The Effect And Mechanism Of Xiaoyaosan On CUMS Blood-brain-barrier Permeability

ObjectiveIn this study,chronic unpredictable mild stress(CUMS)model of depression in male mice were constructed to investigate the effect of chronic stress on blood-brain-barrier(BBB)by observing changes in the expression of glucocorticoid receptor(GR)which is a key factor in HPA axis and Occludin a functional transmembrane proteins of BBB.Base on the previous observations,how Xiaoyaosan(XYS)affect BBB’s structure and function in mice with chronic stress injury would be explored.The study would interpret the mechanism of Shugan Jieyu Therapy’s antidepressant-like effect from this perspective.Methods1.Constructing CUMS model:there are 12 kinds of stressors including feet electric shock(20V-30V,5min),cold swimming(15 ℃-4℃,2-5min),immobilization(4h-8h),water deprivation(24h),food deprivation(24h),wet bedding(4h-24h),blank bedding(4h-24h),empty bottle stimulation(5-6h),stroboscopic lighting(10min-3h),level shaking(5min),cage tilting(4-24h),disturbed light-dark cycle.During the first and second weeks of modeling,one kind of stress stimulation was randomly arranged every day.During the third to sixth weeks of modeling,one or two different kinds of stress stimulation were randomly given every day,and the same stress items were not repeated within three days.2.Experimental groups:subjects were 83 healthy male mice from the C57BL/6J inbred strain(BL6).Upon arrival mice were 12 weeks old.Mice were randomly divided into normal control group,chronic stress model group,low concentration XYS group,moderate concentration XYS group,high concentration XYS group and RU-38486(GR antagonist)group.Eccept for the normal control group,mice in other five groups were exposed to chronic unpredictable mild stress for 6 weeks(42 days).3.Outcome measure:the weight and sucrose preference rate of mice were recorded before and after modeling.Mice were detected the praxeology by open-field test after the modeling.Using enzyme-linked immunosorbent assay(ELISA)to assess the concentrations of serum corticosterone and evans blue method for determination of blood brain barrier permeability.Quantitative real-time PCR(qRT-PCR)and Western blot were performed to detect the expression level of GR and Occludin.Results1.Body weight:after 6 weeks of CUMS modeling,the growth rate of body weight in the stress model group was lower than the normal control group,with statistical difference(P<0.01).2.Sucrose preference test:after 6 weeks of CUMS modeling,the sucrose preference rate of the stress model group was significantly lower than that of other groups(P<0.05).3.Open field test:result show the total distance of horizontal movement,the time and distance of central area and the activity speed of the stress model group were significantly reduced after 6 weeks of modeling,compared with the normal control group(P<0.01);the moderate concentration and the high concentration of XYS could improve the behavior of mice in open field test(P<0.05).4.The concentration of serum corticosterone in mice:compared with the normal control group,the level of serum corticosterone in the stress model group increased significantly(P<0.01);the level of serum corticosterone in the high concentration of XYS group was lower than that in the stress model group(P<0.05).5.Permeability of blood-brain barrier:evans blue of brain tissue in the stress model group was significantly higher than that of the normal control group(P<0.01),and the content of Evans blue in brain tissue of three different concentrations of XYS groups and RU-38486 group were significantly lower than that of the stress model group(P<0.01).6.Expression of GR and Occludin gene in brain:compared with the normal control group,the expression of GR and Occludin mRNA of the stress model group decreased,but there were no statistical significance;compared with the stress model group,the expression of GR mRNA and Occludin mRNA in the brain of the high concentrations of XYS group and RU-38486 groups enhanced(P<0.01).7.Expression of GR and Occludin protein in brain:compared with the normal control group,GR and Occludin protein level of the stress model group significantly reduced(P<0.01);compared with the stress model group,the expression of GR and Occludin protein of mice’s brain were elevated in three different concentrations of XYS groups and RU-38486 group(P<0.01).ConclusionChronic stress can increase the permeability of blood brain barrier,down-regulating the expression of GR and Occludin.Xiaoyaosan can repair the structure and function of BBB and up-regulating the expression of GR and Occludin,which may be one of the mechanisms of Xiaoyaosan in counteracting the damage induced by chronic stress.