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OBJECTIVE@#To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection.@*METHODS@#The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs.@*RESULTS@#A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01).@*CONCLUSION@#DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
Subject(s)
Humans , Autophagy , Cell Death , Cell Nucleus , Human Umbilical Vein Endothelial Cells/virology , Dengue , ProteomeABSTRACT
Objective@#To explore the inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells(HUCMSC) on apoptosis of human umbilical vein endothelial cells(HUVEC) after model group(oxygen-glucose deprivation reoxygenation), and to clarify its possible mechanism.@*Methods@#Human umbilical cord mesenchymal stem cells were cultured. The collected cell supernatant was stored in a centrifugal tube. The exosomes secreted by human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and identified. Human umbilical vein endothelial cells were randomly divided into control group, model group and different concentrations of HUCMSC-EXO(20 μg/ml, 40 μg/ml, 60 μg/ml) treatment groups(adding HUCMSC-EXO into the model group) . The morphological changes of HUVEC cells in each group were observed by inverted phase contrast microscope, and the proliferation inhibition rate of HUVEC in each group was measured by CCK-8 reagent. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3, Bax, Bcl-2 and hypoxia-associated protein hypoxia inducible factor 1α(HIF-1α). Inhibitor(HIF-1α inhibitor) + model group and HUCMSC-EXO + inhibitor + model group were added on the basis of the above experiments. Western blot analysis was performed to observe the effects of HUCMSC-EXO, inhibitor and both of them on HIF-1α and Bax expressions in HUVEC.@*Results@#HUCMSC-EXO was successfully extracted and identified. Compared with the control group, the volume of HUVEC in the model group and the HUCMSC-EXO group with different concentrations decreased, became round, connected and evacuated, and the growth state was poor under the inverted phase contrast microscope.CCK-8 detection showed that the cell viability in the HUCMSC-EXO group was significantly higher than that in the model group, the difference was statistically significant (t=9.23, P<0.05). Western blot analysis showed that compared with the control group, the expression levels of Caspase-3 ((0.296±0.038), (0.879±0.088); t=14.92, P<0.05), Bax((0.234±0.034), (0.762±0.084); t=14.36, P<0.05) of HUVEC in the model group were up-regulated, and the expression level of Bcl-2 was down-regulated ((0.863±0.103), (0.387±0.059); t=9.85, P<0.05), with statistically significant differences. Compared with the model group, the expression levels of Caspase-3( (0.586±0.075); t=6.24, P<0.05), Bax((0.311±0.055); t=11.01, P<0.05) and Bcl-2((0.665±0.071); t=7.45, P<0.05) of HUVEC in the HUCMSC-EXO treatment group were down-regulated and the differences were statistically significant. Inhibitor intervention experiments showed that there were no significant differences between the inhibitor+ model group and HUCMSC-EXO+ inhibitor+ model group in the expression of HIF-1α protein ((0.348±0.055), (0.388±0.077); t=1.04, P>0.05)and Bax protein ((0.363±0.069), (0.370±0.064); t=0.18, P>0.05). But both of them were down-regulated compared with the model group (HIF-1α protein (0.919±0.064), Bax protein (0.902±0.071)), the differences were significant( t=13.56, t=13.03, both P<0.05).@*Conclusion@#HUCMSC-EXO has a protective effect on OGD/R model of HUVEC, and its mechanism may be related to the down-regulation of HIF-1α expression.
ABSTRACT
AIM:To investigate the effects of dehydroepiandrosterone(DHEA)on the expression of intercellu-lar adhesion molecule-1(ICAM-1)induced by high lipid levels in rabbit aorta and human umbilical venous endothelial cells(HUVECs),and the effects of all-trans retinoic acid(ATRA)in this process.METHODS: For in vitro experi-ments,the cultured HUVEC were divided into control group,oxidized low-density lipoprotein(ox-LDL)group,ox-LDL+DHEA group,ox-LDL+DHEA+ATRA group and DHEA group.The HUVECs in all groups were treated with the corre-sponding reagents for 24 h.The expression of ICAM-1 at mRNA and protein levels in all groups were determined by RT-PCR and ELISA,respectively.For in vivo experiments,the rabbits were divided into control group,high lipid group,high lipid+DHEA group,high lipid+DHEA+ATRA group and DHEA group.The rabbits in all groups were fed with the cor-responding diets for 10 weeks.The expression of ICAM-1 in the rabbit aorta at mRNA and protein levels was determined by RT-PCR and immunohistochemistry.RESULTS:The expression of ICAM-1 in the HUVECs in ox-LDL group was signifi-cantly increased compared with control group(P<0.05).Compared with ox-LDL group,the expression of ICAM-1 in ox-LDL+DHEA group was obviously decreased(P<0.05).The expression of ICAM-1 was similar in both control group and DHEA group(P>0.05).The expression of ICAM-1 was similar in both ox-LDL+DHEA group and ox-LDL+DHEA+ATRA group(P>0.05).The expression of ICAM-1 in the rabbit aorta in high lipid group was significantly increased com-pared with control group(P<0.05).Compared with high lipid group, the expression of ICAM-1 in high lipid+DHEA group was obviously decreased(P<0.05).No remarkable difference in the expression of ICAM-1 between control group and DHEA group was observed(P>0.05), so did between high lipid +DHEA group and high lipid +DHEA+ATRA group(P>0.05).CONCLUSION:DHEA inhibits high lipid-induced ICAM-1 expression in rabbit aorta and HUVECs. That may be one of the mechanisms of antiatherosclerotic effect of DHEA.ATRA seems no positive effect on DHEA func-tion.