The Role And Mechanism Of Sirt3 In Ischemic Stroke

Globally,stroke is the second main cause of death and is severely harmful to the health of mankind.Stroke exhibits high mortality,high disability,and high recurrence.Moreover,many surviving stroke patients suffer various chronic disabilities.One of the obstacles to long-term functional recovery after stroke is the limitation of spontaneous neurogenesis and angiogenesis.Therefore,the treatment of stroke needs to focus on reducing the neurological deficit in the early phase and promoting long-term neurological function recovery.Sirt3 belongs to the sirtuin family proteins which display mostly NAD⁺-dependent deacetylase activities.Previous studies have shown that Sirt3 can maintain metabolic homeostasis,promote hematopoietic stem cell function in response to stress,mediate hearing protection under dietary restriction,and act as tumor suppressor via multiple signal pathways.In addition,Sirt3 plays a protective role in cardiac ischemia via regulating mitochondrial function and reducing ROS levels,which is mediated by Foxo3a,MAPK and AKT signaling pathways.Furthermore,Sirt3 can ptrotect neurons against oxygen-glucose deprivation(OGD)and H₂O₂induced oxidative stress injury.However,the role of Sirt3 in ischemic stroke is unknown.Here,we study the role and potential mechanism of Sirt3 in ischemic stroke.In the first part,we studied the role of Sirt3 in the acute phase after ischemic stroke.We used a permanent middle cerebral artery occlusion(p MCAO)model in wild type(WT)and Sirt3 knockout(KO)mice.We found that the level of Sirt3 in infarct region was decreased after ischemic stroke and Sirt3 deficiency aggravated neuronal cell apoptosis and neurological deficits after brain ischemia.In addition,Sirt3 KO mice showed more severe blood-brain barrier(BBB)disruption and inflammatory responses compared with WT group following stroke.Astrocytes are most widely distributed cells in the mammalian brain and play a protective role after brain ischemia via resisting excitotoxicity and releasing neurotrophic factors.We found that specific overexpression of Sirt3 in astrocytes by injecting GFAP-Sirt3 lentivirus in ischemic region showed protective effect.To further investigate the mechanism of Sirt3 in ischemic stroke,we cultured primary astrocytes and performed OGD experiments.The results showed that Sirt3 could regulate VEGF expression by inhibiting HIF-1?after OGD.Therefore,Sirt3 might regulate ischemic injury via HIF-1?/VEGF derived from astrocytes.In the second part,we studied the role of Sirt3 in the recovery phase after ischemic stroke.We found that Sirt3 KO mice showed worse neurobehavioral outcome compared with WT mice,accompanied by decreased neurogenesis and angiogenesis as shown by the reduction in the numbers of DCX⁺/Brd U⁺cells,Neu N⁺/Brd U⁺cells and CD31⁺/Brd U⁺cells in the penumbra region.In addition,Sirt3 deficiency reduced the activation of VEGF,AKT and ERK signaling pathways.Our results indicated that Sirt3 was beneficial to neurovascular and functional recovery following ischemic stroke.In the third part,we studied the role of Sirt3 in astrocyte activation and glial scar formation.In response to stroke-induced injury,astrocytes can be activated and form a scar.Inflammation is an essential component for glial scar formation.Previous study has shown that adjudin,a potential Sirt3 activator,could attenuate lipopolysaccharide(LPS)-and stroke-induced neuroinflammation.To investigate the potential inhibitory effect and mechanism of adjudin on astrocyte activation,we used a transient middle cerebral artery occlusion(t MCAO)model with or without adjudin treatment in WT and Sirt3 KO mice and performed a wound healing experiment in vitro.Both in vivo and in vitro results showed that adjudin reduced astrocyte activation by upregulating Sirt3 expression.In addition,adjudin treatment after stroke promoted functional and neurovascular recovery accompanied with the decreased area of glial scar in WT mice,which was blunted by Sirt3 deficiency.Furthermore,adjudin could increase Foxo3a and inhibit Notch1 signaling pathway via Sirt3.Both suppression of Foxo3a and overexpression of N1ICD could alleviate the in vitro effect of adjudin,indicating that Sirt3-Foxo3a and Sirt3-Notch1 signaling pathways were involved in the inhibitory effect of adjudin in wound healing.In conclusion,we have demonstrated that Sirt3 plays a protective role in both the acute phase and the recovery phase following ischemic stroke,attributable to the inhibition of inflammation and glial scar formation,reduction in BBB destruction,promotion of focal neurogenesis and angiogenesis.Furthermore,the beneficial effects of Sirt3 might also result from the inhibition of HIF-1?and activation of AKT and ERK signaling pathways.This raises a possibility of targeting Sirt3 as a therapeutic strategy for reducing acute ischemia injury and promoting long-term stroke recovery.