ABSTRACT
AIM: To explore whether necroptosis contributes to the high glucose (HG)-induced damage in hu-man umbilical vein endothelial cells (HUVECs).METHODS: The protein levels of receptor-interacting protein 3 (RIP3) and cleaved caspase-3 were detected by Western blot.The intracellular levels of reactive oxygen species (ROS) were deter-mined 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 HG at different concentra-tions (10, 20 and 40 mmol/L glucose) for 24 h gradually enhanced the expression levels of RIP3.Treatment of HUVECs with HG (40 mmol/L glucose) for different time (3 h, 6 h, 9 h, 12 h and 24 h) also up-regulated the expression levels of RIP3, peaking at 9 h.Pretreatment of HUVECs with 20 μmol/L Z-VAD-FMK (an inhibitor of caspase) for 30 min before exposure to HG enhanced the expression level of RIP3.Pretreatment of HUVECs with 100 μmol/L necrostatin-1 (an inhi-bitor of necroptosis) for 1 h before exposure to HG alleviated the HG-induced injuries, such as a decrease in cell viability, an increase in ROS generation and dissipation of MMP, but up-regulated the protein level of cleaved caspase-3.CON- CLUSION: Necroptosis mediates HG-induced injury in HUVECs.There is a negative interacting between necroptosis and apoptosis.
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 .