Hydrogen Sulfide Inhibits High Glucose-induced Senescence Of HT22 Cells Via Improvement Of Autophagic Flux By Upregulation Of SIRT1

Background and ObjectiveHigh glucose?HG?has toxic effects on the central nervous system?CNS?,which can induce nerve cell senescence.Emerging evidences demonstrate that the blockade of autophagic flux plays a critical role in the neurodegenerative diseases.Silent information regulator 1?SIRT1?is an important anti-aging protein and is also a potential regulator of autophagy in vivo.Our previous study has found that H₂S can antagonize the neuronal aging induced by homocysteine and formaldehyde and upregulate the expression of SIRT1 in neurons.Therefore,in the present work we want to investigate whether H2S inhibits HG-induced cellular senescence and whether the underling protective mechanism of H2S is involved in restoring autophagic flux via upreglution of SIRT1.MethodsThe viability of HT22 cells was determined by Cell Counting kit-8?CCK-8?.The senescent cells were observed by SA-?-gal?Senescence Associated acidic-?-Galactosidas?staining.Cell growth curves were generated by using Trypan blue stain assays.The expressions of senescence-associatedproteins(p16^INK4aandp21^CIP1),autophagy-associated proteins?LC3 and p62/SQSTMI?,and SIRT1 were detected by Western blot.The structure of autophagy was observed by Transmission electron microscopy?TEM?.Results1.Pretreatment of NaHS?a donor of H₂S,100,200,400?M?notably attenuated the inhibition of cell viability induced by HG?27mg/mL,48 h?,indicating that H₂S prevented HG-induced cytotoxicity in HT22 cells.2.HT22 cells pretreatment with NaHS?200?M?for 30 min significantly reversed HG-induced increases in the number of SA-?-gal positive cells and the expressions of senescence mark proteins(p16^INK4a and p21^CIP1)and suppression in the growth of cell,which indicates that H₂S reversed the HG-induced cellular senescence.3.HG?27,45 mg/mL?significantly increased the ratio of LC3II/LC3I and the expression of p62/SQSTMI protein in the HT22 cells,which indicates that HG blocked HT22 cell autophagic flux.4.Pretreatment of HT22 cell with 200?M NaHS remarkably reversed the HG-elicited increases in the number of autophagic vacuoles,the ratio of LC3II/LC3I,and the expression of p62/SQSTMI protein,which indicated that H₂S restored HG-blocked autophagic flux.5.Blocking autophagic flux by pretreatment of HT22 cell with CQ?autophagy inhibitor,10?M?significantly abolished the inhibitory roles of H₂S in HG-induced the blockade of autophagic flux and the senescence of HT22 cells,however,pretreatment with 3-MA?autophagy inhibitor,10 m M?has no significantly effect on H₂S improved HG-blocked HT22 cells autophagic flux and inhibited HG-induced celluar senescence,which indicates that autophagic flux mediates the protective activity of H₂S against HG-induced cellular senescence.6.H₂S?200?M?not only increased the expression of SIRT1 protein in HT22 cells,also rescued HG-induced downregulation of SIRT1protein.7.Inhibition of SIRT1 by Sirtinol?15?M?reversed the protection of H₂S against HG-induced cytotoxicity,cellular senescence,and autophagic flux blockade in HT22 cells,indicating that SIRT1 may involve in the autophagy flux-mediated protective effect of H₂S on HG-induced HT22 cells senescence.ConclusionH₂S protects HT22 cells against HG-induced cellular senescence,and the underlying mechanism is involved in the improvement of autophagic flux via upregulation of SIRT1.