Irisin Contributes To Neuroprotection By Promoting Mitochondrial Biogenesis After Experimental Subarachnoid Hemorrhage

Objective: Subarachnoid hemorrhage(SAH)is a serious hemorrhagic stroke with extremely high mortality and disability rates.It causes serious health problems worldwide,and also brings heavy life burden to patients and their families.Irisin is an exercise-related cytokine.After aerobic exercise,the secretion and synthesis of this protein are greatly increased,making it one of the main executors of the benefits brought by exercise.Irisin has attracted much attention in the research field of metabolic regulation,especially in lipid metabolism and mitochondrial homeostasis regulation.However,it is not clear whether this molecule can exert a neuroprotective effect on maintaining mitochondrial homeostasis after SAH.To observe and explore the Irisin’s roles after experimental SAH on our experimental mice and potential mechanisms,we performed neurological score,and evaluated the degree of improvement on neuronal apoptosis,oxidative stress and mitochondrial dysfunction.We devoted to shed light on the neuroprotective effects of Irisin after experimental SAH and its underlying mechanisms related to mitochondria.Methods: Male C57BL/6J mice with average body weight of 18-22 g and age of 8-12 weeks were selected.SAH mouse model was established by endovascular puncture method.All the experimental mice were inductively anesthetized in a hermetic box with 3-4% isoflurane,and then were masked and ventilated with 1-1.5% isoflurane throughout the operation.Intracerebroventricular administration was performed for exosgenous Iirisin treatment.And Genipin,a specific UCP-2 inhibitor,was injected intravenously before the administration of irisin in experimental animals with SAH.The expression level and spatial localization of endogenous Irisin in brain tissues during EBI after SAH were observed by Western blot(WB)and Immunofluorescence(IF),respectively.Secondly,two independent investigators who were blinded to the experimental design information evaluated the Garcia score and Beam balance score were used to evaluate the changes of neurological function scores.Then,the expression levels of apoptosis related proteins,as well as the mitochondrial fusing,fission and biogenisis proteins were evaluated by WB.The transmission electron microscope(TEM)was used to observe the morphological changes of mitochondria.The ATP detection kits were used to evaluate the energy changes.In addition,the conditions of apoptosis and oxidative stress about neurons were observed by TDT-UTP Nick end labeling(TUNEL)staining,dihydroethylene(DHE)staining and IF staining.Results:1.WB results indicated that endogenous Irisin protein in the brain tissues of experimental mice increased to the highest level at 12 hours after SAH,and began to decrease from 24 hours after SAH;Meanwhile,the IF results suggested that Irisin could co-locate with neurons in the mouse brain tissue,and the number of Irisin co-staining positive neurons decreased significantly at 24 h after SAH.2.Compared with the vehicle group,the exogenous Irisin intervention improved the neurological function and behavioral scores of mice significantly after SAH.Meanwhile,the WB and TUNEL staining indicated that neuronal apoptosis was significantly alleviated after Irisin treatment.Moreover,the DHE staining and oxidative stress related kits indicated that the oxidative stress response was significantly reduced by Iirisin administration.3.The intervention of exogenous Irisin significantly improved the mitochondrial imbalance after experimental SAH.TEM observation showed morphological changes in mitochondria,as the number of dilated mitochondria and vacuolation was significantly reduced.WB indicated tmitochondrial biogenesis was increased.4.WB,TUNEL staining,IF staining and neurological score evaluation showed that the UCP-2 inhibitor intervention can reverse the Irisin’s protective roles,involving anti-apoptosis,antioxidant stress,as well as the role of mitochondria homeostasis maintaining.This indicated that the protective effects of Irisin after SAH may be,through the UCP-2 related pathways,at least partially.Conclusion: Firstly,this study revealed that in the EBI phase after experimental SAH,Irisin can exert neuroprotective effects such as anti-apoptosis and reducing oxidative stress,and the neuroprotective effects may be achieved by improving mitochondrial instability.Secondly,the potential mechanisms may be partly related to the UCP-2-related pathway.In addition,taking mitochondrial homeostasis as the research target to improve the pathologic cascade after hemorrhagic stroke is a new research idea,for us to coping with the challenges brought by EBI after stroke.