JAK-STAT/NF-κB Pathway Mediates High Glucose-induced Damage And Inflammatory Reaction In Human Umbilical Vein Endothelial Cells

BackgroundDiabetes mellitus (DM) is a metabolic disease characterized of hyperglycemia. In recent years, with the social development, the economic improvement and the growth of people’s living standard, diabetes prevalence rate rises year by year. Diabetic angiopathy (DA) is the most familiar chronic diabetic complications, and is also the primary reason inducing the diabetes patients blind, disabled and dead, which severely affect patients’ prognosis and life quality.Hyperglycemia causes the dysfunction of vascular endothelial cells directly or indirectly, then activates or aggravates diabetic angiopathy. On the research of mechanisms of hyperglycemia-induced damage in vein endothelial cells, past research focuses on advanced glycation end-products (AGE), polyol metabolic pathway, protein kinase C (PKC) and so on. In recent years, the new research gradually focuses on multiple cell signal pathways. Janus kinase signal transducer and activator of transcription (JAK/STAT) is one of the important signal pathways. New research shows, JAK/STAT signal pathway participates in the development of diabetic complications. Marrero MR et al. found that hyperglycemia activated JAK/ STAT signal pathway to promote the proliferation of the glomerular filtration membranes cells, which induced diabetic nephropathy. In addition, a study established the diabetic rat models, and found that JAK/STAT signal pathway might take part in the development of myocardial fibrosis in diabetic cardiomyopathy (DCM). However, whether the JAK/STAT signal pathway mediates the high glucose (HG)-induced multiple damage in vein endothelial cells is reported rarely.Nuclear factor-KB (NF-κB) signal pathway plays an important regulatory role in inflammatory response, immune reactions and so on. Some researches show that hyperglycemia can activate NF-κB signal pathway to increase the production of inflammatory cytokines and immune factors in vein endothelial cells, then advances the progression of vessel inflammation which is an important stage in the development of diabetic angiopathy. It has been shown that JAK/STAT signal pathway and NF-κB signal pathway have interaction. Digicaylioglu M proved that NF-κB signal pathway was involved in the neural protection by erythropoietin (EPO), and this was regulated by JAK/STAT signal pathway. So, whether the JAK/STAT signal pathway can modulate NF-κB signal pathway to mediate HG-induced inflammatory response in vein endothelial cells needs a further discussion.So the aim of this study is to investigate whether the JAK/STAT signal pathway mediates the high glucose (HG)-induced multiple damage in vein endothelial cells and whether the JAK/STAT signal pathway can modulate NF-κB signal pathway to mediate HG-induced inflammatory response in vein endothelial cells, which may provide new evidence for further research of mechanisms of hyperglycemia-induced damage in vein endothelial cells.Research methods1 Human umbilical vein endothelial cells (HUVECs) cultureHUVECs were cultured in DMEM (Low Glucose) medium containing 10% fetal bovine serum,100U penicillin and 100mg streptomycin, placed in the incubator with 37 ℃ and 5% CO2. When cells grew nearly 80%, we used 0.25% Trypsin-EDTA to digest, and used complete medium to stop digestion. Then we made cells suspended, and centrifuged cells at 1200 r/min for 5 min, removed the supernatant and divided cells as 1:3.2 Groups of the experimentThis experiment was divided into six groups:(1) Control group; (2) High glucose (HG) group; (3) AG490 (JAK/STAT signal pathway inhibitor) group+HG group; (4) AG490 group; (5) PDTC (NF-κB signal pathway inhibitor) group+HG group; (6) PDTC group.3 The cell viability was examined by Cell Counting Kit-8 (CCK-8) assay.HUVECs were cultured in 96-well plates, When cells grew nearly 80%, we gave cells different treatments and added 1 Oul CCK-8 solution in every well plate, placed it in the incubator with 37 ℃ for 2 h. Microplate reader was applied to examine the absorbancy of every well plate cell viability, which we set it’s wavelength at 450nm. And we used the formula:Cell viability (%)=Treatment group OD/Control group OD×100% to get the cell viability.4 The expression levels of JAK2, STAT3, caspase-9, endothelial nitric oxide synthase (eNOS) and NF-κB p-65 were detected by Western blottingHUVECs were cultured in 60 mm culture dishes. We gave each group a different treatment according to different experimental objectives when we found that cells grew nearly 80%. Then the cells were washed with cold PBS for two times, added 80ul cell lysate, placed in a refrigerator at 4℃ for 30 min, and then centrifugated at 12 000 r/min for 15 min. The supernatant was collected, and then proteins were quantified by the BCA assay kit. Total proteins were separated by the SDS-PAGE, and were transferred to PVDF membrane. After blocking the proteins with 5% skim milk for 90 min, p-JAK2 and JAK2 (1:2000), p-STAT3、 STAT3 and caspase-9 (1:5000), NF-κB p65、p-NF-κB p65、eNOS (1:1000) were added at 4℃ overnight. Then membranes were washed with TBST for 3 times (each time 10 min), incubated with the secondary antibody(1:10000) at room temperature for 60 min, and then were washed with TBST for 3 times again. We made the color by the ECL angent. Membranes were exposured in the darkroom, scanned by gel imaging system, and finally we analysed the results.5 Detection of the intracellular levels of reactive oxygen species (ROS) by DCFH-DA staining followed by photo fluorographHUVECs were cultured in 6-well plates. We gave each group a different treatment according to different experimental objectives when we found that cells grew nearly 80%. Then the cells were washed with PBS for three times, added 10 μmol/L DCFH-DA, placed in a incubator at 37℃ for 30 min, then washed with PBS for three times again.5 non-repeated regions were selected randomly under fluorescence microscope and their average green fluorescence intensity was analyzed by ImageJ1.41 software.6 Detection of Mitochondrial membrane potential (MMP) levels by rhodamine 123 staining followed by photo fluorographHUVECs were cultured in 6-well plates. We gave each group a different treatment according to different experimental objectives when we found that cells grew nearly 80%. Then the cells were washed with PBS for three times, added 10 μg/L Rh123, placed in a incubator at 37℃ for 45 min, then washed with PBS for three times again.5 non-repeated regions were selected randomly under fluorescence microscope and their average green fluorescence intensity was analyzed by ImageJ1.41 software.7 The levels of IL-1β、IL-6、TNF-α secretion were detected by ELISAHUVECs were cultured in 96-well plates. We gave each group a different treatment according to different experimental objectives. Taking 100 μL culture medium into assay plates which coated by IL-1β、IL-6、TNF-a antibody, then followed the introduction of ELISA kits. Microplate reader was applied to record the absorbancy in the wavelength at 450nm.8 Statistical processingData were expressed as mean ± standard deviation (x ± s) to indicate, using SPSS 20.0 statistical software for statistical analysis, comparison between experiment groups using SNK-q test, comparison between experiment groups and control groups using Dunnett-t test. p<0.05 was said that the difference was statistically significant.Results1 High glucose up-regulated the protein levels of phosphorylated (p-)JAK2 and p-STAT3 in the HUVECsTreatment of HUVECs with 40 mmol/L glucose (HG) for 6-12 h enhanced the protein level of p-JAK2(P<0.05, P<0.01), peaking at 9 h. Treatment of the cells with HG for 6-12 h also increased the protein level of p-STAT3 (P<0.01) with the peak value at 12 h.2 JAK/STAT signaling pathway mediated the high glucose-induced cytotoxicity in HUVECsTreatment of HUVECs with 40 mmol/L glucose (HG) for 24h could produce cytotoxicity, getting the cell viability down to (68.6±2.4)%. Before exposure to HG, we used 5、10、20、40、80 μmol/L AG490(the inhibitor of JAK/STAT signaling pathway) pretreat HUVECs 1 h, found that 10、20、40、80 μmol/L AG490 could inhibit HG-induced cytotoxicity with the increase of cell viability. Treatment of HUVECs with 80μmo1/L AG490 alone had no impact on cell viability.3 JAK/STAT signaling pathway participated in high glucose-induced apoptosis in HUVECsTreatment of HUVECs with 10～40 mmol/L glucose (HG) for 24h, the expression levels of caspase-9 (an indicator of cell apoptosis) increased with the rise of glucose. Compared with control group, experiment groups all had statistical significance (P<0.05, P<0.01),and in 40 mmol/L glucose, the expression of caspase-9 reached the highest level. Meanwhile, treatment of HUVECs with 40 mmol/L glucose (HG) for 6-24h enhanced the caspase-9 level obviously with the peak value at 12 h (P<0.01). However, before exposure to HG, we used 10 μmol/L AG490 pretreat HUVECs 1 h, found that 10 μmol/L AG490 could inhibit HG-induced up-regulation of caspase-9, which had statistical significance compared with HG group (P<0.05) Treatment of HUVECs with 1Oμmol/L AG490 alone had no impact on the expression levels of caspase-9.4 JAK/STAT signaling pathway was implicated in high glucose-induced oxidative stress in HUVECs.Treatment of HUVECs with HG for 24h could enhance the mean fluorescence intensity (MFI) of intracellular 2’,7’-Dichlorofluorescein (DCFH) obviously, which had statistical significance compared with control group (P<0.05).However, before exposure to HG, we made a pretreatment with 10 μmol/L AG490 for 1 h, the intracellular levels of ROS reduced significantly, which had statistical significance compared with HG group (P<0.05). Treatment of HUVECs with AG490 alone had no impact on the production of ROS (P>0.05)5 JAK/STAT signaling pathway was involved in high glucose-induced dissipation of MMP in HUVECsTreatment of HUVECs with HG for 24h could reduce MFI (an indicator of MMP) of intracellular rhodamine 123 (Rh123) from (9.4±1.2)%(control group) to (3.5±0.7)%, which had statistical significance (P<0.05).However, after pretreatment with 10 μmol/L AG490 for 1 h, the MFI could rise to (6.1±1.1)%,which had statistical significance compared with HG group (P<0.05). Treatment of HUVECs with AG490 alone had no impact on the level of MMP in HUVECs.6 JAK/STAT signaling pathway mediated the high glucose-induced the decline of the expression of endothelial nitric oxide synthase (eNOS) in HUVECs.Treatment of HUVECs with 10～40 mmol/L glucose (HG) for 24h, we found that 40 mmol/L glucose had inhibition effect on the level of eNOS, which was statistically significant compared with control group (P<0.01). Meanwhile, treatment of HUVECs with 40 mmol/L glucose (HG) for 3-24h,it showed that 12 h and 14 h could down-regulate the eNOS level (P<0.01) with the minimum value at 24 h. Before exposure to HG, we used 1Oμmol/L AG490 pretreat HUVECs 1 h, found that AG490 could inhibit HG-induced down-regulation of eNOS, which had statistical significance compared with control group (P<0.05). Treatment of HUVECs with AG490 alone had no impact on the expression levels of eNOS.7 JAK/STAT signaling pathway mediated the high glucose-induced the increase of the expression of p-NF-KB p65 in HUVECs.Treatment of HUVECs with 10,20,40 mmol/L glucose (HG) for 24h, we found that both 20 mmol/L and 40 mmol/L glucose could increase the expression levels of p-NF-κB p65,which had statistical significance compared with control group (P<0.01). Meanwhile, treatment of HUVECs with 40 mmol/L glucose (HG) for 9-24h enhanced the p-NF-KB p65 level obviously with the peak value at 24 h(P<0.05, P<0.0\). However, before exposure to HG, we used 10 μmol/L AG490 pretreat HUVECs 1 h, found that 10 μmol/L AG490 could inhibit HG-induced up-regulation of p-NF-κB p65, which had statistical significance compared with HG group (P<0.05). Treatment of HUVECs with lOμmol/L AG490 alone had no impact on the expression levels of p-NF-KB p65.8 The inhibitor of NF-κB p65 signaling pathway attenuated the high glucose-induced cytotoxicity in HUVECsTreatment of HUVECs with 40 mmol/L glucose (HG) for 24h could produce cytotoxicity, getting the cell viability down to (70.1±3.7)%, which had statistical significance compared with control group (P<0.05).Before exposure to HG, we used 25、50、100、200 μmol/L PDTC(the inhibitor of NF-κB p65 signaling pathway) pretreat HUVECs 1 h, found that both 100 μmol/L and 200 μmol/L PDTC could inhibit HG-induced cytotoxicity with the increase of cell viability. Treatment of HUVECs with 200μmo1/L PDTC alone had no impact on cell viability.9 JAK-STAT/NF-κB signaling pathway mediated the high glucose-induced inflammatory reaction in HUVECsTreatment of HUVECs with 40 mmol/L glucose (HG) for 24h could increase the secretion of inflammatory factor interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), which had statistical significance compared with control group (P<0.05).Before exposure to HG, we used 10 μmol/L AG490 and 100 μmol/L PDTC pretreat HUVECs 1 h, found that both 10 μmol/L AG490 and 200 μmol/L PDTC could attenuate HG-induced increase of IL-1β、IL-6、TNF-α (P<0.05).Treatment of HUVECs with 10 μmol/L AG490 and 200 μmol/L PDTC alone had no impact on secretion level of IL-1β、IL-6、TNF-α.Conclusion1 JAK-STAT signaling pathway is involved in the high glucose-induced damage and inflammatory reaction in HUVECs.2 JAK-STAT/NF-κB signal pathway can mediate HG-induced damage and inflammatory reaction in HUVECs.