| Objective:The function of vascular endothelial cells decrease with aging.Many studies have confirmed that the injury of vascular endothelial cells is closely related with serum or serum components,however,the fine adjustment of the mechanism remains unclear.In this study,we compared the cell biological activity,intracellular ROS,inflammatory mediators and adhesion molecules levels of umbilical vein endothelial cells(HUVECs)incubated with the serum of elderly and young people.Then we further studied the regulation of NF-?B-Jmjd3 signaling pathway on the inflammatory mediators and adhesion molecules genes to analyze the possible underlying epigenetic mechanisms.The aim of this study was to explore the mechanisms underlying the effects of aging serum on endothelial dysfunction,so as to provide new insights and new targets for the diagnosis and treatment of endothelial dysfunction related diseases.Methods:1.The Selection and grouping of the research objects: The young group: 16 cases of 20~30 years old healthy subjects,aged 25.60 + 1.97 years;The old group: 16 cases of 60~70 years old healthy subjects,aged 66.60 + 4.78 years;The senior group of 16 cases: healthy senior individuals whose average age is 85.86 + 5.79 years.5ml of fasting venous blood was taken and then serum was separated by centrifugation,aliquoted and stored in-80? freezer until use.3 sample pools named25 y,6y and 85 y were made by mixing the serum from the young group,the old group and the senior group respectively.2.HUVECs isolation,culture and grouping: The 3~5 generation of cells in logarithmic growth phase were randomly divided into 25 y,65y and 85 y group,with the treatment of conditioned medium including serum from the pools of 25 y,6y and85 y,respectively.HUVECs and serum samples were collected at different time points according to the detections,each experiment was repeated 3 times.3.Cell viability analysis: CCK8 method was used to detect the effect of serum treatment on HUVECs activity.4.Cell aging analysis: the number of aging cells of each group was counted by beta galactosidase Staining Kit.5.Cell cycle analysis: Flow cytometry was used to analyze the effect of serum on cell cycle of the HUVECs cells.6.Analysis of reactive oxygen species:DCFH-DA probe method was used to detect the level of oxidative stress in endothelial cells.7.The repair ability of HUVECs was analyzed: After HUVECs was induced by the serum in each group,the repair ability of endothelial cells was detected by scratch test.8.The expression of inflammatory mediators in Serum pools was measured by Luminex.9.The expression of inflammatory response and functional moleculars: the expression of inflammatory mediators and adhesion molecules in endothelial cells was measured by real-time quantitative PCR(qRT-PCR)(IL-1?,IL-6,TNF-?,,IL-12,TGF-?,VCAM,ICAM,eNOS).10.Analysis of the activity of NF-kappa B/p65-Jmjd3 signaling pathway in the expression of inflammatory mediators and adhesion molecules in vascular endothelial cell: Immunofluorescence staining was used to observe the location and expression of NF-kappa B/p65 in endothelial cells,the expression of Jmjd3 and the level of lysine methylation of histone H3K27 in the serum of each group after the induction of HUVECs.Results1.The changes of cell viability after the treatments of aging serum: Compared with the young group(25y),after HUVECs was induced by the elderly group(65y)and the senior group(85y)serum,the HUVECs growth was restrained,the difference was statistically significant,respectively,P < 0.05,P < 0.01.2.The aging serum induced cell senescence: compared with 25 y group,the HUVECs induced by 65 y group serum,beta galactosidase staining positive cells significantly increased,P<0.05;the HUVECs induced by the 85 y group serum,betagalactosidase staining positive cells were significantly increased,P<0.0001.3.The effects of the serum treatments on cell cycle: compared with 25 y group,the percentage of S phase cells in 65 y group decreased,while the number of S phase cells increased in 85 y group.It is speculated that with the increase of age,serum induces the endothelial cell cycle disorder,and the percentage of cells of S phase reduced in 65 y group,while in the 85 y group,the percentage is increased abnormally.4.Analysis of HUVECs oxidative stress induced by different serum: the mean fluorescence intensity of 65 y group was significantly higher than that of 25 y group,P<0.0001;The mean fluorescence intensity of 85 y was also significantly higher than that of 25 y,P<0.0001.5.The effect of aging serum on the repair ability of HUVECs: after 48-hour observation,65 y and 85 y group showed a significant reduction in the migration and repair ability of HUVECs.6.The activation of NF-kappaB/p65-Jmjd3 signaling pathway which regulating the expression of inflammatory mediators and adhesion molecules: after 2h-serum incubation,the expression of NF-?B/p65,Jmjd3 and H3K27me2 in 65 y and 85 y group were up-regulated,H3K27me3 downregulated,furthermore,the fluorescence intensity of NF-?B/p65 in the nuclei gradually increased.7.The levels of inflammatory media in serum pools : when compared with the25 y group,the levels of TNF-alpha,IL-12,IL-6 and IL-1? in the elderly group(65y)in were more higher;However,in 85 y group,The expressions of TNF-alpha,IL-12,IL-6 and IL-1? were lower8.The expression of inflammatory and functional molecules: after 24-hour incubation,when compared with the 25 y group,the expression of TNF-alpha,interleukin-12(IL-12)and VCAM-1 gene in the elderly group(65y)was significantly higher,P <0.01,P <0.001,P < 0.001 respectively;and the level of eNOS was down-regulated,P <0.001;the levels of IL-6 and IL-1? were significantly down regulated,P <0.0001,P <0.0001.However,in 85 y group,The expressions of IL-6,IL-1 beta,TGF-beta and eNOS were significantly lower than those in 25 y group,P <0.0001,P <0.0001.ConclusionThe aging serum restrain the activity of HUVECs,promote the aging of endothelial cell significantly,and finally result in cell cycle disorders and weakened repairing ability.At the same time,aging serum increase oxidative stress reaction of HUVECs.The levels of inflammatory mediators and adhesion molecule are found abnormally expressed in HUVECs induced by aging serum,which might be associated with decreased of H3K27 methylation caused by abnormal activation of the NF-?B /p65-Jmjd3 signaling.In sum,The NF-?B /p65-Jmjd3 signaling pathways involved in activation,aging and dysfunction of endothelial cell induced by aging serum. |
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