased the viability and decreased the apoptosis of HUVECs exposed to LPS. Inflammation and endothelial cell adhesion were improved after Sev addition. Besides, Sev alleviated LPS-induced endothelial cell permeability damage in HUVECs. RORα served as a potential protein that bound to Sev. Importantly, Sev upregulated RORα expression and inhibited endoplasmic reticulum (ER) stress in LPS-treated HUVECs. RORα silencing reversed the impacts of Sev on ER stress. Moreover, RORα deficiency or tunicamycin (ER stress inducer) treatment restored the effects of Sev on the viability, apoptosis, inflammation and endothelial permeability damage of HUVECs exposed to LPS. Taken together, Sev ameliorated LPS-induced endothelial cell damage by targeting RORα to inhibit ER stress.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Células Endoteliais da Veia Umbilical Humana , Inflamação , Lipopolissacarídeos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares , Sevoflurano , Regulação para Cima , Humanos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sevoflurano/farmacologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Inflamação/patologia , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Regulação para Cima/efeitos dos fármacos , Permeabilidade/efeitos dos fármacosRESUMO
One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis-Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins.
Assuntos
Benzaldeídos , Glucosiltransferases , Cinética , Glucosiltransferases/metabolismo , Ácidos Graxos , Especificidade por SubstratoRESUMO
BACKGROUND: NOS1 expression predicts poor prognosis in patients with melanoma. However, the molecular function of NOS1 in the type I IFN response and immune escape of melanoma is still unknown. METHODS: The CRISPR/Cas9 system was used to generate NOS1-knockout melanoma cells and the biological characteristics of NOS1-knockout cells were evaluated by MTT assay, clonogenic assay, EdU assay, and flow cytometric assay. The effect on tumor growth was tested in BALB/c-nu and C57BL/6 mouse models. The gene expression profiles were detected with Affymetrix microarray and RNA-seq and KEGG (Kyoto Encyclopedia of Genes and Genomes) and CLUE GO analysis was done. The clinical data and transcriptional profiles of melanoma patients from the public database TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus, GSE32611) were analyzed by Qlucore Omics Explorer. RESULTS: NOS1 deletion suppressed the proliferation of melanoma A375 cells in culture, blocked cell cycling at the G0/G1 phase, and decreased the tumor growth in lung metastasis nodes in a B16 melanoma xenograft mouse model. Moreover, NOS1 knockout increased the infiltration of CD3+ immune cells in tumors. The transcriptomics analysis identified 2203 differential expression genes (DEGs) after NOS1 deletion. These DEGs indicated that NOS1 deletion downregulated mostly metabolic functions but upregulated immune response pathways. After inhibiting with NOS1 inhibitor N-PLA, melanoma cells significantly increased the response to IFN[Formula: see text] by upregulation expression of IFN[Formula: see text] simulation genes (ISGs), especially the components in innate immune signaling, JAK-STAT, and TOLL-LIKE pathway. Furthermore, these NOS1-regulating immune genes (NOS1-ISGs) worked as a signature to predict poor overall survival and lower response to chemotherapy in melanoma patients. CONCLUSION: These findings provided a transcriptional evidence of NOS1 promotion on tumor growth, which is correlated with metabolic regulation and immune escape in melanoma cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma Experimental , Animais , Perfilação da Expressão Gênica , Humanos , Interferons , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo IRESUMO
To explore the potential role of Lin28a in the development of restenosis after percutaneous transluminal angioplasty, double-balloon injury surgery and mono-balloon injury surgery were used to establish restenosis and atherosclerosis models, respectively, so as to better distinguish restenosis from atherosclerotic lesions. Immunohistochemical analysis revealed that significantly higher expression of Lin28a was observed in the iliac arteries of restenosis plaques than that of atherosclerosis plaques. Immunofluorescence studies showed the colocalization of Lin28a with α-smooth muscle actin in restenosis plaques, rather than in atherosclerosis plaques, which suggested that Lin28a might be related to the unique behaviour of vascular smooth muscle cells (VSMCs) in restenosis. To further confirm above hypothesis, Lin28a expression was up-regulated by transfection of Lenti-Lin28a and inhibited by Lenti-Lin28a-shRNA transfection in cultured VSMCs, and then the proliferation and migration capability of VSMCs were detected by EdU and Transwell assays, respectively. Results showed that the proliferation and migration of VSMCs were significantly increased in accordance with the up-regulation of Lin28a expression, while above behaviours of VSMCs were significantly suppressed after inhibiting the expression of Lin28a. In conclusion, the up-regulation of Lin28a exerts its modulatory effect on VSMCs' proliferation and migration, which may play a critical role in contributing to pathological formation of restenosis.
Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Proteínas de Ligação a RNA/genética , Regulação para Cima/genética , Angioplastia/métodos , Animais , Aterosclerose/genética , Células Cultivadas , Masculino , Placa Aterosclerótica/genética , Ratos , Ratos Sprague-Dawley , Transfecção/métodosRESUMO
BACKGROUND: Hyperlipidaemia causes kidney damage over the long term. We investigated the effect of the administration of endothelial progenitor cells (EPCs) on the progression of kidney damage in a mouse model of hyperlipidaemia. METHODS: Apolipoprotein E-knockout (ApoE-/-) mice were treated with a high-cholesterol diet after spleen resection. Twenty-four weeks later, the mice were divided into two groups and intravenously injected with PBS or EPCs. Six weeks later, the recruitment of EPCs to the kidney was monitored by immunofluorescence. The lipid, endothelial cell, and collagen contents in the kidney were evaluated by specific immunostaining. The protein expression levels of transforming growth factor-ß (TGF-ß), Smad2/3, and phospho-Smad3 (p-smad3) were detected by western blot analysis. RESULTS: ApoE-/- mice treated with a high-fat diet demonstrated glomerular lipid deposition, enlargement of the glomerular mesangial matrix, endothelial cell enlargement accompanied by vacuolar degeneration and an area of interstitial collagen in the kidney. Six weeks after EPC treatment, only a few EPCs were detected in the kidney tissues of ApoE-/- mice, mainly in the kidney interstitial area. No significant differences in TGF-ß, p-smad3 or smad2/3 expression were found between the PBS group and the EPC treatment group (TGF-ß expression, PBS group: 1.06 ± 0.09, EPC treatment group: 1.09 ± 0.17, P = 0.787; p-smad3/smad2/3 expression: PBS group: 1.11 ± 0.41, EPC treatment group: 1.05 ± 0.33, P = 0.861). CONCLUSIONS: Our findings demonstrate that hyperlipidaemia causes basement membrane thickening, glomerulosclerosis and the vascular degeneration of endothelial cells. The long-term administration of EPCs substantially has limited effect in the progression of kidney damage in a mouse model of hyperlipidaemia.
Assuntos
Apolipoproteínas E/metabolismo , Células da Medula Óssea/citologia , Células Progenitoras Endoteliais/transplante , Hiperlipidemias/terapia , Rim/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Western Blotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Esplenectomia , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Sulfonylurea receptor 1 (SUR1) is primarily responsible for glucose regulation in normal conditions. Here, we sought to investigate the glucose metabolism characteristics of SUR1-/- rats. METHODS: The TALEN technique was used to construct a SUR1 gene deficiency rat model. Rats were grouped by SUR1 gene knockout or not and sex difference. Body weight; glucose metabolism indicators, including IPGTT, IPITT, glycogen contents and so on; and other molecule changes were examined. RESULTS: Insulin secretion was significantly inhibited by knocking out the SUR1 gene. SUR1-/- rats showed lower body weights compared to wild-type rats, and even SUR1-/- males weighed less than wild-type females. Upon SUR1 gene knockout, the rats showed a peculiar plasma glucose profile. During IPGTT, plasma glucose levels were significantly elevated in SUR1-/- rats at 15 min, which could be explained by SUR1 mainly working in the first phase of insulin secretion. Moreover, SUR1-/- male rats showed obviously impaired glucose tolerance than before and a better insulin sensitivity in the 12th week compared with females, which might be related with excess androgen secretion in adulthood. Increased glycogen content and GLUT4 expression and the inactivation of GSK3 were also observed in SUR1-/- rats, which suggested an enhancement of insulin sensitivity. CONCLUSIONS: These results reconfirm the role of SUR1 in systemic glucose metabolism. More importantly, our SUR1-/- rat model might be applied in other fields, such as for exploring other hypoglycaemic functions of sulfonylureas.
Assuntos
Glucose/metabolismo , Receptores de Sulfonilureias/genética , Animais , Peso Corporal , Feminino , Insulina/metabolismo , Masculino , Pâncreas/metabolismo , Ratos Sprague-Dawley , Ratos TransgênicosRESUMO
BACKGROUND/AIMS: Diabetes affects the entire vascular system and accelerates atherosclerosis and ischemia. Percutaneous transluminal angioplasty with or without stenting is the main therapeutic strategies; however, the restenosis rate is high in diabetics. Sulfonylureas (SUs) are widely prescribed agents for the treatment of type 2 diabetes (T2DM) and function by interacting with sulfonylurea receptors (SURs), which also exists in vascular smooth muscle cells (VSMCs), give rise to the potential that SUs influence VSMCs. The proliferation and migration of VSMCs play important roles in the formations of primary stenosis and restenosis, especially the latter. However, there are no data about the exact effects of SUs on these processes. METHODS: Human aortic smooth muscle cells (HASMCs) were exposed to SUs prior to exposure to 30mM glucose. Cell proliferation was detected by CCK8 assay. Cell migration was detected by wound healing assay and transwell assay. Protein expression was determined by immunofluorescence and western blot. Diazoxide was used to evaluate the role of KATP channel in these processes. RESULTS: The proliferation and migration of HASMCs were significantly aggravated by glibenclamide and glimepiride, and their effects were reversed by diazoxide. In contrast, above characteristics of HASMCs were apparently inhibited by gliclazide, and this was maintained after opening the KATP channel. CONCLUSION: These results imply that KATP channels play an important part in proliferation and migration of VSMCs induced by glibenclamide and glimepiride. In contrast, the inhibitory effect of gliclazide on VSMCs appeared to have more potential for the prevention of vascular obstructive diseases in T2DM.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Compostos de Sulfonilureia/farmacologia , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologiaRESUMO
BACKGROUND: Sulfonylureas (SUs) are widely prescribed for the treatment of type 2 diabetes (T2DM). Sulfonylurea receptors (SURs) are their main functional receptors. These receptors are also found in kidney, especially the tubular cells. However, the effects of SUs on renal proximal tubular epithelial cells (PTECs) were unclear. METHODS: Three commonly used SUs were included in this study to investigate if different SUs have different effects on the apoptosis of PTECs. HK-2 cells were exposed to SUs for 24 h prior to exposure to 30 mM glucose, the apoptosis rate was evaluated by Annexin/PI flow cytometry. Bcl-2, Bax and the ratio of LC3II to LC3I were also studied by western blot in vitro. Diazoxide was used to evaluate the role of KATP channel in SUs-induced apoptosis of PTECs. A Student's t-test was used to assess significance for data within two groups. RESULTS: Treatment with glibenclamide aggravated the apoptosis of HK-2 cells in high-glucose, as indicated by a significant decrease in the expression of Bcl-2 and increase in Bax. Additionally, the decreased LC3II/LC3I reflects that the autophagy was inhibited by glibenclamide. Similar but less pronounced change was found in glimepiride group, however, nearly opposite effects were found in gliclazide group. Further, the effects of glibenclamide on apoptosis promotion and the decreased LC3II/LC3I were ameliorated obviously by treatment with 100uM diazoxide. The potential protection effect of gliclazide was also inhibited after opening the KATP channel. CONCLUSION: Our results suggest that, the effects of glibenclamide and glimepiride on PTECs apoptosis, especially the former, were achieved in part by closing the KATP channel. In contrast to glibenclamide and glimepiride, therapeutic concentrations of gliclazide showed an inhibitory effect on apoptosis of PTECs, which may have a benefit in the preservation of functional PTECs mass.
Assuntos
Células Epiteliais/efeitos dos fármacos , Gliclazida/farmacologia , Glibureto/farmacologia , Canais KATP/fisiologia , Compostos de Sulfonilureia/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/fisiologia , Humanos , Túbulos Renais Proximais/citologiaRESUMO
BACKGROUND: A low-protein diet (LPD) is believed to be beneficial in slowing the progression of kidney disease. It is reported that low protein diet can improve protein, sugar and lipid metabolism, and reduce the symptoms and complications of renal insufficiency. However, there has been controversial regarding the effects of protein restriction on diabetic nephropathy (DN). OBJECTIVE: To investigate the efficacy of LPD on renal function in patients with type 1 or 2 DN by meta-analysis of randomized controlled trials (RCTs). DESIGN: PubMed, MEDLINE, EMBASE and China National Knowledge Infrastructure databases were searched. Eleven randomized controlled trials met the inclusion criteria, of which 10 were English and 1 was Chinese. The primary outcome was a change in glomerular filtration rate (GFR). The secondary outcome was a change in proteinuria. Random-effects models were used to calculate the standardized mean difference (SMD) and the corresponding 95% confidence intervals (CI). Subgroup analyses were also performed. RESULTS: Our research indicated that LPD was not associated with a significant improvement in GFR (1.59 ml · min-1 · 1.73 m-2, 95% CI -0.57, 3.75, I2 = 76%; p = 0.15). This effect was consistent across the subgroups regardless of type of diabetes, course of diabetes and intervention period. Our results also showed that there was no significant difference on improvement of proteinuria in patients of LPD and those in normal-protein diet groups (- 0.48, 95%CI-1.70, 0.74, I2 = 94%, p = 0.44). Subgroup analysis revealed that LPD resulted in increased excretion of proteinuria in patients with type 2 diabetes (1.32, 95% CI 0.17, 2.47, I2 = 86%, p = 0.02). CONCLUSION: The present research showed that LPD was not significantly associated with improvement of renal function in patients with either type 1 or 2 diabetic nephropathy. Although these results do not completely eliminate the possibility that LPD is beneficial for patients with diabetic nephropathy, it does not seem to be significant benefit to renal function.
Assuntos
Diabetes Mellitus Tipo 1/dietoterapia , Diabetes Mellitus Tipo 2/dietoterapia , Nefropatias Diabéticas/dietoterapia , Dieta com Restrição de Proteínas , Modelos Estatísticos , Adulto , Índice de Massa Corporal , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Nefropatias Diabéticas/fisiopatologia , Feminino , Taxa de Filtração Glomerular/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteinúria/dietoterapia , Proteinúria/fisiopatologiaRESUMO
Uridine diphosphate-dependent glycosyltransferases (UGTs) mediate the glycosylation of plant metabolites, thereby altering their physicochemical properties and bioactivities. Plants possess numerous UGT genes, with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics, but the biological function and molecular basis of these phenomena are not fully understood. The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition (Ki) at 4 µM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 µM. We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs. A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reduced scopoletin substrate inhibition, whereas its monolignol glycosylation activity was almost unaffected. Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation, leading to enhanced promiscuity, which was accompanied by substrate inhibition. Studies of 3D structures identified open and closed UGT conformers, allowing visualization of the dynamics of conformational changes that occur during catalysis. Previously postulated substrate access tunnels likely serve as drainage channels. The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release. Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions. The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.
Assuntos
Fitoalexinas , Escopoletina , Escopoletina/metabolismo , Glicosilação , Glicosiltransferases/química , Plantas/metabolismoRESUMO
BACKGROUND: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are the main causes of restenosis (RS) in diabetic lower extremity arterial disease (LEAD). However, the relevant pathogenic mechanisms are poorly understood. METHODS: In this study, we introduced a "two-step injury protocol" rat RS model, which started with the induction of atherosclerosis (AS) and was followed by percutaneous transluminal angioplasty (PTA). Hematoxylin-eosin (HE) staining and immunohistochemistry staining were used to verify the form of RS. Two-step transfection was performed, with the first transfection of Lin28a followed by a second transfection of let-7c and let-7g, to explore the possible mechanism by which Lin28a exerted effects. 5-ethynyl-2Î-deoxyuridine (EdU) and Transwell assay were performed to evaluate the ability of proliferation and migration of VSMCs. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect the expression of Lin28a protein and let-7 family members. RESULTS: Using a combination of in vitro and in vivo experiments, we discovered that let-7c, let-7g, and microRNA98 (miR98) were downstream targets of Lin28a. More importantly, decreased expression of let-7c/let-7g increased Lin28a, leading to further inhibition of let-7c/let-7g. We also found an increased level of let-7d in the RS pathological condition, suggesting that it may function as a protective regulator of the Lin28a/let-7 loop by inhibiting the proliferation and migration of VSMCs. CONCLUSION: These findings indicated the presence of a double-negative feedback loop consisting of Lin28a and let-7c/let-7g, which may be responsible for the vicious behavior of VSMCs in RS.
Assuntos
Aterosclerose , MicroRNAs , Ratos , Animais , Regulação para Baixo , MicroRNAs/genética , MicroRNAs/metabolismo , Retroalimentação , Proliferação de Células/genéticaRESUMO
Gliquidone was suggested to exert hypoglycemic effect through enhancing hepatic insulin sensitivity. However, inadequate in vivo evidences make this statement controversial. The aim of the present study was to clarify the insulin-sensitizer role of gliquidone in liver and muscle, so as to confirm its extra-pancreatic effects in vivo. TALEN technique was used to create Sur1 knockout (Sur1-/-) rats. Diabetic Sur1-/- rat models were established by high-fat diet combined with streptozotocin, and which were randomly divided into three groups: gliquidone, metformin and saline, treated for 8 weeks. Fasting blood glucose (FBG) and body mass were tested each week. IPGTT, IPITT and hyperinsulinemic-euglycemic clamp tests were used to evaluate glucose tolerance and insulin sensitivity, respectively. Key mediators of glucose metabolism in liver and skeletal muscle and the activity of AKT and AMPK in these tissues were further analyzed. We found that gliquidone decreased FBG and increased insulin sensitivity without increasing insulin secretion in diabetic Sur1-/- rats. Further exploration implied that gliquidone mainly increased hepatic glycogen storage and decreased gluconeogenesis, which were accompanied with activation of AKT, but not enhanced muscle GLUT4 expression. However, both these effects were still weaker than that of metformin. These results suggested that gliquidone could exerts an extra-pancreatic hypoglycemic effect by improving insulin sensitivity, which might be largely attributes to its additional insulin sensitizer role in hepatic glucose metabolism.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Compostos de Sulfonilureia/farmacologia , Animais , Glicemia/análise , Glicemia/biossíntese , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/metabolismo , Técnicas de Inativação de Genes , Gluconeogênese/efeitos dos fármacos , Humanos , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Transgênicos , Estreptozocina/administração & dosagem , Estreptozocina/toxicidade , Compostos de Sulfonilureia/uso terapêutico , Receptores de Sulfonilureias/genéticaRESUMO
One of the malignant transformation hallmarks is metabolism reprogramming, which plays a critical role in the biosynthetic needs of unchecked proliferation, abrogating cell death programs, and immunologic escape. However, the mechanism of the metabolic switch is not fully understood. Here, we found that the S-nitrosoproteomic profile of endogenous nitrogen oxide in ovarian cancer cells targeted multiple components in metabolism processes. Phosphofructokinase (PFKM), one of the most important regulatory enzymes of glycolysis, was S-nitrosylated by nitric oxide synthase NOS1 at Cys351. S-nitrosylation at Cys351 stabilized the tetramer of PFKM, leading to resist negative feedback of downstream metabolic intermediates. The PFKM-C351S mutation decreased the proliferation rate of cultured cancer cells, and reduced tumor growth and metastasis in the mouse xenograft model. These findings indicated that S-nitrosylation at Cys351 of PFKM by NOS1 contributes to the metabolic reprogramming of ovarian cancer cells, highlighting a critical role of endogenous nitrogen oxide on metabolism regulations in tumor progression.
Assuntos
Carcinoma Epitelial do Ovário/genética , Glicólise/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Animais , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , CamundongosRESUMO
OBJECTIVE: The goal of this systematic review was to provide guidelines for treatment parameters regarding electrical stimulation by investigating its efficacy in improving muscle strength and decreasing pain in patients with knee osteoarthritis. DESIGN: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses standard, three electronic databases (CINAHL, PubMed, and PEDro) and gray literature were used. Randomized control trials comparing electrical stimulation and conservative physical therapy were critically appraised using the 2005 University of Oxford standard. RESULTS: Nine randomized control trials were included in our review. First, our review confirmed that neuromuscular electrical stimulation is the most effective electrical stimulation treatment in the management of knee OA, and its efficiency is higher when combined with a strengthening program. Second, frequency of at least 50 Hz and no more than 75 Hz with a pulse duration between 200 and 400 µs and a treatment duration of 20 mins is necessary for successful treatment. CONCLUSIONS: For the first time, our review provides standardized clinical treatment parameters for neuromuscular electrical stimulation to be included in a strengthening program for the adult patient with knee OA. TO CLAIM CME CREDITS: Complete the self-assessment activity and evaluation online at http://www.physiatry.org/JournalCME CME OBJECTIVES: Upon completion of this article, the reader should be able to: (1) Recall the impact of quadriceps femoris weakness on joint stability; (2) Summarize the mechanism of action of neuromuscular electrical stimulation (NMES) on reducing pain and increasing muscle strength; and (3) Plan the clinical treatment parameters of NMES to be included in a strengthening program for an adult patient with knee osteoarthritis. LEVEL: Advanced. ACCREDITATION: The Association of Academic Physiatrists is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.The Association of Academic Physiatrists designates this Journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.
Assuntos
Terapia por Estimulação Elétrica , Osteoartrite do Joelho/reabilitação , Terapia por Estimulação Elétrica/métodos , Humanos , Força Muscular/fisiologia , Osteoartrite do Joelho/fisiopatologia , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Nitric oxide (NO), an important chemical messenger, serves a dual role in tumor progression. Nitric oxide synthase isoform 1 (NOS1) was observed to be increasingly expressed in various types of cancer, and its expression has been associated with tumor progression. However, the level of NOS1 expression and the associated functions of NOS1 in human ovarian cancer remain undefined. Using gene expression profiles of ovarian cancer from the Gene Expression Omnibus (GEO) database, the present study revealed that NOS1 was increasingly expressed in ovarian cancer tissues. The present study investigated the level of NOS1 expression and its effects on in vitro cell function, including proliferation, migration and invasion as well as chemoresistance to cispatin (DDP) treatment in OVCAR3 cells. Reverse transcription-quantitative polymerase chain reaction demonstrated that the level of NOS1 mRNA expression varied in different ovarian cancer lines. However, immunoblotting indicated that the level of NOS1 protein expression was constitutively high in ovarian cancer cell lines. Treatment with NOS inhibitor NG-nitro-L-arginine methyl ester or transfection with NOS1 short hairpin RNA significantly inhibited cell proliferation, migration and invasion compared with the control, whereas the sensitivity of OVCAR3 cells to DDP treatment was increased. The results of the present study indicated that NOS1 promoted the function of ovarian cancer cells, including proliferation, invasion and chemoresistance, providing a potential target for ovarian cancer therapeutic.
RESUMO
BACKGROUND: Differentiated thyroid cancer involves thyroid follicular carcinoma (FTC) and papillary thyroid carcinoma (PTC). Patients with FTC have a worse prognosis than those with PTC for early metastasis through blood of FTC. However, the mechanism of poor prognosis of FTC is still unclear. Here, we aim to evaluate the role of DUSP5 in the prognostic evaluation of FTC. METHOD: We searched the Gene Expression Omnibus (GEO) database for the differentially expressed genes (DEGs) between FTC and PTC, and then combined with survival analysis of cBioPortal database to locate the gene significantly related to prognosis. Tissue microarrays and clinical tissues were used to examine DUSP5 expression by immunohistochemical (IHC) staining between FTC and PTC tissues. In vitro experiment, proliferation, migration and invasion of FTC were observed after regulation of DUSP5 by transfection of siRNA and plasmids, respectively. RESULTS: After searching the GEO database, 26 DEGs were found. DUSP5 was significantly associated with prognosis of FTC in combination with survival analysis. Data of IHC staining showed lower expression of DUSP5 in FTC compared to PTC tissues. Furthermore, overexpression of DUSP5 inhibited the proliferation, migration and invasion accompanied with low level of MMP9 and Vimentin and high level of E-cadherin. Nevertheless, inhibition of DUSP5 ameliorated above damaging effect on the proliferation, migration and invasion. CONCLUSION: DUSP5 was differentially expressed in FTC and PTC tissues. Low level of DUSP5 in FTC participates in the high frequency of metastasis, and further contributes to poor prognosis of FTC. DUSP5 could be served as a novel therapeutic target for FTC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Papilar/patologia , Fosfatases de Especificidade Dupla/metabolismo , Neoplasias da Glândula Tireoide/patologia , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Movimento Celular , Proliferação de Células , Fosfatases de Especificidade Dupla/genética , Perfilação da Expressão Gênica , Humanos , Prognóstico , Taxa de Sobrevida , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais CultivadasRESUMO
A rapid and accurate diagnosis of acute myocardial infarction (AMI) is crucial for saving lives. For this purpose, we have been developing a rapid, automatic, point-of-care, biosensing system for simultaneous four cardiac marker quantification. This system performs a fluorophore mediated immuno-sensing on optical fibers. To improve the sensitivity of the sensor, novel nanoparticle reagents enhancing fluorescence were implemented. Micro-electro-mechanical system (MEMS) technology was applied in the sensing chip development and automatic sensing operation was implemented to ensure a reliable and user-friendly assay. The resulting system is a point-of-care, automatic four cardiac marker sensing system with a 2 x 2.5 cm sensing chip. An assay requires a 200 microL plasma sample and 15-minute assay time.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Sistemas Microeletromecânicos/instrumentação , Sistemas Microeletromecânicos/métodos , Infarto do Miocárdio/diagnóstico , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Doença Aguda , Biomarcadores , Infarto do Miocárdio/metabolismo , Peptídeo Natriurético Encefálico/análiseRESUMO
Nitric oxide synthase 1 (NOS1) has been reported to promote various cancer processes including chemoresistance. However, the role of NOS1 in chemoresistance has remained unclear. ATP-binding cassette, subfamily G, member 2 (ABCG2) has been identified as a molecular cause of multidrug resistance in a number of cancer types, including ovarian cancer. The present study observed that in ovarian cancer cells, the expression of ABCG2 was significantly upregulated in response to cis-diamminedichloroplatinum (cisplatin/DDP) treatment, in addition the expression of NOS1 exhibited an increasing trend. Additionally, the levels of NOS1 and ABCG2 in chemoresistant ovarian cancer profiles in Gene Expression Omnibus datasets (GSE26712 and GSE51373) were higher than in chemosensitive profiles. Furthermore, overexpression of NOS1 could upregulate ABCG2 expression, and expression of ABCG2 was inhibited by NOS1 selective inhibitor (N-PLA). In assays of cell survival, NOS1 appeared to increase the potential for DDP resistance, and this effect was reversed by addition of ABCG2 inhibitor (verapamil). The present study indicated that NOS1-induced chemoresistance was partly mediated by the upregulation of ABCG2 expression. This result suggests a link between the expression of NOS1 and the ABCG2-associated chemoresistance in ovarian cancer.
RESUMO
BACKGROUND AND PURPOSE: Sulfonylureas (SUs) have been suggested to have an insulin-independent blood glucose-decreasing activity due to an extrapancreatic effect. However, a lack of adequate in vivo evidence makes this statement controversial. Here, we aimed to evaluate whether glimepiride has extrapancreatic blood glucose-lowering activity in vivo. EXPERIMENTAL APPROACH: Sulfonylurea receptor 1 deficient (SUR1-/- ) rats were created by means of transcription activator-like effector nucleases (TALEN)-mediated gene targeting technology. Type 2 diabetic models were established by feeding a high-fat diet and administering a low-dose of streptozotocin. These rats were then randomly divided into four groups: glimepiride, gliclazide, metformin and saline. All rats were treated for 2 weeks. KEY RESULTS: Glimepiride decreased blood glucose levels and increased insulin sensitivity without elevating insulin levels. Gliclazide showed similar effects as glimepiride. Both agents were weaker than metformin. Further mechanistic investigations revealed that glimepiride increased hepatic glycogen synthesis and decreased gluconeogenesis, which were accompanied by the activation of Akt in the liver. Moreover, glimepiride increased both total and membrane glucose transporter 4 (GLUT4) levels in muscle and fat, which might be attributed to insulin receptor-independent IRS1/Akt activation. CONCLUSION AND IMPLICATIONS: Glimepiride possesses an extrapancreatic blood glucose-lowering effect in vivo, which might be attributed to its direct effect on insulin-sensitive tissues. Therefore, the combination of glimepiride with multiple insulin injections should not be excluded per se.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Compostos de Sulfonilureia/farmacologia , Receptores de Sulfonilureias/antagonistas & inibidores , Receptores de Sulfonilureias/deficiência , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Hipoglicemiantes/administração & dosagem , Ratos , Compostos de Sulfonilureia/administração & dosagem , Receptores de Sulfonilureias/metabolismoRESUMO
It was brought to the attention of the Editors that there had been an accidental duplication of the western blot images during the preparation of Figs. 1b and c. The authors were notified about the error and have supplied the correct image for Fig. 1c (below). We apologize for any inconvenience this may have caused readers.