ABSTRACT
AIM: To explore whether exogenous hydrogen sulfide (H2S) depresses high glucose (HG)-in-duced injury by modulating the Janus kinase/signal transducer and activator of transcription ( JAK/STAT) pathway in hu-man umbilical vein endothelial cells (HUVECs).METHODS:The protein levels of JAK2, STAT3 and cleaved caspase-3 were determined by Western blot.The cell viability was measured by CCK-8 assay.Mitochondrial membrane potential ( MMP) was detected by rhodamine 123 staining followed by photofluorography.The intracellular level of reactive oxygen species (ROS) was analyzed by DCFH-DA staining followed by photofluorography.The activity of superoxide dismutase (SOD) was also measured.RESULTS:Pretreatment of the HUVECs with 400 μmol/L NaHS (a donor of H2S) for 30 min prior to exposure to 40 mmol/L glucose ( HG) markedly attenuated HG-induced upregulation of the phosphorylation of JAK2 and STAT3.Pretreatment with 400μmol/L NaHS for 30 min or with 20μmol/L AG490 (inhibitor of the JAK/STAT pathway) for 30 min attenuated the injury of HUVECs induced by HG, as indicated by the increases in cell viability and SOD activity, and decreases in the protein level of cleaved caspase-3, ROS generation and dissipation of MMP.CONCLU-SION:Exogenous H2 S protects HUVECs against HG-induced injury by inhibiting JAK/STAT pathway.
ABSTRACT
AIM: To investigate whether Janus kinase/signal transducer and activator of transcription ( JAK/STAT) signaling pathway mediates high glucose-induced damage in human umbilical vein endothelial cells ( HUVECs ) . METHODS:The cell viability was examined by CCK-8 assay.The expression levels of JAK2, STAT3, caspase-9 and en-dothelial nitric oxide synthase ( eNOS) were detected by Western blot .The intracellular levels of reactive oxygen species ( ROS) were tested by DCFH-DA staining followed by photofluorography .Mitochondrial membrane potential ( MMP) was measured by rhodamine 123 staining followed by photofluorography .RESULTS:Treatment of HUVECs with high glucose (40 mmol/L glucose) for 6~12 h enhanced the protein level of phosphorylated JAK 2, peaking at 9 h.Treatment of the cells with high glucose for 6~12 h also increased the protein level of p-STAT3 with the peak value at 12 h.Pretreatment with the inhibitor of JAK/STAT pathway AG490 for 1 h before exposure of the HUVECs to high glucose significantly inhibi-ted the high glucose -induced injury , as evidenced by an increase in the cell viability , decreases in the expression of caspase-9 and the intracellular ROS production , and increases in MMP and the expression of eNOS .CONCLUSION:JAK/STAT signaling pathway is involved in the high glucose-induced damage in HUVECs .
ABSTRACT
AIM: To explore whether strontium ranelate ( Sr ) promotes osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) through the Hedgehog/Gli1 signaling pathway.METHODS:BMSCs were isolated from 4-week-old rats by adherent culture and induced to differentiate into osteoblasts .According to the experimental purposes , the cells were exposed to different concentrations of Sr , cyclopamine ( Cy, an inhibitor of Hedgehog receptor ) or Gli1-siR-NA.The expression of Gli1 and Runx2 in the cells was detected by Western blotting .The activity of alkaline phosphatase ( ALP) was measured by the method of colorimetry , and the mineralized nodules were observed under microscope with aliz-arin red staining .RESULTS:Exposure to Sr at concentrations of 0.1 to 5 mmol/L for 7 d markedly increased the expres-sion of Gli1 in the BMSCs , and the increase in Gli1 expression was the most obvious following Sr exposure at concentration of 3 mmol/L.Cy at concentration of 10 μmol/L inhibited Sr-induced up-regulation of Gli1 expression.Transfection of the BMSCs with Gli1-siRNA not only obviously inhibited Sr-induced up-regulation of Gli1 and Runx2 ( a downstream protein of Gli1) expression, but also antagonized Sr-induced enhancement of ALP activity and the formation of mineralized nodules . CONCLUSION:The Hedgehog/Gli1 pathway is involved in Sr-induced osteogenic differentiation of rat BMSCs .