Effects Of CHI3L1 On White Matter Lesions In Hypertension-related Vascular Cognitive Impairment

Objective:Vascular cognitive impairment(VCI)is a syndrome ranging from mild cognitive impairment to dementia caused by cerebrovascular disease and its risk factors.VCI is a common cause of chronic progressive cognitive decline in the elderly population,causing a great burden on society.White matter Lesions(WMLs)are important pathological changes in VCI.Oligodendrocytes(OLs)are myelin-forming cells in the central nervous system and play an important role in maintaining white matter homeostasis and normal cognitive function.They are also the main target cells attacked in the process of white matter lesions of VCI.Hypertension is the most significant risk factor for WMLs and has attracted much attention because of its treatability.Cerebral small vessel lesions caused by hypertension secondary to hypoperfusion WMLs is the classical mechanism of WMLs,but more and more evidence shows that the intensity of antihypertensive treatment is not parallel to the degree of WMLs and cognitive decline,suggesting that hypertension may not only be caused by vascular-related WMLs,but other mechanisms may also exist.The purpose of this study was to preliminarily explore the key pathogenic factors of VCI caused by hypertension,and to explore the role and related mechanism of OLs and myelin injury,in order to provide a new intervention target for the prevention and treatment of VCI.Part one:The establishment of hypertensive-related VCI rat model and the screening and acquisition of key genes of VCIObjective:The aim is to screen and obtain the key genes which caused VCI in hypertensive rats(SHR rats)by transcriptome sequencing of brain tissues.Methods:In this study,we collected the data of blood pressure and cognitive function performances of spontaneous hypertensive rats(SHR rats)and wild Kyoto West rats(WKY rats)at 6,12 and 18 months age by using the methods of caudal artery manometry and cognitive behavioral testing.The lesions of white matter and cerebrovascular were detected in SHR rats and WKY rats of the same age by multimodal magnetic resonance.In addition,the multi-region brain tissues of the frontal cortex,hippocampus and corpus callosum of SHR rats with naturally occurring VCI and WKY rats of the same month were sequenced by RNA-seq.After that,the data were analyzed by bioinformatics analysis method and Atlas database.Results:(1)SHR rats showed a stable hypertension phenotype at 6 months of age,and the systolic blood pressure,diastolic blood pressure and mean arterial pressure were significantly higher than those of WKY rats at the same month of age;(2)Compared with WKY rats of the same age,SHR rats showed no cognitive impairment at 6 months,while began to show cognitive decline at 12 months,and the cognitive impairment was more prominent at 18 months;(3)Compared with WKY rats of the same age,the WMLs in SHR rats with naturally occurring VCI were present,and WMLs preceded vascular lesions such as microbleeds;(4)Combined with transcriptome sequencing data and various bioinformatics analysis methods,CHI3L1 was selected as the most significantly differentially expressed gene in VCI-SHR rats compared with WKY rats;(5)Bioinformatic analysis suggested that CHI3L1 was closely related to tumor,inflammation,immune response and other biological processes;(6)Through Atlas human single cell sequencing database,CHI3L1 was predicted to mainly exist in astrocytes in the brains of SHR rats.Conclusion:In this part,we conducted long-term cognitive behavioral monitoring of SHR rats,and established an animal model of hypertensive spontaneous VCI.Combined with RNA-seq and a series of biological analysis methods,CHI3L1 was screened as the key gene of naturally occurring VCI phenotype in SHR rats,which may mainly come from astrocytes in the CNS.CHI3L1 is believed to be related to biological processes such as tumor,inflammation and immune response.Part two:The expression level and cell origin of CHI3L1 in hypertensive patients with VCI and VCI-SHR rats.Objective:The aim is to verify the expression level of CHI3L1 from different perspectives,including patients,animals and cells,and to identify the cell origin and upstream regulatory factor of CHI3L1 in the brain of VCI-SHR rats.Methods:In this part of the study,we observed CHI3L1~+cell types in hippocampus by Immunofluorescence double-label staining.The primary cultured astrocytes and microglia were stimulated by Ang-Ⅱ,and the expression level of CHI3L1 protein was detected by western blotting to verify the main cell source of CHI3L1 in the brain of VCI-SHR rats.Serum ELISA was used to detect CHI3L1 in peripheral blood of hypertensive VCI patients and VCI-SHR rats,and Western blotting was used to detect CHI3L1 protein expression in brain tissue of VCI-SHR rats.Results:(1)Compared with WKY group of the same age,there were no significant differences in the number of GFAP~+CHI3L1~+cells in hippocampus and CA1,CA3 and DG subregions of 6-month-old SHR group;The number of GFAP~+CHI3L1~+cells in the whole hippocampus of 12-month-old SHR group was higher than that of WKY group(p<0.05),but there was no difference in the number of GFAP~+CHI3L1~+co-expressing cells in the subregions of hippocampus.The number of GFAP~+CHI3L1~+cells in the whole hippocampus and three subregions of hippocampus in SHR group at 18 months of age were significantly increased compared with WKY control group at the same age(p<0.001,p<0.001,p<0.001,p<0.001).There was no difference in the number of Iba1~+CHI3L1~+cells in the whole hippocampus and the subregions of the hippocampus at 6,12 and 18 months of age between SHR group and WKY group;(2)AngⅡcould stimulate the synthesis and secretion of CHI3L1 in primary cultured astrocytes,but it was not observed that AngⅡcould stimulate the synthesis of CHI3L1 in microglia;(3)With the occurrence and development of VCI,CHI3L1 level in peripheral blood of SHR rats aged 6 months,12months and 18 months showed a gradually increasing trend,and the CHI3L1 expression level in peripheral blood and hippocampus of SHR rats aged 12 months and 18 months were significantly increased compared with WKY control rats of the same age(p<0.05,p<0.05,p<0.001,p<0.001);(4)The serum CHI3L1 level in hypertension with VCI group was significantly higher than that in hypertension without VCI group and healthy control group(p<0.05,p<0.01).Conclusions:In this part of the study,CHI3L1 levels in peripheral blood of patients with hypertension related VCI and VCI-SHR rats were significantly increased.The expression level of CHI3L1 protein in the hippocampus of VCI-SHR rats was also significantly increased.In the background of hypertension,CHI3L1 is mainly derived from astrocytes in the central nervous system,and AngⅡmay be an important upstream regulatory molecule that promotes the secretion of CHI3L1 by astrocytes,but has no similar effect on microglia.Part three:The effects of CHI3L1 on cognitive function and myelin injury in VCI-SHR rats.Objective:The aim is to observe the leision of myelin and related cells in the brain of VCI-SHR rats,to observe the effect of myelin repairment by antagonizing CHI3L1,and to further explore the possible mechanism.Methods:In this part of the experiment,immunohistochemical staining was performed for different neuronal cells and myelin sheaths by stained with GFAP,Iba1,Neu N,GST-pi and MBP in hippocampus,and LFB staining was also used to observe the white matter lesions of corpus callosum.At the same time,Western blots were adopted to test the expression of CHI3L1 and MBP in the hippocampus of VCI-SHR and WKY rats of the same age.Furthermore,the specific antagonist of CHI3L1 with K284-6111 was used to observe the influence of CHI3L1 on cognitive function and MBP expression in the hippocampus of SHR rats.What’s more,western blotting was also used to observe the phosphorylation level of P65 in NF-κB signaling pathway,an inflammatory pathway in VCI-SHR rats after using CHI3L1 antagonist.Results:(1)The number of astrocytes,microglia,neurons and oligodendrocytes in the hippocampus of NVCI-SHR rats at 6 months of age was not significantly increased compared with that of WKY control rats at the same month of age,and there was no difference in myelin staining,either.In contrast,the number of astrocytes in hippocampus of 18-month-old VCI-SHR rats was increased compared with that of WKY control rats,with increase prominently in CA3 and DG regions were increased significantly(p<0.05,p<0.05).The number of microglia cells showed an increasing trend,but there was no statistical difference.The number of neurons did not change significantly.Additionally,the number of oligodendrocytes and the level of MBP staining were decreased,with a significant in DG region was decreased significantly(p<0.05,p<0.05);(2)There was no significant difference in LFB staining in corpus callosum of NVCI-SHR rats at 6 months of age compared with WKY rats at the same month of age;The IOD value of LFB staining in corpus callosum of 18-month-old VCI-SHR rats was significantly decreased compared with WKY rats of the same month(p<0.05);(3)Western blotting showed that there were no significant changes in CHI3L1 or MBP proteins in hippocampus of 6-month-old NVCI-SHR rats compared with WKY control rats of the same age,while the expression of CHI3L1 protein in 18-month-old VCI-SHR rats was significantly increased compared with WKY control rats(p<0.001,p<0.05);(4)After antagonism of CHI3L1 by K284-6111,cognitive impairment of SHR rats was significantly improved.Meanwhile,the expression of CHI3L1 protein in hippocampus was decreased,while the expression of GST-pi and MBP protein was increased(p<0.01,p<0.05,p<0.05);(5)The phosphorylation level of P65 in the NF-κB pathway in the hippocampus of VCI-SHR rats was increased(p<0.05),indicating that the inhibition of CHI3L1 could reduce the phosphorylation level of P65(p<0.05).Conclusion:VCI-SHR rats had decreased oligodendrocytes in the hippocampus,while significant myelin lesions and neuro-inflammatory in the hippocampus and corpus callosum.The inflammation was mainly originated from astrocytes in the brain of VCI-SHR rats,which resulted to higher expression of CHI3L1.CHI3L1 inhibitor improved cognitive function and alleviate white matter lesions in SHR rats,which might related to P65 phosphorylation of NF-κB pathway.