| Vascular cognitive impairment(VCI)is a clinical syndrome with a complex etiology.In recent years,with the increasing aging of the population and the high prevalence of cerebrovascular diseases,the incidence of VCI is increasing and has become the second most common type of dementia after Alzheimer’s disease.There are many hypotheses about the pathogenesis of VCI,including neurovascular unit dysregulation,blood-brain barrier dysfunction,mitochondrial dysfunction,over-activation of microglia,neuroinflammation,and oxidative stress injury.Since the concept of ferroptosis was proposed in 2012,it has been proven to be involved in the pathological process of various neurological diseases,including cerebral hemorrhage,subarachnoid hemorrhage,cerebral ischemia/reperfusion injury and other cerebrovascular diseases,which indirectly confirms that ferroptosis is involved in the pathological damage of VCI.However,relatively few studies have been conducted on whether ferroptosis is directly involved in the pathogenesis of VCI.Thus,we constructed the rat model of chronic cerebral hypoperfusion(CCH)by permanent bilateral common carotid artery occlusion(BCCAO or two vessel occlusion,2VO)to simulate VCI pathological events,and to investigate whether ferroptosis is involved in the CCH-induced cognitive impairment.It is well known that an unrestricted increase in ROS is a central terminal event in ferroptosis,and a large proportion of ROS in vivo originates from mitochondria,so it is crucial to remove mitochondrial-derived ROS.Mitoquinone mesylate(mitoQ)is a drug developed on this basis and is now widely used as an antioxidant targeting mitochondrial-derived ROS.Therefore,in my study,mitoQ was chosen as a therapeutic agent to interfere with the oxidative stress injury associated with ferroptosis in CCH.We would like to further explore the molecular targets and pathways through which mitoQ exerts its neuroprotective effects,thus opening up new ideas and research directions for the treatment of VCI.Part Ⅰ: Ferroptosis is involved in the pathogenesis of chronic cerebral hypoperfusionObjective: To investigate whether ferroptosis is involved in the pathogenesis of CCH caused by 2VO in rats.Methods: Male Sprague-Dawley(SD)rats weighing 250-300 g and aged 8-10 w were selected.We used the 2VO method to construct a rat model of CCH.The rats for the experiment were divided into 3 groups: control,sham,and 2VO groups.Morris water maze experiment was performed at the 6th week after 2VO surgery,and then hippocampal tissues were isolated and extracted after cardiac perfusion.Paraffin slices were subjected to HE and Nissl staining to observe the structural changes of neurons in CA1 and CA3 regions of hippocampal tissue.The total iron,SOD,MDA and GSH contents of hippocampal tissues were measured by the corresponding kits.The protein levels of GPX4 and COX2 in hippocampal tissues were detected by western blot.ROS fluorescence staining of frozen slices was used to observe the ROS content in CA1 and CA3 regions of hippocampal tissues.Mitochondrial isolation kit was used to extract mitochondria from fresh hippocampal tissues,and mitochondrial membrane potential JC-1 kit was used to detect mitochondrial membrane potential.Transmission electron microscopy was used to observe the ultrastructural changes of mitochondria.Results:(1)The CCH rat model was constructed by the 2VO method,with a surgical success rate of 87%.Morris water maze experiment was performed at the 6th week after 2VO surgery,and the results suggested that the rats in the 2VO group showed significant cognitive dysfunction.(2)The neuronal structure in hippocampal tissue of rats in the 2VO group was significantly damaged,with the most obvious damage in the CA1 area.(3)The rats in the 2VO group experienced ferroptotic events: compared to the control and sham groups,hippocampal tissue total iron content was significantly increased(P<0.01)(iron overload);a significant increase in hippocampal ROS content,MDA content,COX2 protein level(P<0.01),and a significant decrease in SOD content(P<0.001)(oxidative stress injury);and a significant decrease in GPX4 protein level and GSH content(P<0.05)(GPX4/GSH axis dysregulation)(4)Mitochondrial structure and function were impaired in the 2VO group,as shown by: smaller and denser mitochondria,localized matrix cavitation,dilated or even disappeared cristae,and some mitochondrial inner and outer membrane structures disappeared;compared with the control and sham groups,the mitochondrial membrane potential was significantly reduced in the 2VO group(P<0.05).Conclusions:(1)CCH caused cognitive dysfunction in rats.(2)Ferroptosis led to pathological impairment of CCH.Part Ⅱ: mitoQ inhibits ferroptosis-mediated cognitive impairment in chronic cerebral hypoperfusion via modulation of Nrf2/AREs/GPX4 pathway.Objective: To investigate the protective effects of mitoQ on CCH rats.We wanted to clarify whether it could improve cognitive dysfunction in CCH rats;whether it could attenuate ferroptosis-related oxidative stress damage in CCH rats;whether it could protect the integrity of mitochondrial structure and function,and further investigate whether it exerts neuroprotective effects through modulation of Nrf2/AREs/GPX4 pathway.Methods:(1)In vivo experiments: The 2VO method was used to construct a CCH rat model.The experimental rats were divided into three groups: sham+0.9% Na Cl,2VO+0.9% Na Cl,and 2VO+mitoQ groups.We set mitoQ into 3 dose subgroups:2mg/kg,4mg/kg and 6mg/kg.mitoQ was administered to 2VO rats by intraperitoneal injection from the 1st day after surgery,once every other day for 6 weeks.Novel object recognition test and morris water maze experiments were performed in the 6th week after surgery.After completion of behavioral experiments,cardiac perfusion was performed under anesthesia followed by isolation and extraction of hippocampal tissue.The neuronal structural changes in the CA1 and CA3 regions of hippocampal tissue were observed by HE and nissl staining.The SOD,MDA and GSH contents of hippocampal tissue were measured by the corresponding kits.The nucleus and cytoplasm protein separation kits were used to extract the nucleus and cytoplasm fractions,and western blot was used to detect Nrf2 protein in the nucleus fraction and Nrf2,Keap1 and HO-1 protein in the cytoplasm fraction.Western blot was used to detect the protein levels of Nrf2,GPX4,Keap1,HO-1,NQO1 and COX2 in hippocampal tissues.Immunohistochemical staining of paraffin slices was used to observe the number of positive Nrf2 and GPX4 cells in hippocampal tissues.ROS fluorescence staining of frozen slices was used to observe the ROS content in CA1 and CA3 regions of hippocampal tissues.Mitochondrial isolation kit was used to extract mitochondria from fresh hippocampal tissues.Mitochondrial membrane potential JC-1 kit was used to detect mitochondrial membrane potential.Mitochondrial ultrastructural changes were observed by transmission electron microscopy.(2)In vitro experiments: Since detection of mitochondrial ROS and superoxide content in fresh animal tissues is difficult to achieve,in this study,SH-SY5 Y cells were induced to be ischemic and hypoxic by the combination of Co Cl2·6H2O and low glucose/serum-free DMEM/F12 medium.Mito SOX red mitochondrial superoxide indicator was used to detect mitochondrial superoxide content in the ischemic-hypoxic cells to observe whether mitoQ could target mitochondria to reduce the production of toxic oxidative products and thus exert neuroprotective effects.Results:(1)Exploring mitoQ therapeutic doses and adverse drug reactions: the 4mg/kg dose group was always lower in weight than the sham+0.9% Na Cl group,2VO+0.9% Na Cl group and mitoQ+2mg/kg group during the experiment,some of them showed diarrhea,nasal bleeding,and most of them had swelling of the intestinal canal at the time of sampling;the 6mg/kg dose group,after giving the 1st injection of mitoQ,successively death occurred,while the 2mg/kg dose was safer and no significant adverse effects were seen,so the 2mg/kg dose was finally selected and denoted as the 2VO+mitoQ group.(2)Novel object recognition test and morris water maze were performed at the6 th week after surgery,and the results suggested that mitoQ treatment significantly improved cognitive dysfunction in 2VO-treated rats.(3)HE and nissl staining of paraffin slices: the mitoQ group significantly improved the degenerative damage of hippocampal neurons.(4)mitoQ ameliorates oxidative stress injury: the levels of ROS,MDA and COX2 in hippocampal tissue were significantly reduced in the mitoQ group compared with the 2VO group(P<0.05).(5)mitoQ promotes Nrf2 nuclear translocation: compared with the 2VO group,Nrf2 protein levels were increased in the nucleus of mitoQ group,and Nrf2 and Keap1 protein levels in the cytoplasm were significantly decreased(P<0.01),suggesting that mitoQ promotes Nrf2 nuclear translocation.(6)mitoQ activates Nrf2/AREs/GPX4 pathway: the levels of GPX4 and Nrf2 proteins were significantly higher in the mitoQ group than in the 2VO group(P<0.01).(7)mitoQ protects the structural and functional integrity of mitochondria: mitoQ treatment significantly improved the damage of mitochondrial structure and function after 2VO treatment.(8)mitoQ reduces mitochondrial superoxide production(in vitro experiments):mitochondrial superoxide production was significantly increased in the Co Cl2·6H2O model group,and significantly more red fluorescence was observed under inverted fluorescence microscopy than in the control group.mitoQ treatment attenuates the damage to mitochondria caused by ischemia and hypoxia and reduces the production of mitochondrial superoxide.Conclusions:(1)mitoQ ameliorates cognitive dysfunction of CCH rats.(2)mitoQ attenuates oxidative stress injury and protects the structural and functional integrity of mitochondria in CCH rats.(3)mitoQ promotes Nrf2 nuclear translocation and attenuates ferroptosis-related oxidative stress injury in CCH rats by activating the Nrf2/AREs/GPX4 pathway. |
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