Spermidine Alleviates LPS-induced HUVECs Senescence Via Enhancing Autophagic Flux

Background:Ageing is an independent risk factor for cardiovascular disease(CVD).The growing of age is often accompanied with interruption of hemostasis of endothelial cell of vessels,which in turn causing endothelial dysfunction.This process plays crucial role in the initiation and development of CVD.Cellular senescence,is defined as cell cycle arrest under injury condition such as oxidative stress and inflammation.Also,cellular senescence is characterized a special phenotype called senescence-associated secretory phenotype(SASP)which delivers pro-inflammatory cytokines in an autocrine manner.Researched have demonstrated that physiological and pathological process of senescence is associated with depression level of autophagy,and in contrast with this,longevity benefits from enhancing autophagic flux which altogether suggest the potential role of autophagy in suspending life span.Spermidine is a polyamine which is rich in nature,existed study showed that addition of spermidine is capable of increasing life span of yeast and other animal models through manipulating autophagy.Moreover,oral supplement of spermidine to mice received cardio-protection and increasing life span effect.So far,no research on the involvement of spermidine in endothelial cell is reported.Thus,we hypothesize that spermidine may exert its anti-ageing effect through enhancing autophagic flux and decreasing the level of oxidative stress.Objectives:To investigate whether spermidine could effectively reverse lipopolysaccharide(LPS)induced human umbilical vein endothelial cells(HUVECs)senescence as well as improve endothelial cell function.Furthermore,to illuminate the underlying mechanism of spermidine on anti-senescence of endothelial cell.Methods:1.Spermidine was added to interfere with LPS-induced HUVECs senescence,β-gal staining and protein level of p53 and p16 were compared between SPD group and control group.2.Autophagic flux inhibitor chloroquine was furthered added to investigate whether spermidine exerted its-anti-ageing effect via enhancing autophagic flux.Autophagic flux associated protein LC3 Ⅱ and P62 were measured for the evaluation of autophagic flux.3.ROS immunofluorescence staining and JC-1 assay were conducted to determine if spermidine could reduce oxidative stress by autophagy-dependent clearance of injury mitochondrial.4.CCK-8 and scratch assay were conducted for the observation of function improvement of HUVECs.Results:1.In comparison with control group,senescence-associated β-gal staining revealed that LPS group showed significant elevated ratio of blue-staining endothelial cell while Western Blot results further demonstrated the increase expression of senescence related cell cycle protein p53 and p16.In contrast with LPS group,the result of SA-β-gal staining of SPD+LPS group displayed with decrease level of blue-staining endothelial cell while Western Blot followed the tendency with drop expression of p53 and p16.2.In evaluation of autophagic flux,we utilized autophagy maker LC3-Ⅱ and p62.CQ+SPD+LPS co-treated group showed significant elevation of p62 protein,indicative of autophagic flux block with significant increased senescence associated protein p53 and p16.3.In comparison with LPS group,JC-1 staining in evaluation of mitochondrial membrane potential in LPS+SPD group showed significant increase of red/green fluorescence cell ratio while ROS immune-fluorescence staining showed drastic drop of oxidative stress.Comparing CQ+SPD+LPS co-treated group and SPD+LPS co-treated group,we discovered that adding CQ would blunt the mitochondrial protective effect with decreased level of red/green fluorescence cell ratio and increased oxidative stress.4.CCK-8 assay results showed no significant differences among control group,LPS group,SPD+LPS group and CQ+SPD+LPS group.The scratching test revealed that the migration function was improved by SPD treatment which the addition of CQ blunt the effect.Conclusions:1.Continued LPS stimulation to HUVECs successfully establish endothelial cellular senescence model.Spermidine was capable of alleviating endothelial cellular senescence which was supported by drastic drop of p53 and p16 and decreased number of blue-stain cell in β-gal staining2.The addition of chloroquine reversed anti-senescence effect of spermidine indicated that spermidine might exerts its anti-ageing function via enhancing autophagic flux.3.The reduced level of oxidative stress observed in spermidine treated group was in accord with the repairment of mitochondrial,which conclusively suggest spermidine might soften oxidative stress by decreasing mitochondrial injury.4.Spermidine improved migration ability of senescence HUVECs with no effect on proliferation.