Effects Of ?-Asarone On Step-down Test And Expressions Of Akt, GSK3? In Hippocampus Of Fmr1 Knockout Mouse

?Background?Fragile X syndrome(fragile X syndrome, FXS) is one of the most common X-linked hereditary mental retardations,which is caused by absence of fragile X mental retardation protein(FMRP). Fmr1 knockout mouse(KO mouse) is an ideal model of FXS. It was generated with phenotypes simila to those observed in human FXS patients such as deficit in learning and memory, macro-orchidism and hyperactivity. ?-asarone, one of the main components of volatile oil extraction of Acorus tatarinowii Schott., is wildly used for treating hyperlipoidemia, epilepsy and Alzheimer Disease in clinical medicine. It has been proved that ?-asarone could ameliorate the spatial memory in a rat model of learning and memory disorder.Acorrding to our previous research, cortical and hippocampal expression of inhibitory phospho-Ser9-GSK3?(P-GSK3?) were reduced in FVB KO mice. And it has been demonstrated that increased expression of P-GSK3? can improve the hippocampal memory of KO mouse. Therefore, the decreased level of P-GSK3? might contribute to deficit in learning and memory of KO mice. Since ?-asarone could improve spatial memory, it was worth doing further study on the effect of ?-asarone towards to treating learning and memory disorder of KO mice.Akt, also known as protein kinase B(PKB), is an upstream factor of GSK3?. Activation of Akt could inhibit GSK3? activity. Meanwhile, Akt activation might be involved in the formation of long term potentiation(LTP) and memory. However, the expression of Akt in hippocampus of FXS remains controversial. Moreover, in hippocampus of KO mouse, relationship between GSK3? activity and Akt activation has not been elucidated.?Objective?In present study, we observed the learning and memory behavior and expressions of Akt/GSK3? in both hippocampi of KO and WT mice after ?-asarone administration. We determined the therapeutic effect of ?-asarone towards to learning and memory disorder of KO mice and the possible mechanism of it.?Materials and Methods?1. AnimalsFVB Fmr1 knock mouse(KO) and wild type mouse(WT)(4-weeks-old, 18~22g) were used.2. Gene identificationPCR was applied to identify the KO mice.3. GroupsKO and WT mice were randomly divided into seven groups, respectively: blank control group, negative control group(normal saline group) and 5 ?-asarone treatment groups, they were 3mg/kg group, 6mg/kg group, 9mg/kg group, 12 mg/kg group and 24 mg/kg group.4. Experimental methods and observationsMouse was intraperitoneal injected with ?-asarone for 6 days continuously. And the same volume of normal saline was intraperitoneal injected to the control group. On the 7th and 8th day, 30 minutes after injection, step-down behavior was tested. And on day 9, hippocampus were rapidly removed 30 minutes after drug injection. Hippocampal tissue would be well prepared for detecting the expressions of Akt, P-Akt, GSK3? and P-GSK3? with Western blot.5. Statistical methodsAll of the results were performed as mean±SEM. Statistical software SPSS18.0 was used for data analysis. Independent-Samples t Test was applied to comparing data between groups. For group analysis, one-way ANOVA was used. P<0.05 was statistically significant.?Results?1. Step-down test: Compared to the KO blank control group, 3mg/kg, 6mg/kg, 9mg/kg and 12mg/kg ?-asarone treatment groups had significant increasing in step-down latencies(P<0.05), while in 24mg/kg group, only increasing tendency of latency was observed, but no statistically significant difference(P>0.05). Error counts of 3mg/kg and 9mg/kg treatment groups were remarkably less than that of blank control group(P<0.05). And there was no significant difference on error count between blank control group and other treatment groups(P>0.05), but just decreasing tendencies were found. To WT mice, there was no significantly difference of step-down latency and error count among all of the groups(P>0.05).2. Western blot: Compared the KO blank control to the WT one, expressions of KO hippocampal P-Akt and P-GSK3? were remarkable less than those of WT mice(P<0.05). However, there was no significant difference of Akt or GSK3? expression between KO and WT blank control mice(P>0.05). After ?-asarone administration, there was no significant difference of Akt expression among all of the KO groups(P>0.05). Compared with blank control group, merely 3mg/kg ?-asarone significantly increased hippocampal P-GSK3? expression(P<0.05). For the expressions of Akt and GSK3?,no significant difference was detected among all of the KO groups. To WT mice, no significant difference was observed in expressions of Akt/GSK3? after ?-asarone administration(P>0.05).?Conclusions?1. The hippocampal Akt/GSK3? pathway of KO mouse is impaired, which might be one of the mechanisms contributing to the defected learning and memory in KO mouse.2. The step-down behavior of KO mice could be corrected by ?-asarone in doses ranging from 3 to 12mg/kg.3. The hippocampal P-GSK3? expression could be increased by ?-asarone with a dosage of 3mg/kg, which may be one of the mechanisms of ?-asarone improving step-down behavior of KO mouse.