The Function And Mechanism Of Actin Polymerization Dysfunction In Dementia Caused By Abnormal Cerebral Microcirculation

Background Dementia refers to chronic acquired progressive cognition disability syndrome,which mainly characterized by slow decline in intelligence and varying degrees of personality change.Dementia is a common disease among the elderly over 65 years old.By 2015,about 47 million people worldwide are suffering from dementia,and the number is expected to increase to 130 million by 2050.However,the pathogenesis of dementia is still not very clear.With the acceleration of the world’s aging,the number of dementia patients will further increase,so it is particularly important to study the pathogenesis of dementia.In recent years,studies have shown that the mutation of ADD3 gene is related to dementia.The protein expressed by ADD3 gene is an adducin.In cells,the primary function of adducin is to bind to the fast-growing end of F-actin,to maintain the actin’s polymerization state.The polymerization state of actin is the structural basis of the contraction function of vascular smooth muscle cells in microcirculation.The abnormality of microcirculation function is an important cause of dementia.However,whether the polymerization change of actin is involved in the formation of dementia is not clear at present,and there are few related studies at home and abroad.Therefore,we propose the hypothesis that the dysfunction of actin polymerization leads to the impairment of contractility of vascular smooth muscle cells,then leads to the abnormality of microcirculation function,the impairment of cerebral blood flow autoregulation,and causes the injury of endothelial cells,the leakage of blood-brain barrier,inflammatory and neuron apoptosis,and finally forms dementia.This hypothesis was proposed through cell experiments and in vitro experiments,and animal experiments to verify.Objective(1)At the molecular level,we investigate the effects of actin polymerization and depolymerization on the quantities and ratios of F-actin and G-actin in vascular smooth muscle cells(VSMCs).(2)At the cellular level,we aim to study the effects of actin polymerization and depolymerization on the contractibility of VSMCs.(3)In vitro experiments,we observe the effects of actin polymerization dysfunction on the pressure-induced myogenic response of rat middle cerebral arteries(MACs).(4)In animal experiments,we try to study the effects of actin polymerization dysfunction on spatial memory and learning ability in rats.(5)We are intent to explore the possible mechanism of actin polymerization dysfunction in dementia caused by abnormal cerebral microcirculation.Method(1)Immunohistochemistry experiments: Cell culture was carried out with a rat-derived VSMC line,and then the cells were planted in 6-well culture plates.At the bottom of each well of the culture plate were slides for cell attachment and growth.VSMCs were treated with Dimethyl sulfoxide(DMSO),Cytochalasin(Cyt-D)and Jasplakinolied(JK),and then stained with F-actin and G-actin to make slides.Finally,a fluorescence microscope was applied to observe the structure and quantify F-actin and G-actin.(2)Cell contraction experiments: Rat-derived VSMCs were cultured and mixed with collagen gel solution and then planted in 24-well culture plates.After VSMCs were treated with DMSO,Cyt-D and JK,the contraction of collagen gel was recorded and quantified.(3)Myogenic response experiments: The M2 segments of the MCAs of wild-type(WT)rats and Add3 gene knock out(Add3 KO)rats were installed in the Living System for myogenic response experiments.After stabilization,vessels were treated with DMSO,Cyt-D and JK,respectively,and changes in the inner and outer diameter of blood vessels induced by pressure changes were recorded.(4)Animal experiments: We measured the body weight,brain weight,and MAP of wild-type rats and Add3 KO rats to observe if there is any difference between them.We repeated eight-arm water maze experiments for 4 days to observe the difference in the time the rats escaped.Results(1)Immunohistochemistry experiments: The normalized fluorescence intensities of F-actin were higher than G-actin at all groups.The F-actin normalized fluorescence intensity of Cyt-D treatment group was lower than that of the control group,while JK treatment group was higher than the control group.The G-actin normalized fluorescence intensity of Cyt-D treatment group was higher than that of the control group,while JK treatment group was lower than that of the control group.The F / G-actin ratio of the Cyt-D treatment group was lower than that of the control group,while F / G-actin ratio in JK treatment group was higher than the control group.(2)Cell contraction experiments: The contraction percentage of collagen matrix in the Cyt-D treatment group was smaller than the control group,while the contraction percentage of collagen matrix in the JK treatment group was bigger than the control group.(3)Myogenic response experiments: The degree of contraction of the inner diameter of Add3 KO MCAs was significantly smaller than WT.There was no significant difference in the percentage of MCAs dilation between the two groups.There was no significant difference in the percentage of vasoconstriction between the blank control group and the DMSO treatment group.Cyt-D treatment and DMSO treatment both in WT and Add3 KO MCAs reduced the contraction percentage of vascular inner diameter,specifically,WT rats reduced more.Compared with the DMSO group,JK-treated group both in WT and Add3 KO displayed increased contraction percentage of vascular inner diameter,specifically,the Add3 KO rats increased more.(4)Animal experiments: There is no significant difference in body weight,brain weight,and MAP between Add3 KO rats and WT rats.Add3 KO rats took a longer time to escape in the first two days and the first two repeated experiments on the third day during the eight-arm water maze experiments.There was no significant difference in the time it took for the two groups of rats to escape in the eight-arm water maze in the last two repeated experiments on the third day and all experiments on the fourth day.Conclusion Actin could change F-actin and G-actin in vascular smooth muscle cells through polymerization changes.When the conversion of G-actin to F-actin increased,the contractility of vascular smooth muscle cells increased.In contrast,when the conversion of F-actin to G-actin increased,the contraction ability of vascular smooth muscle cells decreased.When the contractile function of vascular smooth muscle cells was weakened,the cerebral blood vessels pressure-induced myogenic response was impaired and thus caused abnormal autoregulation of cerebral blood flow,which in turn led to blood-brain barrier leakage,inflammatory exudation,hippocampal neurons injury and eventually led to the formation of dementia or accelerated the process of dementia.