Capsaicin Inhibits Intermittent High Glucose-induced Vascular Endothelial Cell Senescence Via TRPV1/SIRT1 Pathway

Objective: High blood glucose induces vascular endothelial aging,which is an important pathological mechanism for vascular complications in diabetic patients.Clinical studies have shown that intermittent high glucose damages vascular endothelial cells more than persistent high glucose.Capsaicin(Cap)is the main active ingredient of capsicum,and its mechanism is mainly related to transient receptor potential vanilloid 1(TRPV1),which has recently attracted more and more attention in metabolic diseases.Our previous study found that rutaecarpine,another natural agonist of TRPV1,up-regulates the expression of SIRT1 in vascular endothelial cells and vascular smooth muscle cells,and inhibits cell senescence induced by high glucose or Ang II.Therefore,the aim of this study is to investigate whether Cap(a classical agonist of TRPV1)has the protective effect on vascular endothelial cell senescence induced by intermittent high glucose,and further explore whether the mechanism is related to increase the expression of SIRT1 by activating TRPV1 signaling pathway.Methods:(1)To observe the effect of intermittent high glucose and persistent high glucose on endothelial cell senescence.Intermittent high glucose group: the vascular endothelial cell injury model was induced by alternately culturing human umbilical vein endothelial cells(HUVECs)with normal glucose medium(5.5 mM)or high glucose medium(33 mM)for 6 days and changed the medium every 24 hours.Continuous high glucose group: high glucose medium(33 mM)continuously cultured HUVECs for 6 days.In addition,SIRT1 inhibitor EX527(10 ?M)was added in advance to determine the effect of SIRT1 on high glucose-induced endothelial cell senescence.(2)In the endothelial cell injury model induced by intermittent high glucose,HUVECs were incubated with different concentrations of Cap(0.3 ?M,1?M,3 ?M)to observe the effect of Cap on endothelial cell senescence and SIRT1 level.(3)To explore the mechanism of Cap inhibiting endothelial cell senescence: we pretreated with TRPV1 antagonist Capsazepine(CAPZ,1 ?M),intracellular calcium chelating agent BAPTA-AM(10 ?M),calmodulin CaMK? blocker KN62(5 ?M),and AMPK blocker Compound C(1 ?M)to observe whether these can block the up-regulation SIRT1 expression and anti-aging effect of Cap.(4)To further determine the role of TRPV1 in endothelial cell senescence.We constructed a TRPV1 low expression stable cell line(TRPV1 shRNA)by lentiviral transfection to observe whether low expression of TRPV1 can affect endothelial cell senescence and attenuate the anti-aging effect of Cap(1 ?M).Related indicators of cell senescence:The cell viability was detected by CCK-8,the number of senescent cells was detected by ?-galactosidase staining,the cell cycle was detected by PI staining,the fluorescent probe DCFH-DA method was used to detect the level of ROS,the expression of TRPV1,SIRT1,p21 and p-AMPK was detected by Western blotting,and RT-qPCR was used to detect mRNA level of TRPV1,p53 and p21.Results:(1)Intermittent high glucose and persistent high glucose could induce vascular endothelial cell senescence.The result showed that the cell viability of HUVECs was inhibited,while the number of senescent cells and the level of intracellular reactive oxygen species increased.However,compared with persistent high glucose,intermittent high glucose could significantly induce endothelial cell senescence and inhibit SIRT1 expression and increase the expression of p21.The SIRT1 inhibitor EX527 aggravated cellular senescence induced by intermittent high glucose.(2)Cap dose-dependently inhibited intermittent high glucose-induced cell senescence.The specific performance was as follows: Cap increased cell viability of HUVECs,reduced the number of senescent cells,inhibited the level of ROS,up-regulated the expression of SIRT1 protein,and inhibited the level of p53 and p21 mRNA.Flow cytometry results showed that intermittent high glucose can increase cell cycle in G0/G1 phase and decrease the percentage of cell proliferation phase(S and G2/M).Moreover,the different concentrations of Cap relieved the inhibition of intermittent high glucose.(3)The anti-aging effect of Cap was partially abolished by pretreatment with TRPV1 antagonist CAPZ,intracellular calcium chelating agent BAPTA-AM,calmodulin-activated protein kinase II(CaMKII)blocker KN62,and AMPK blocker Compound C.At the same time,intermittent high glucose inhibited AMPKphosphorylation in HUVECs.Pre-addition of Cap increased the expression of p-AMPK protein,which can be abolished by CAPZ.(4)Cap dose-dependently increased the expression of TRPV1 protein and mRNA.Compared with normal endothelial cells and cells transfected with control negative lentivirus,TRPV1 shRNA could reduce endothelial cell viability,increase the number of senescent cells,down-regulate the expression of SIRT1 and p-AMPK.TRPV1 shRNA aggravate endothelial cell senescence induced by intermittent high glucose,but it failed to exert the anti-aging effect treated with Cap.Conclusion: Cap can prevent the senescence of vascular endothelial cells induced by intermittent high glucose.Its anti-aging mechanism is related to up-regulate the expression of SIRT1 by activating TRPV1/Ca2+/CaMKII /AMPK signaling pathway,thereby inhibiting the expression of p53 and p21 protein.