| Purpose:Sirtuins are a class of histone deacetylases(HDACs),including seven mammalian Sirtuins(SIRT1-7),that have been shown to regulate a range of pathophysiological processes such as cellular aging,inflammation,metabolism,and cell proliferation,and during which SIRT1 attracts the most attention.Recent studies have pointed out that SIRT1 and its activator,resveratrol,were shown to enhance the survival of retinal ganglion cells(RGCs)following ischemia-reperfusion(I/R)injury for glaucoma.However,the location and function of Sirtuins in retina are not well defined,and the precise mechanisms for resveratrol’s protective effects are still unclear.The aim of this study is to determine the expression features of sirtuins in mice,rat,humans,and define the critical protein among of sirtuins in retinal injury and age-related dysfunction,furtherly demonstrate whether resveratrol can inhibit RGC apoptosis,retinal gliosis,and inflammation,all of which are critical events in retinal degeneration following I/R injury.Methods: This study assessed the retinal expression of Sirtuins in mice,rats,and humans and measured the expression of Sirtuins in aged and injured retinas.Expression of all 7 Sirtuins was confirmed by Western blot and qRT-PCR analysis in all three species.In the further study,right retinal ischemia was induced in adult male SD rats by increasing intraocular pressure to 110 mmHg for 60 mins,and the left eyes maintained at normal pressure serve as the control.Intraperitoneal injection of resveratrol or control buffer was performed continuously for three days from pre-to post-I/R injury and the protective effects were evaluated and compared.RGCs were retrogradely labeled with Fluoro-Gold by injection into superior colliculi.Apoptosis was detected by TUNEL staining,western blot and immunostainings were used to explore the associated pathway,Bax/Bcl-2-caspase 3.Gliosis was assessed by western blot and immunostaining of retinal cross sections with glial fibrillary acidic protein(GFAP).Results: In this study,SIRT1 is highly expressed in mouse,rat,and human retina,whereas SIRT2-7 expression was relatively lower in human retinas.Immunofluorescence was also used to examine the expression and localization of Sirtuins in rat retinal neurons.Importantly,we demonstrate a marked reduction of SIRT1 expression in aged retinal neurons as well as retinas injured by acute ischemia-reperfusion.On the other hand,none of the other Sirtuins exhibit any significant age-related changes in expression except for SIRT5,which was significantly higher in the retinas of adults compared to both young and aged rats.Resveratrol treatment significantly reduced retinal damage and RGC loss as demonstrated by the relatively intact tissue structure in H&E staining at day 7 and increased Fluoro-Gold labeling of RGCs at day 14,respectively.We found that resveratrol exhibited an anti-apoptotic effect as assessed by reduced TUNEL staining,inhibition of the early upregulated expression of the apoptosis-related protein Bax,and decreased subsequently cleaved caspase-3.However,it did not affect Bcl-2 levels.Moreover,in our I/R injury model,the combined response of reactive gliosis and related inflammation,which were demonstrated byan early induction of pro-inflammatory mediators and subsequently increased GFAP level,,were significantly attenuated after resveratrol treatment.Conclusion: Our work presents the first composite analysis of Sirtuins,and have defined their expression features among mice,rat and humans.We also find SIRT1 was down-regulated obviously in ischemia-reperfusion injured or aged rat retinas.The further research has demonstrated that resveratrol can prevent RGC death by blocking the Bax-caspase-3-dependent apoptotic pathway and suppressed gliosis-related inflammation in the retina after I/R injury.Together our results support that SIRT5,especially SIRT1 may be critical regulator in retinal degeneration,and the use of SIRT1 activator,resveratrol should be a possible therapeutic strategy for glaucoma. |
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