ABSTRACT
OBJECTIVE@#To investigate whether ginsenoside Rb1 (Rb1) can protect human umbilical vein endothelial cells (HUVECs) against high glucose-induced apoptosis and examine the underlying mechanism.@*METHODS@#HUVECs were divided into 5 groups: control group (5.5 mmol/L glucose), high glucose (HG, 40 mmol/L) treatment group, Rb1 (50 µ mol/L) treatment group, Rb1 plus HG treatment group, and Rb1 and 3-(@*RESULTS@#Rb1 ameliorated survival in cells in which apoptosis was induced by high glucose (P<0.05 or P<0.01). Upon the addition of Rb1, mitochondrial and intracellular reactive oxygen species generation and malondialdehyde levels were decreased (P<0.01), while the activities of antioxidant enzymes were increased (P<0.05 or P<0.01). Rb1 preserved the mitochondrial membrane potential and reduced the release of Cyt-c from the mitochondria into the cytosol (P<0.01). In addition, Rb1 upregulated mitochondrial biogenesis-associated proteins (P<0.01). Notably, the cytoprotective effects of Rb1 were correlated with SIRT3 signalling pathway activation (P<0.01). The effect of Rb1 against high glucose-induced mitochondria-related apoptosis was restrained by 3-TYP (P<0.05 or P<0.01).@*CONCLUSION@#Rb1 could protect HUVECs from high glucose-induced apoptosis by promoting mitochondrial function and suppressing oxidative stress through the SIRT3 signalling pathway.
ABSTRACT
AIM:To explore the role of Sirt1/eNOS signalling pathway in the protective effect of hydrogen sul-phide(H2S)against endothelial cell senescence induced by high glucose.METHODS:High glucose(33 mmol/L)was applied to induce senescence in primary human umbilical vein endothelial cells(HUVECs).The cell viability,the propor-tion of senescence-associated β-galactosidase(SA-β-Gal)positive cells and the plasminogen activator inhibitor 1(PAI-1) expression were detected to assess the senescence model.Mean while,Sirt1 siRNA was used to examine the effect of Sirt 1 on eNOS expression and the senescence-related parameters.RESULTS: Treatment of HUVECs with high glucose de-creased the cell viability slowly with a larger proportion of the cells stained with SA-β-Gal, and the protein expression of PAI-1 was dramatically increased.The increased cell viability,reduced SA-β-Gal positive cells and decreased protein ex-pression of PAI-1 were detected after sodium hydrosulfide(NaHS,100 μmol/L)treatment.Furthermore,NaHS treatment upregulated the protein expression of Sirt 1 and eNOS,and eventually increased the production of nitric oxide(NO).CON-CLUSION:Exogenous H2S modulates Sirt1/eNOS/NO pathway to prevent HUVECs against high glucose-induced senes-cence.
ABSTRACT
<p><b>OBJECTIVE</b>To label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF 1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits.</p><p><b>METHODS</b>GFP cDNA was inserted into pcDNA3.1 hIGF 1 to label the expression vector. The recombinant vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418-resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hIGF 1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type II collagen.</p><p><b>RESULTS</b>The expression of hIGF 1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF 1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF 1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type II collagen.</p><p><b>CONCLUSIONS</b>The hIGF 1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF 1. The labeled articular chondrocytes overexpressing hIGF 1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.</p>
Subject(s)
Animals , Rabbits , Cartilage, Articular , Metabolism , Cells, Cultured , Chondrocytes , Metabolism , Flow Cytometry , Genetic Markers , Genetic Therapy , Genetic Vectors , Green Fluorescent Proteins , Genetics , Insulin-Like Growth Factor I , Genetics , Metabolism , Luminescent Agents , RNA, MessengerABSTRACT
<p><b>OBJECTIVE</b>To identify the biomechanical feasibility of the thoracic extrapedicular approach to the placement of screws.</p><p><b>METHODS</b>Five fresh adult cadaveric thoracic spine from T1 to T8 were harvested. The screw was inserted either by pedicular approach or extrapedicular approach. The result was observed and the pullout strength by pedicular screw approach and extrapedicular screw approach via sagittal axis of the vertebrale was measured and compared statistically.</p><p><b>RESULTS</b>In thoracic pedicular approach, the pullout strength of pedicle screw was 1001.23 N+/-220 N (288.2-1561.7 N)ls and that of thoracic extrapedicular screw approach was 827.01 N+/-260 N when screw was inserted into the vertebrae through transverse process, and 954.25 N+/-254 N when screw was inserted into the vertebrae through the lateral cortex of the pedicle. Compared with pedicular group, the pullout strength in extrapedicular group was decreased by 4.7% inserted through transverse process (P larger than 0.05) and by 17.3% inserted through the lateral cortex (P less than 0.05). The mean pullout strength by extrapedicular approach was decreased by 11.04% as compared with pedicular approach (P less than 0.05).</p><p><b>CONCLUSIONS</b>It is feasible biomechanically to use extrapedicular screw technique to insert pedicular screws in the thoracic spine when it is hard to insert by pedicular approach.</p>