| Objective: The present research is aimed at exploring the dynamic changes of ERs and GSK-3β in the different ages of APP/PS1 double ovariectomized transgenic AD mouse model,and to observe the effect of valproic acid sodium salt combined with estradiol on the expression of ERs and GSK-3β in APP/PS1 double ovariectomized transgenic AD mouse model,and discuss their mechanisms.The results of this study will provide new laboratory basis for studying the pathlogenesis and clinical treatment of AD.Methods: The present study is divided into two parts.The first part: The 3-month-old APP/PS1 double transgenic femal mice were choosen in this study.They were randomly divided into sham-operated group(AD-Sham)(n=10)and ovariectomized group(n=10),and age-matched wild type mice were as controls(WT)(n=10).Mice were examined at 1 month and 5 months after OVX or sham operation.The Morris water maze test,radioimmunoassay,Western blot and immunofluorescent staining were used to detect the learning and memory ability and the serum estrogen levels of mice,and senile plaques,ERβ and GSK-3β protein levels in the brain of mice.The second part: APP/PS1 double transgenic mice at the age of 1 month after ovariectomized were chosen as the experimental objects.The subjects were randomly grouped into 6 groups: VPA-treated(30mg/kg.d),VPA combined E2-treated(VPA: 30mg/kg.d,E2: 2.4ug/d),E2-treated(2.4ug/d),Liq-treated(30mg/kg.d),VPA combined Liq-treated(VPA: 30mg/kg.d,Liq: 30mg/kg.d)and saline-treated groups(10 for each group).The drugs were regularly injected into mice for 4 weeks.After 4-week drug treatment,the behavioral test was performed.In order to detect depressive emotions in mice;we used the elevated plus maze In order to detect the spatial learning and memory abilities of mice,we used the morris water maze.Western blot were performed to examine the expression level of GSK-3β and ERs in the mice brain.Results1.The first part: Radioimmunoassay showed that the serum estradiol levels was dropped significantly at 1 month and 5 months after OVX,and the uterine began to atrophy at 1 month after OVX,and the sizes of uterine further atrophy at 5 months after OVX.Morris water maze test showed that compared with those of AD-Sham group,the escape latency and the path length were increased significantly and the passing times were decreased significantly of AD-OVX group at 1 month after OVX(P<0.05).With the time following after OVX,the escape latency,the path length and the passing times were showed a further decline(P<0.05).IF results showed that compared with the AD-Sham group,the senile plaques numbers was begun to increase in the cortex in the AD-OVX group at 1 month after OVX,as the incidence of OVX,the number of senile plaques was increased significantly in the hippocampus and cortex(P<0.05).Western blot showed that compared with that of WT group,the ERα and ERβ was abnormally expressed in AD-Sham group(P<0.05).The expression of ERα and ERβ was down regulated at 1 month and 5 months after OVX(P<0.05),while a similarly consistent trend was found in the expression of p-GSK-3β(ser9)(P<0.05),although the expression of total GSK-3β showed no differences.The second part:1)Behavioral tests: 1.In the EMP test: The E2,VPA and Liq group showed no significant different on the open arm duration and the open arm entries when compared with the saline group(P>0.05).However the combined group showed a significantly increased on them(P<0.05).2.In the Morris water maze test: There was no significant difference in the escape latency and path length in the visible platform among the saline,VPA,E2,Liq and combined treatment group(P>0.05).In the hidden platform experiment,there was a significant decrease in the escape latency and the path length among the VPA,E2,Liq and combined treatment group when compared with the saline group(P <0.05),and the combined treatment group also showed a significantly decreased compared with the single medication group(P<0.05).In the probe test,the platform-crossing times in the VPA,E2,Liq and combined treatment group were significantly more than the saline group(P <0.05),and the allied treatment group also express significantly increased compared with the single medication group(P <0.05).2)The weight and morphology of uterus in the mice showed that: the uters weight in the VPA and Liq group showed no significantly different compared with the saline group(P>0.05),and the weight of uters in the E2 and combined treatment group showed a significant increase when compared with the other groups(P<0.01).3)The result of western blot: 1.ERα and ERβ,the expression of ERα in the cortex showed significantly increased in the combined treatment group when compared with the saline group(P<0.01),however the level of ERα in other groups showed no differences with the saline group(P>0.05).The expression of ERα in the hippocampus showed no differences in the VPA group when compared with the saline group(P>0.05),but the level of ERα in the other groups showed an obvious increase(P<0.05).The expression of ERβ also showed a similar trend with ERα.2.GSK-3β and pSer9-GSK-3β,no matter in the cortex or in the hippocampus,the level of total GSK-3β showed no obvious difference compared with the saline group,but the combined treatment group showed significantly decreased in the cortex(P<0.05)while showed no obvious difference in the hippocampus(P>0.05)compared with the saline group.The level of pSer9-GSK-3β decided the activity of GSK-3β,from the present study,The level of p Ser9-GSK-3β in the cortex,compared with the saline group,the VPA and E2 group showed no obvious different(P>0.05)however the Liq and the combined treatment group showed an obvious increased(P<0.05).The level of pSer9-GSK-3β in the hippocampus also showed a similar result.Conclusions:1.Ovariectomy induced the serum estradiol levels dropped and the uterine atrophy in APP/PS1 double transgenic mice after with the time following.2.Ovariectomy lead to the decrease of spatial learning memory in APP/PS1 double transgenic mice since the time following.3.Ovariectomy increased Aβ deposition and SP formation,the underlying mechanism may relate to the signaling pathway of ERs/GSK-3β.4.Drug treatment significantly improved the depression and anxiety behaviors,reduced the decline in spatial learning and memory ability in APP/PS1 double transgenic ovariectomy mice.And the effect of combined treatment was better than the single medication.5.Drug treatment significantly increase the level of estrogen receptors in APP/PS1 double transgenic ovariectomy mice brain,and the effect of combined treatment was better than the single medication.6.Drug treatment significantly increased the level of pSer9-GSK-3β,further depressed the activity of GSK-3β,and the effect of combined treatment was better than the single medication. |
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