Effects Of Nap-NO-Curcumin Hydrogel On LPS-induced Human Umbilical Vein Endothelial Cell Senescence

Background:Aging is one of the major risk factors for cardiovascular disease(CVD)development.Advancing age of blood vessels can increase the susceptibility of many CVDs,including atherosclerosis,hypertension and vascular calcification.Percutaneous coronary intervention(PCI)is one of the most important treatment to reconstruct myocardial perfusion,but restenosis and delay re-endothelialization after PCI are still urgent problems to be solved.It has been reported that endothelial dysfunction caused by endothelial cell senescence participates in the pathological process and anti-endothelial cell senescence may be a therapeutic strategy to promote re-endothelialization after PCI and to prevent restenosis.Peptide small molecule hydrogels have been widely used in the treatment of various biological diseases,but no anti-aging treatment has been reported.Our group independently developed a small molecule hydrogel that carries both NO donor and curcumin.This Nap-NO-CUR hydrogel can increase the level of NO in the endothelial cells,and decrease the degree of inflammation of the senescent endothelial cells.This hydrogel has a potential of anti-cellular senescence.Objective:To investigate whether Nap-NO-CUR hydrogel intervention can delaylipopolysaccharide-induced endothelial cell senescence and its effect on senescent endothelial cells function,and whether Nap-NO-CUR hydrogel intervention can inhibit NF?B phosphorylation and the expression of SASP.Methods:1.Human umbilical vein endothelial cells(HUVECs)were cultured and continuously stimulated with low concentrations of lipopolysaccharide(LPS)for 6 days.Senescence associated ?-galactosidase staining(SA-?-Gal staining)were used to measure the degree of cellular senescence and the expression levels of p16,p21 and p53 in HUVECs were detected by real-time fluorescence quantitative PCR to establish the senescent cell model of HUVECs.2.HUVECs induced by LPS were treated with Nap-NO-CURcumin hydrogel(0.1,0.5,1,2.5,5 ?M).The level of cellular senescence was detected by?-galactosidase staining to obtain the optimal intervention concentrations of the hydrogel.NO molecular probe was used to detect whether Nap-NO-CUR hydrogel intervention could increase NO level in LPS-induced HUVECs.To compare the anti-aging effect of the NO hydrogel(LPS+NO group),curcumin hydrogel(LPS+Nap-CUR group)and NO-curcumin hydrogel(LPS+Nap-NO-CUR group)on LPS-induced HUVECs,the expression of p16,p21 and p53 were qualified by western blot.The proliferation,migration and angiogenesis abilities of LPS-induced HUVECs were examined by cck-8 test,scratch assay and tube formation test,respectively.3.To understand whether Nap-NO-CUR hydrogel delay the senescence of HUVECs by regulating SASP,ROS level were tested by ROS fluorescence probe,the protein expression of NF?B and p-NF?B were measured by western blot and the expression of TNF-?,IL-10,IL-6 and IL-8 were detected by qPCR.Results:1.After 6 days of LPS stimulation,the proportion of senescent cells of LPS groups of all concentration were close to 80%,compared with the control group.The expression of p16 and p21 of HUVECs stimulated by LPS at the concentration of 0.5?g/ml were increased by 2.45-fold and 1.7-fold,respectively.The expression of p53 in each group had no significant difference.2.Accroding to the results of SA-P-GAL staining,the intervention concentration of NO-curcumin hydrogel at 1?M showed the most significant anti-aging effect.NO molecular probe test showed that,the basal NO level between the control group and the LPS group was no significant difference.After addition of Nap-NO-CUR hydrogel,NO fluorescence intensity in the LPS group was increased compared to the control group.After adding Nap-NO-CUR hydrogel and ?galactosidase,NO fluorescence intensity was significantly increased in both groups.All three hydrogel intervetions reduced the proportion of senescent cells.Compared with LPS group,curcumin hydrogel and Nap-NO-CUR hydrogel could down-regulate the protein p16,p21 and p53 in LPS-induced HUVECs,but the down-regulation of Nap-NO-CUR hydrogel was more significant.3.As shown in the scratching test and tube formation test,the migration rate between control group and LPS group was no difference,but the function of HUVECs tube formulation was significantly impaired in the LPS group.After the intervention of NO,Nap-CUR and Nap-NO-CUR hydrogel,the migration rates of LPS-induced HUVECs in each intervention group were enhanced,as well as the angiogenesis ability.CCK-8 test showed that there was no significant difference in cell proliferation between each group.4.All three hydrogel intervetions reduced the production of ROS in LPS-induced HUVECs.Nap-CUR and Nap-NO-CUR hydrogel decreased p-NF?B/NF?B ratio compared with LPS group,while NO hydrogel intervention didn't affect NF?B phosphorylation.qPCR results showed that the expression of TNF-?,IL-1?,IL-6 in Nap-CUR and Nap-NO-CUR group was lower than that in LPS group.However,the expression of IL-8 between LPS group and each intervention group had no statistical difference.Conclusions:1.Low concentrations of LPS stimulation can lead to human umbilical vein endothelial cell senescence.2.Nap-NO-CUR hydrogel increases NO level of LPS-induced HUVECs.Nap-CUR and Nap-NO-CUR hydrogel reduce the protein expression of p16,p21 and p53 in LPS-induced HUVECs.4.The intevetion of NO,Nap-CUR and Nap-NO-CUR hydrogels can enhance the migration ability and angiogenesis ability of HUVECs.5.Nap-CUR and Nap-NO-CUR hydrogels may exert anti-cell senescence by reducing ROS generation,inhibiting NF?B phosphorylation and decreasing SASP secretion.