Rutaecarpine Inhibits High Glucose-induced Endothelial Cell Senescence Via TRPV1/[Ca²⁺]i/Sirt1 Pathway

Objective: Diabetes vascular complications induced by hyperglycaemia account for significant morbidity and mortality of Diabetes.Endothelial senescence plays crucial roles in diabetic vascular complication.Sirt1,a histone deacetylase,has been demonstrated to mediate high glucose-induced endothelial senescence.Hyperglycemia accelerates senescence of the vascular ECs via downregulation of Sirt1.Sirt1 could directly promote the deacetylation of P53,and thereby inhibited P21-mediated endothelial senescence.Rutaecarpine（Rut）is the main active ingredients of traditional Chinese medicine Evodia rutaecarpa.Rut can activate transient receptor potential vanilloid 1（TRPV1）to promote calcium influx,which mediates most cardiovascular protection effects of rut.Previous study has found that Rut inhibited the production of ROS.It was reported that Rut inhibited senescence of endothelial progenitor cell induced by hypertension and Ang II.The aim of our study was to observe the anti-aging effect of Rut in the human umbilical vein endothelial cells（HUVECs）induced by high glucose and to explore whether the mechanism is related to activation of TRPV1/[Ca²⁺]/Sirt1 pathway.Methods: The endothelial cells injury was induced by incubation HUVEC with30 mM glucose for 24 h.Pretreatment with different concentrations of Rut(10^-6mol/L,3×10^-6mol/L,10^-5mol/L)to observe the effects of Rut on endothelial cell exposure to high glucose.CCK-8 detected cell viability and flow cytometry detects cell apoptosis by AnnexinV/PI.Western blotting detected autophagy related protein LC3II/LC3 I to estimate the effects of Rut on autophagy.Furthermore,to observe high glucose-induced endothelial cell senescence,flow cytometry detected cell cycle by PI,senescence β-galactosidase staining detected the number of senescent cells,Western blotting detected the protein expression of Sirt1 and P21,and fluorescence probe detected the production of ROS.To explore whether TRPV1/[Ca²⁺]i pathway are involved in the anti-aging mechanism of Rut,we pretreated with TRPV1 competitive antagonist Capsazepine(10^-5mol/L),calmodulinCaM blocker W-7(10^-5mol/L)or intracellular calcium chelating agent BAPTA-AM(10^-5mol/L)10 min before treatment with Rut.Meanwhile flow cytometry detected intracellular Ca²⁺ concentration by Fluo-3 AM to determine whether Rut can activate TRPV1 to promote the influx of Ca²⁺.Results: As shown by CCK-8 and Annexin V-PI,exposure HUVEC to high glucose（30mM）for 24 h significantly decreased the cell viability and increased apoptosis.Treatment with Rut recovered cell viability and inhibited cell apoptosis induced by high glucose,which was partly abolished by CAPZ.However,the autophagy related protein LC3II/LC3 I keeped unchanged,indicating Rut had no obvious influence on autophagy of HUVEC exposure to high glucose.Flow cytometry detecting cell cycle revealed that Rut remove G0/G1 cell cycle arrest induced by high glucose,and increased the percentage of cell proliferation phase（S and G2/M）.The SA-β-gal assay showed Rut dose-dependently reduced the percentage of SA-β-gal positive cells in high glucose-treated HUVECs.Rut up-regulated the protein level of Sirt1 whereas inhibited P21 expression and the ROS production in a dose-dependence manner.These anti-aging effects of Rut were abolished by pretreatment with TRPV1 receptor blockers CAPZ,calmodulin CaM blocker W-7 or intracellular calcium chelating agent BAPTA-AM.Additionally,Flow cytometry result showed that Rut increased intracellular Ca²⁺ concentration mildly,which was blocked by CAPZ.Conclusion: Rut prevents the endothelial cell senescence induced by high glucose via activation of TRPV1/[Ca²⁺]i pathway,causing up-regulation of Sirt1,and down-regulation the level of P21 and ROS.