Resveratrol Inhibits Microglialactivation And Inflammation Via Mediation Of Sirt1/sonic Hedgehog Signaling After Ischemic Cerebral Stroke

Background: Microglial activation and neuroinflammation play a pivotal role in damage and repair of ischemic cerebral stroke.Resveratrol has a potential effect of anti-inflammation and regulating microglial activation.However,it is unclear how resveratrol affects microglial activation and inflammation after stroke.Earlier studies have shown that the activated Shh signaling has anti-inflammatory properties.Moreover,resveratrol,an activator of Sirt1,can activate the Shh signaling.It is unknown whether resveratrol activates the Shh signaling via Sirt1.Therefore,in this study,we tested whether resveratrol could suppress microglia activation and neuroinflammation by Sirt1/Shh Signaling in vitro and in vivo.Methods: Oxygen-glucose deprivation/reoxygenation(OGD/R)N9cells(a microglial cell line)and middle cerebral artery occlusion/reperfusion(MCAO/R)male C57BL/6 mice were used as model cells and animals in vitro and vivo and treated with drugs.1.To investigate concentration effects of resveratrol on microglial viability after OGD/R injury in vitro,the experimental groups were studied:(1)normal(Nor)group,(2)control(Ctrl)group,(3)vehicle(Veh)group,(4)different concentrations of resveratrol group(1,5,20,40,80μmol/L),(5)blank group.2.To determine whether effects of resveratrol on activation and inflammation of microglias after OGD/R or MCAO/R injury were mediated by the Shh signaling,the experimental groups were studied:(1)normal(Nor)group or sham(Sham)group,(2)control(Ctrl)group,(3)resveratrol(Res)group,(4)resveratrol combined with cyclopamine(R+C)group,(5)cyclopamine(Cyc)group.3.To determine whether Sirt1 is associated with Shh signaling,microglias were divided into four groups:(1)normal(Nor)group,(2)control(Ctrl)group,(3)resveratrol(Res)group,(4)sirtinol(Sirtinol)group.Cell viability,cerebral infarct volume and functional outcome were assessed by Cell Counting Kit 8,TTC staining,Longa,modified Bederson,and modified Neurological Severity Score(mNSS),respectively.Apoptosis,activation and inflammatory responses of microglia,expressions and activity of Shh signaling pathway proteins were detected by Flow Cytometry,immunofluorescence,ELISA,and Western blotting,respectively.Results:1.the optimal concentration effect of resveratrol on viability of microglia after OGD/R injury in vitro.The results of CCK-8 assay showed that microglial viability was significantly reduced in the control,vehicle,and 1,5,20,40 and 80μmol/L resveratrol groups compared with the normal group(P <0.05).However,microglial viability was significantly higher in the 5,20 and 40μmol/L resveratrol groups than those of the control and vehicle groups,and the highest viability was observed in 20μmol/L resveratrol group(P <0.05).2.Shh signaling mediated resveratrol to reduce injury and inhibit microglial activation and inflammation after OGD/R or MCAO/R injury.Resveratrol enhanced viability(P <0.05),inhibited apoptosis(P<0.05),down-regulated the expression of Iba1,Caspase3,Bax,TNF-α and IL-1β proteins(P <0.05),up-regulated the expression of IL-10,Shh,Ptc-1,Smo and Gli-1 proteins(P<0.05),and promoted Smo protein translocation to primary cilia and Gli-1 nuclear translocation after OGD/R injury in vitro.At the same time,resveratrol reduced cerebral infarction volume(P<0.05),improved neurological functional outcome such as Longa,Bederson and mNSS Scores(P <0.05),inhibited the expression of Iba1,TNF-α and IL-1βproteins and promoted the expression of anti-inflammatory factor IL-10 after MCAO/R injury in vivo(P <0.05).In contrast,the Shh antagonistcyclopamine eliminated the above effects of resveratrol(P <0.05).3.Sirt1 might mediate effect of resveratrol on the Shh signaling.Western blotting showed that Sirt1 antagonist Sirtinol could inhibit the expression of Shh,Ptc-1,Smo and Gli-1 proteins(P <0.05).Meanwhile,immunofluorescence staining showed that Sirtinol could inhibit Gli-1nuclear translocation.Conclusions: This study suggests in the first time that effects of resveratrol-inhibited microglial activation and neuroinflammation after OGD/R or MCAO/R injury may be,at least partly,mediated by the Sirt1/Shh Signaling.