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AIM:To study the protective effect of anti-aging Klotho protein on human umbilical vein endothe-lial cells (HUVECs) treated with high glucose (HG).METHODS:HUVECs were cultured in vitro, and divided into PBS control group, 5.5 mmol/L glucose group, 33.3 mmol/L glucose group, 0.1 μmol/L Klotho +33.3 mmol/L glucose group, 1 μmol/L Klotho+33.3 mmol/L glucose group , and 10μmol/L Klotho+33.3 mmol/L glucose group .The viabili-ty of the HUVECs was measured by MTT assay .The content of malondialdehyde ( MDA) , and the activities of lactate de-hydrogenase (LDH), superoxide dismutase (SOD) and glutathione (GSH) in cell culture supernatants were observed . The production of reactive oxygen species ( ROS) in HUVECs was analyzed by flow cytometry .The levels of nitric oxide ( NO) , endothelin ( ET-1 ) , intercellular adhesion molecule-1 ( ICAM-1 ) in HUVEC culture medium were detected by ELISA.The protein expression of nuclear factor-kappa B (NF-κB) in the HUVECs was determined by Western blot .RE-SULTS:Compared with PBS control group , 33.3 mmol/L glucose significantly decreased the HUVEC viability , increased ROS, LDH and MDA levels , reduced the activities of SOD and GSH , decreased the NO secretion , and induced the ET-1 and ICAM-1 secretion and the protein expression of NF-κB in HUVECs.When HUVECs were treated with Klotho protein at different concentrations combined with 33.3 mmol/L glucose, the cell viability was increased significantly , the ROS, LDH and MDA levels were decreased significantly , the antioxidant SOD and GSH activities were significantly increased , the se-cretion of NO was increased , but ET-1 and ICAM-1 releases and protein expression of NF-κB were significantly reduced . CONCLUSION:Anti-aging Klotho protein promotes the viability of HUVECs treated with HG , reduces the oxidative dam-age and ROS production , and restores the normal secretory function of HUVECs , thus playing a protective role in vascular endothelial cells through reducing the protein expression of NF-κB.
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[ ABSTRACT] AIM:To study the effects of anti-aging Klotho protein on neonatal rat myocardial cells with hypo-xia/reoxygenation ( H/R) injury.METHODS:The cardiomyocytes of neonatal SD rats were cultured to establish hypoxia/reoxygenation model.The myocardial cells were divided into normal control group, H/R model group, different concentra-tions of Klotho protein (0.1μmol/L, 1μmol/L and 10μmol/L) pretreatment groups.The myocardial cells pulse frequen-cy was observed before and after H/R.The cell viability was measured by MTT assay.The leakages of LDH, CK and AST, the content of MDA and the activity of SOD were detected.The apoptotic rate of the myocardial cells was analyzed by flow cytometry.The mRNA expression of endoplasmic reticulum stress markers and apoptosis-related molecules GRP78, CRT, CHOP and caspase-12 was measured by real-time PCR.The protein levels of CHOP, caspase-12 and phosphorylated Akt in the myocardial cells were determined by Western blot.RESULTS: Compared with normal control group, the pulse fre-quency, cell viability rate and SOD activity of myocardial cells were significantly decreased, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were increased in H/R model group.The mRNA expressions of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and caspase-12 were increased, whereas p-Akt level was decreased obviously.Compared with H/R model group, the pulse frequency, cell viability rate and SOD activity were in-creased significantly, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were decreased in Klotho pretreated group.The mRNA expression of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and caspase-12 were decreased, while p-Akt level increased significantly.CONCLUSION:Anti-aging Klotho protein improves the myocardial cell survival and inhibits the apoptosis by increasing the resistance of the cells to oxidative stress and exces-sive endoplasmic reticulum stress response, which is related with the activation of Akt phosphorylation in H/R-injured my-cardial cells.
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AIM:To explore the effects of transforming growth factor-α( TGF-α) in the monoclonal formation , proliferation, migration and adhesiveness of human endothelial progenitor cells ( EPCs).METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α(final concentrations of 1, 5, 10μg/L, respectively).At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10μg/L TGF-αplus 1∶1 000 EGFR-TKI) were set.The effects of TGF-αon monoclonal formation , proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment , thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays , respectively.The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor ( VEGF) were measured by Western blotting .RESULTS:Different concentrations of TGF-αall significantly induced the monoclonal formation , proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI.The results of Western blotting showed that TGF-αalso induced the expression of EGFR and VEGF with a cer-tain concentration effect ( P<0.01) .CONCLUSION:By combining with EGFR induced the expression of VEGF , TGF-αsignificantly promotes the related cell function of monoclonal formation , proliferation, migration, adhesiveness in EPCs.
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Objective To assess the clinical diagnostic value of left atrial function index (LAFI) for heart failure with normal left ventricular ejection fraction (HFNEF).Methods One hundred and ten patients with HFNEF were divided into 3 groups by New York Heart Association (NYHA) cardiac functional grading:NYHA Ⅱ grade group (43 cases),NYHA Ⅲ grade group (40 cases) and NYHA Ⅳ grade group (27 cases),and another 33 healthy subjects were selected as control group.The 4 groups were examined by ultrasoundcardiogram,LAFI and E/E' were calculated,and the plasma brain natriuretic peptide (BNP)levels were determined.The relation between LAFI and NYHA cardiac functional grading and the correlation between LAFI and plasma BNP,E/E' were observed.Results The worse heart function,the higher plasma BNP and E/E',and the lower LAFI,and there were statistical differences [control group:(40.9 ± 26.1) ng/L,6.4 ± 2.0,0.73 ± 0.10,NYHA Ⅱ grade group:(230.1 ± 85.9) ng/L,10.0 ± 3.7,0.46 ± 0.30,NYHA Ⅲ grade group:(398.6 ±98.7) ng/L,16.9 ±4.0,0.26 ± 0.30,NYHA Ⅳ grade group:(680.3 ± 146.6) ng/L,23.8 ± 3.9,0.17 ± 0.20,P ＜ 0.05].The correlation analysis results showed that LAFI and plasma BNP,E/E' were negatively correlated in patients with HFNEF (r =-0.868,-0.873,P ＜ 0.01).Conclusions In HFNEF patients,there is significant correlation between LAFI and left ventricular diastolic dysfunction.LAFI can well diagnose diastolic heart failure early and evaluate the diastolic function of the left ventricle.
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ObjectiveTo estimate the clinical efficacy and immunomodulatory effect of polysaccharidenucleic acid fraction of bacillus calmette guerin(BCG-PSN) combined with a traditional Chinese medicine in patients with vitiligo.MethodsThis study recruited 99 patients with vitiligo aged from 13 to 65(35,6 ± 5.8) years.The patients were classified into 3 groups to be treated with BCG-PSN and a traditional Chinese medicine (Baidianfeng granules) alone or in combination.BCG-PSN was intramuscularly injected at a dose of 2 ml every other day and baidianfeng granules were given orally thrice a day,for 3 months.Peripheral blood samples were obtained from the patients at the baseline and after the end of treatment and from 30 healthy controls.Flow cytometry and enzyme linked immunosorbent assay(ELISA) were performed to detect T cell subsets and expression level of Fas and Fas ligand(FasL),respectively.Data were analyzed by t test and chi-square test.Results The response rate was significantly higher in patients treated with BCG-PSN combined with Baidianfeng granules than in those with BCG-PSN alone(82.86％ vs.40.63％,P ＜ 0.01 ).Before the treatment,patients showed a lower percentage of CD3+ cells,CD3+CD4+ cells and CD3+CD8+ cells in peripheral blood(all P ＜0.01 ),weaker expression of Fas (P ＜ 0.01 ),but a higher CD4/CD8 ratio (P ＜ 0.01 ) compared with the controls.The treatment with BCG-PSN and Baidianfeng granules alone or in combinatiou all induced an increase in the percentage of CD3+ cells,CD3+CD4+ cells and CD3+CD8+ cells in peripheral blood(P ＜ 0.05) and in the expression of Fas(P ＜ 0.01),but a decrease in CD4/CD8 ratio(P ＜ 0.05).ConclusionsBCG-PSN may induce the normal apoptosis in lymphocytes via reversing the abnormality in the expression of Fas/FasL by peripheral blood lymphocytes,and the effect of BCG-PSN may be enhanced by a traditional Chinese medicine,Baidianfeng granules.
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Objecave The effects of PGF2α on the reactive oxygen species generation and cardiomyocyte hypertrophy were examined in experiments on the cultured neonatal rat cardiomyocytes.It is to study the role of ROS in the signaling pathway of cardiomyocyte hypertrophy induced by PGF2α.Methods The level of intracellular ROS wag measured by the ROS-specific probe 2',7'-dichlorofluorescin diacetate (DCF-DA).Cardiomyocyte hypertrophy was determined by total protein content of the cells and the cell diameter.Results In the cardiomyocytes treated with PGF2α(1nmol/l,10nmol/l,100nmol/l),the fluorescence intensity of intracellular DCF-DA increased by 38.99%,61.76% and 93.55% respectively compared with control group(F=195.69,P＜0.01).It is indicated that PGF2α can induce intracellular ROS generation on the cultured neonatal rat cardiomyocytes in a dose-dependent manner.Compared with control group,the total protein increased by 39.51%, 69.93%and 139.06%respectively(F=74.014,P＜0.01),and the cell diameter increased by 29.02%,60.79%and 127.40%respectively(F=721.02,P＜0.01).It is indicated that PGF2α induce cardiomyocyte hypertrophy on the cultured neonatal rat cardiomyocytes in a dose-dependent manner.Conclusion PGF2α can induce intracellular ROS generation and cardiomyocyte hypertrophy on the cultured neonatal rat cardiomyocytes in a dose-dependent manner.
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Objective To investigate the effects and mechanisms of extract of ginkgo biloba(EGb)on the cell of cardiocyte hypertrophy induced by prostaglandin F2 alpha.Methods normal cells were used as negative control,and spontaneously PGF2? and EGb were used as experimental groups.The cells were isolated and cultured on cultured neonatal rat cardiocyte by PGF2?,cardiocyte hypertrophy was determined by the cell surface and the total protein of cells;the level of intracellular ROS measured by the fluorescence microscope.Results In cardiocyte hypertrophy,the cell surface,the level of intracellular ROS and the total protein of cells increased significantly in cutured neonatal rat cardiac treated with PGF2?.Compared with cells treated with PGF2?,EGb(40?g/ml,80?g/ml,100?g/ml)inhibited cardiocyte hypertrophy by PGF2?-induced,decreased in the cell surface by 19%,27%,33% and in the total protein of cells by 21%,39%,47% respectively(all P