Effects Of Vitamin D Receptor In OPCs Ferroptosis And White Matter Lesion In Hippocampus Of Mice With Vascular Cognitive Impairment

Objectives:Vascular Cognitive Impairment(VCI)is a type of cognitive impairment caused by cerebrovascular diseases or their risk factors,once VCI occurs,there are no effective targeted treatments,and it has caused a great burden to society.White matter injury is one of the typical pathological changes in VCI,the white matter hyperintensity in magnetic resonance T2-weighted images is the most common imaging manifestation of VCI.Oligodendrocytes(OLs)form the myelin sheath of the central nervous system,they play an important role in maintaining myelin integrity and stabilizing white matter function.OLs are transformed from their precursor cells,which are called oligodendrocyte precursor cells(OPCs).Studies have shown that the number of mature OLs in the brain decreases when VCI occurs,suggesting that OLs impairment and OPCs differentiation disorders may be key links in VCI.Ferroptosis is a novel type of programmed cell death,which is iron-dependent,participating in the occurrence and development of various neurological diseases,ferroptosis is closely related to oxidative stress levels,and because of the insufficiency of antioxidant enzymes and abundant iron in oligodendrocytes,it is more susceptible to oxidative stress than other neural cells.Persistent chronic cerebral hypoperfusion in patients with VCI causes oxidative stress in the brain,which may induce the ferroptosis of oligodendrocytes.Previous studies have shown that fat-soluble Vitamin D(VitD)can inhibit the occurrence of ferroptosis through its antioxidant effect and regulation of iron metabolism,and clinical studies have shown that VitD supplementation could prevent cognitive impairment,so,will VitD regulate the ferroptosis of OPCs to improve white matter lesion of VCI through vitamin D erceptor(VDR)?This study aims to preliminarily explore the role of ferroptosis in VCI and the effects of regulating VDR in OPCs ferroptosis and white matter lesion,furthermore,our study will focus on exploring the underlying mechanism of these effects.Methods:Part Ⅰ:(1)Bilateral carotid artery stenosis(BCAS)model was constructed using a microcoil,mice were randomly divided into two groups:Sham and BCAS;(2)The cognitive function were assessed by morris water maze(MWM)on day 30;(3)The white matter of corpus callosum and hippocampus in the two groups was evaluated by LFB staining,immunohistochemical staining and Western blotting(WB);(4)the expression of ferroptosis-related proteins in hippocampus was detected by WB.Part Ⅱ:In order to research the role of VitD/VDR in the onset of VCI,(1)we constructed BCAS model of wild type(WT)and vitamin D receptor knockout mice(VDR-/-),and divided the mice into 4 groups,which were WT-Sham,VDR-/--Sham,WT-BCAS and VDR-/--BCAS groups;(2)VCI mice were injected with paricalcitol(Pari),and mice were randomly divided into 4 groups,namely Sham,BCAS+Vehicle,BCAS+low-dose paricalcitol(L-Pari),BCAS+high-dose paricalcitol(H-Pari);(3)The cognitive function of mice in different groups were evaluated by MWM,Y maze,and Novel Object Recognition(NOR)on day 30 after modeling;(4)LFB and immunohistochemical staining were used to evaluate the white matter lesion in the corpus callosum and hippocampus of mice,and WB was used to evaluate the expression of MBP in the hippocampus.Part III:To study the relevant mechanisms of how VitD/VDR influences VCI,we conducted in vivo and in vitro experiments.In vivo experiments:(1)The mice were subdivided into 4 groups randomly,namely Sham,BCAS+Vehicle,BCAS+L-Pari,BCAS+H-Pari;(2)The production of malondialdehyde(MDA),reactive oxygen species(ROS)and other substances in peripheral blood and hippocampus of mice were measured by Elisa to observe the oxidative stress levels;(3)The expression of the related pathway proteins in the hippocampus of mice were assessed by WB;(4)The Nrf2 expression of the oligodendrocyte line cells in the hippocampus was observed by immunofluorescence staining;In vitro experiments:(1)Oligodendrocyte precursor cells(OPCs)were extracted and cultured in vitro,then immunofluorescence staining was used to identify Olig2 positive cells;(2)An in vitro chronic hypoxia model was constructed by adding 100mmol/L cobalt dichloride(CoCl2)into culture medium,the chronic hypoxic OPCs were dealt with Pari at the same time,the cells were divided into Control,CoCl2+Vehicle,CoCl2+2nM Pari,CoCl2+10nM Pari,and CoCl2+50nM Pari groups;(3)The oxidative stress level of cells in each group was evaluated by ROS staining;(4)The differentiation of OPCs was observed by immunofluorescence double staining and WB;(5)Then we selected the specific concentration of Pari(50nM)that can effectively inhibit the level of oxidative stress and promote the differentiation of OPCs,to study the effect of Pari on ferroptosis of the chronic hypoxia OPCs.The morphological changes of mitochondria in cells were observed by transmission electron microscope,and the expression of 4-HNE in cells was assessed by immunofluorescence staining;(6)The mRNA and protein changes of ferroptosis pathway-related molecules were measured by RT-PCR and WB respectively.ResultsPart Ⅰ:(1)In the MWM tests,the escape latency of BCAS mice significantly prolonged starting from the fourth day,in addition,the platform crossing times and the time in the target quadrant of BCAS mice significantly decreased after 30 days,indicating obvious cognitive impairment in the BCAS group;(2)The LFB staining results demonstrated that myelin fibers in the corpus callosum became loose and the myelin basic protein(MBP)level in the hippocampus decreased significantly,showing the white matter injury in the corpus callosum and hippocampus;(3)In addition,the western blotting(WB)results displayed that the expression of CD71 increased while that of GPX4 decreased,suggesting the initiation of ferroptosis in the hippocampus of VCI mice.Part Ⅱ:(1)The cognitive behaviors of VDR-/-mice was injured significantly after BCAS operation,what’s more,compared with WT-BCAS mice,VDR-/--BCAS group displayed worsen cognitive faction in the NOR test and Y maze alternation test;and the white matter injury aggravated in VDR-/--BCAS group,especially in CA1 area ofhippocampus than that of WT-BCAS mice;(3)After being treated with Pari,the cognitive function of VCI mice improved in MWM,Y maze and NOR test significantly,in addition,the white matter injury of them was also ameliorated,the expression of MBP in the hippocampus elevated obviously after the treatment of Pari;Part Ⅲ:In vivo experiments:(1)Pari decreased the oxidative stress level in serum and the hippocampus of VCI mice constrcucted by BCAS to raise the antioxidation ability;(2)H-Pari could increase the levels of VDR and GPX4 proteins and decrease the level of CD71 protein significantly in the hippocampus of VCI mice;(3)The immunostaining showed that the number of Olig2+-Nrf2+ cells decreased in the hippocampus of VCI mice significantly,while H-Pari reversed the tendency;In vitro experiments:(1)In cultured OPCs,the Olig2+cells counted for more than 90%of total cells,indicating high purity of the primary cells;(2)Pari could reduce the oxidative stress level of chronic hypoxic OPCs;(3)Pari treatment upregulated the MBP level and downregulated the O4 level,which promoted the differentiation and maturation of OPCs after chronic hypoxia;(4)50nM Pari could recover the mitochondria morphology,inhibit the expression of 4-HNE to inhibit ferroptosis in OPCs suffering from chronic hypoxia;(5)RT-PCR and WB results indicated that 50nM Pari could increase the mRNA and protein expressions of VDR,GPX4 and Nrf2 while decrease those of CD71 in hypoxic OPCs.ConclusionThe VCI mice constructed from BCAS showed cognitive impairment,demyelination in hippocampus,OLs injury and the abnormal expression of ferroptosis-related indicators.VDR knock-out aggravated the white matter injury and the cognitive impairment of VCI mice,the VDR agonist Pari could improve the cognitive function of VCI mice,repair white matter injury of hippocampus;in the VCI mice,Pari could reduce the level of oxidative stress and inhibit the occurrence of ferroptosis in the hippocampus.According to the experiments carried out in primarily cultured OPCs,Pari could raise the expression level of VDR in OPCs significantly,restrain the ferroptosis of chronic hypoxic OPCs and subsequently motivate the differentiation and maturation of OPCs.Thus,this study suggests that VDR could inhibit ferroptosis of OPCs and promote the recovery of white matter injuries in VCI mice.