The Role Of Sirt1 In Ferroptosis After Ischemic Brain Injury

Background and Purpose:Ischemic stroke is one of the important problems to be solved in the field of health.Neuronal death after ischemic stroke is the key node leading to irreversible brain damage.Therefore,clarifying the regulatory pathway of neuronal death has great clinical significance.There are many different forms of cell death,including necrosis,apoptosis,autophagy and mitotic disaster related death.Ferroptosis is a new type of cell death mediated by iron and characterized by accumulation of intracellular lipid reactive oxygen species(ROS),which plays an important role in the pathophysiological process after ischemic brain injury.Inhibition of ferroptosis alleviates ischemic brain injury,but how ferroptosis is initiated and regulated remains to be elucidated.Sirtuin 1(Sirt1)and Sonic Hedgehog(Shh)signalings are involved in regulating the development,damage and repair of organs and tissues.Activation of Sirt1 or Shh can play a strong neuroprotective and repair role,but whether Sirt1 and Shh regulate the occurrence and development of ferroptosis after cerebral ischemia injury remains unclear.Previous studies of our group showed that Sirt1 may be an upstream regulator of Shh signaling,and Resveratrol(Res),a Sirt1 agonist,can enhance the activation of Shh signaling pathway,promote neurogenesis after cerebral ischemia injury,reduce neuronal death,and improve neurological function.However,whether Sirt1 can regulate ferroptosis process after ischemic brain injury through Shh signaling and thus reduce ischemic tissue injury needs to be elucidated urgently.Therefore,this study combined in vivo and in vitro experiments to explore whether Sirt1 regulates the ferroptosis after ischemic brain injury through Shh signaling.It will lay the experimental foundation for the early development of drugs to intervene the ferroptosis process after ischemic brain injury and promote the recovery of brain function.Methods: The experiments were divided into in vivo and in vitro experiments.In vivo,Sprague Dawley(SD)rats were used to construct the middle cerebral artery occlusion reperfusion(MCAO/R)model.DMSO,Ferrostatin-1,Sirt1 agonist resveratrol and adenovirus overexpressing Sirt1 and Shh or Shh knockout adenovirus were injected via intracerebroventricular or intraperitoneal injection.In vitro,experiments focused on primary cortical neurons.Primary cortical neurons were extracted from the cerebral cortex of newborn SD rats and used to construct oxygen glucose deprivation/reoxygenation(OGD/R)model.Then neurons were treated with Sirt1 agonist resveratrol,Ferrostatin-1,DMSO,erastin,RSL3 and cyclipamine,a smo receptor inhibitor of the Shh pathway,respectively.Hematoxylin-eosin(HE)staining was used to detect the changes of tissue structure;Prussian blue(Perl’s)staining was used to determine iron deposition;Glutathione Peroxidase 4(GPX4),Ferritin and acyl-Co A synthetase long-chain family member 4(ACSL4)were detected by western blotting(WB);DCFH-DA staining was used to determine the expression of Reactive oxygen species(ROS);GSH kit was used to detect glutathione(GSH)levels in tissues and cells;Mitochondrial changes were observed by Transmission electron microscopy;2,3,5-Triphenyl-2H-Tetrazolium Chloride(TTC)staining was used to determine infarct volume;Neuronal structure was detected by Nissl staining;Fluoro-jade C(FJC)staining was used to detect the degeneration of neurons in and around infarcts;The expression of Sirt1 and Shh,and the purity and structure of primary cortical neuron were determined by immunofluorescence;Cell Counting Kit 8(CCK-8)was used to detect Cell survival rate;Iron colorimetric method was used to detect the level of iron in cells;Flow cytometry was used to determine intracellular ROS levels by DCFH-DA fluorescence.Results:(1)MCAO/R injury can induce ferroptosis related changes in SD rats,such as ferroptosis-protein(GPX4,Ferritin and ACSL4)expression changes,brain structural damage,iron deposition,ROS increase,mitochondrial vacuolation,etc.,and may reach the peak at MCAO/R 7d(P<0.05).(2)Inhibition of ferroptosis by Fer-1,a ferroptosis inhibitor,can reduce neuronal injury and infarct volume after MCAO/R(P<0.05).(3)Sirt1 can regulate ferroptosis after MCAO/R.a,Sirt1 expression was down-regulated in ischemic brain tissue on day 7 after MCAO/R injury.b,Sirt1 agonist Resveratrol pretreatment can affect the expression of ferroptosis-related proteins(GPX4,Ferritin and ACSL4)after MCAO/R,reduce ROS level and neuronal degeneration,and alleviate brain tissue injury and infarct volume after MCAO/R.c,Overexpression of Sirt1 significantly increased the expression of ferroptosis protective protein and inhibited the expression of ferroptosis injury protein(P<0.05).(4)Shh can regulate the ferroptosis after MCAO/R.a,MCAO/R injury can increase Shh expression in infarct,that is,activate Shh signaling pathway.b,Overexpression of Shh can increase the level of ferroptosis protective protein and inhibit the expression of ferroptosis injury protein in ischemic brain tissue,but knockout of Shh can promote ferroptosis(P<0.05).(5)After Shh knockout,the effect of Res preconditioning on ferroptosis-related proteins after MCAO/R was significantly inhibited.Only ferroptosis injury protein ACSL4 was slightly decreased,while other ferroptosis protective proteins were not significantly increased(P<0.05).(6)Effect of Sirt1 on ferroptosis after OGD/R in primary cortical neurons in vitro.a,OGD/R injury can induce ferroptosis in primary cortical neurons cultured in vitro.b,Res preconditioning has a similar effect to Fer-1,which both inhibits ferroptosis-related injury after OGD/R of primary cortical neurons and improves neuronal survival.c,Res preconditioning inhibited ferroptosis-related damage in primary cortical neurons induced by ferroptosis inducers Erastin and RSL3,and increased neuronal activity.d,Res preconditioning alleviates ferroptosis after OGD/R injury of primary cortical neurons aggravated by inhibition of Shh pathway,and improves neuronal survival(P<0.05).Conclusion: Sirt1 can inhibit ferroptosis process by activating Shh pathway,and help reduce OGD/R injury of primary cortical neurons and ischemia-reperfusion injury of SD rats brain tissue.