The Role And Mechanisms Of Cerebral Microvascular Injury Meditated By MiRNA-451/MIF Pathway In Cognitive Impairment Of Parkinson's Disease

Objectives:The aim of the present study is to investigate the role of miRNA-451/MIF and its downstream signaling pathway in cerebral microvascular disease and angiogenesis,and to demonstrate its role in neuroinflammation and autophagy.Meanwhile,the role and mechanism of miRNA-451/MIF pathway in the pathogenesis of cognitive impairment in Parkinson's disease were clarified in the present study.Methods:Exoquick kit was used to isolate exosomes from plasma in PD patients.The morphology of exosomes was identified by transmission electron microscopy(TEM)and nano particle analyzer(nanosight).The expression of exosomes surface proteins CD9 and CD63 was detected by western blot.The expression of exosomes miRNA was detected by high channel sequencing.Expression of biomarkers in cerebrospinal fluid(CSF)of PD patients was identified by liquid chip and ELISA.The damage and abnormal changes patterns of cerebral white matter were analyzed by TBSS technology.The mechanism of PD cognitive impairment was discussed,and biochemical and imaging markers were provided.The cultured mouse brain microvascular endothelial cells(bend.3)were transfected with miRNA-451 and miRNA-451 inhibitor lentivirus to overexpress or silence miRNA-451,respectively.The results were as follows:(1)the proliferation and colony-forming ability of bend.3 cells in each group were detected by CCK-8 and clone formation assay;(2)transwell and scratch test were performed to detect the migration ability of bend.3 cells in each group;(3)the tube formation of bend.3 cells in each group was tested in vitro;(4)western blot was used to detect the expression of MIF,its downstream molecules and autophagy-related molecules in bend.3 cells of each group.60 male C57BL/6 were randomly divided into 4 groups:Control group,Sham group,miRNA-451 group and miRNA-451 inhibitor group.After stereotactic injection of 2 p.1 of lentiviruses into hippocampus,the morphological changes of hippocampus were detected by H-E and Nissel staining,and the expression of ZO-1,CD34 and Iba-1 was detected by immunohistochemistry.The expression of miRNA-451/MIF pathway related signaling molecules was detected by western blot,qRT-PCR was used to detect the expression of inflammatory factors in hippocampus of mice in each group.Results:(1)Exosomes were isolated from PD patients' plasma.the diameter of exosomes was mainly in the range of 40-100 nm.The expression of CD9 and CD63 on the surface of exosomes were detected by western blot.Compared with the healthy control,miRNA-451 was found to be up-regulated in PD-MCI and PD-NC by miRNA microarray screening.The results of liquid chip and ELISA showed that there was a significant positive correlation between inflammatory factors IL6,IL8,MCP-1,vascular injury factor and endothelial cell activation factor in CSF.(2)Compared with PD-NC group,FA values in PD-MCI group were different in bilateral superior longitudinal fasciculus,bilateral external capsule,knee of corpus callosum,body of corpus callosum,left fornix,bilateral superior corona radiata,bilateral posterior corona radiata,bilateral cerebral peduncles and bilateral posterior thalamic radiation.(3)Knockdown of miRNA-451 could significantly increase the proliferation,migration and tube formation of bend.3 cell in vitro,which was related to the change of p38MAPK signal pathway;(4)The results of animal experiments showed that ?H-E staining showed that the hippocampal structure of miRNA-451 inhibitor group was damaged and angiogenesis was observed.The expression of ZO-1,CD34 and Iba-1 in hippocampus of miRNA-451 inhibitor group was shown by immunohistochemistry.? Western blot showed that the expression of p38 MAPK protein in hippocampus of miRNA-451 inhibitor group was higher than that of control group.? qRT-PCR showed that the expression of inflammatory factors in hippocampus of miRNA-451 inhibitor group was significantly higher than that of control group(P<0.05).Conclusions:Expression of miRNA-451 in the plasma-derived exosomes of PD patients is expected to be a biomarker of PD,especially,combining with miRNAs,exosomes and CSF proteomics and radiomics in the early diagnosis of PD and PD cognitive impairment.MiRNA-451/MIF is involved in brain micro vascular injury,angiogenesis and inflammatory response by regulating p38 MAPK,and then involved in the development of PD cognitive impairment.