The Role And Mechanism Of SIRT1 Regulating The Oxidative Stress-induced Senescence Of Rat Nucleus Pulposus Cell

Objective:The aim of this study was to investigate the effects of SIRT1 on oxidative stress-induced senescence in rat nucleus pulposus cells,and to explore the mechanism by which Akt-FoxO1 axis regulates SIRT1 during the process of oxidative stress-induced senescence,which might provide the new straegy for efficient targeted drug screening and combined therapy after intervertebral disc degeneration.Methods:(1)Identification of primary rat nucleus pulposus cells by HE staining,toluidine blue staining and type II collagen immunofluorescence staining;(2)The sub-lethal concentration of H2O2 was determined by ROS detection,CCK-8 assay,Hoechst staining and flow cytometry,which was going to establish a senescent model of rat NP cells;(3)The effect of oxidative stress on SIRT1 expression was analyzed by QT-PCR,Western blot and immunofluorescence;(4)Activation of SIRT1 by specific drugs to elucidate its regulatory mechanism in oxidative stress-induced senescence in rat NP cells;(5)Western blot,immunofluorescence and specific drugs were used to elucidate the effect and regulation mechanism of Akt-FoxO1-SIRT1 axis on oxidative stress-induced senescent rat NP cells;(6)QT-PCR,Western blot and immunofluorescence techniques were used to investigate the effects of resveratrol on oxidative stress-induced senescent rat NP cells.Results:(1)The primary cultured cells were identified as rat nucleus pulposus cells by three kinds of relative specific staining related to nucleus pulposus cells;(2)H2O2 acted on rat NP cells in the form of ROS with a sublethal concentration of 50-150μM;Sublethal concentration of H2O2 activated p53-p21-pRb and pl6-pRb pathways,promoted the expression of TNF-α,IL-1β,IL-6,IL-8 inflammatory related factors,blocked cells in G0/G1 phase,and induced β-gal positive senescent rat NP cells;(3)Oxidative stress led to the decrease of SIRT1 expression at both gene and protein levels,but did not affect SIRT1 activity through nucleocytoplasmic shuttling;(4)Activation of SIRT1 stimulated by SRT1720 inhibited the activation of aging-related indicators during oxidative stress(p53-p21-pRb and pl6-pRb pathways,inflammatory related factors,cell cycle arrest andβ-gal positive cells);(5)Oxidative stress inhibited the expression of FoxO1 and activated p-Akt(Ser473).And the activation of Akt promoted the phosphorylation of FoxO1 on Ser256 site,and led to cytoplasmic localization of FoxO1,which inhibited the activity of FoxO1;Inhibiting p-Akt or FoxO1 by specifical durgs,it was found that p-Akt promoted the phosphorylation of FoxO1 and further inhibited the regulation of SIRT1 expression in rat NP cells.(6)Resveratrol could inhibit p-Akt,decrease phosphorylation of FoxO1 and increase its activity,then promote the expression of SIRT1 to play an anti-senescence role ultimately.Conclusions:This study demonstrates staining and flow cytometry,which was going to establish a senescent model of rat NP that SIRT1 acts as an anti-senescence factor that attenuates oxidative stress-induced senescence in rat NP cells,which is regulated by the Akt-FoxO1 pathway.And resveratrol exerts anti-senescence function by activating SIRT1.These findings suggest that the Akt-FoxO1-SIRT1 axis might be a potential therapeutic target for delaying the progression of intervertebral disc degeneration.