Objective T

o observe the effect of

Guben Jiannao Anshen Formula

（固本健脑安神方， GJAF） on learning and memory in aging insomnia rats， and to explore the possible mechanism.

Methods

2

Sixty SD rats were subcutaneously injected with D-galactose on the back of the neck for 40 days to establish the aging rat model. After modelling， the rats were randomly divided into aging group， model group， GJAF high dose group， GJAF low dose group and estazolam group， with 12 rats in each group. Except for the aging group， the rats in the other groups were intraperitoneally injected with 400 mg/（kg·d） of DL-4-chlorophenylalanine suspension for two consecutive days to establish the aging insomnia model. After modelling， GJAF at the dose of 1.8 g/（kg·d） and 0.9 g/（kg·d） were given into the rats in the high- and low-dose GJAF group by gavage， respectively； 0.09 mg/（kg·d） of estazolam was given into the estazolam group by gavage， while 0.1 ml/kg of normal saline was given to the aging group and model group by gavage. All the interventions were administered once daily for seven consecutive days. Afterwards， Morris water maze test was used to detect the learning and memory ability of rats， including positioning navigation test for five consecutive days to record the daily escape latency， and space exploration experiment on the 7th day to record the times that the rats crossed the platform area in the third quadrant within 60 seconds after a day of rest. HomeCageScan was used to analyze the sleep time and sleep rhythm of rats， while RT-PCR and immunohistochemical methods were used to detect the mRNA and protein expression levels of Clock and Bmal1 in the CA1 region. Nissl staining was used to observe the pathological changes of Nissl bodies in the CA1 region of the hippocampus.

Results

2

The escape latency of the GJAF high-dose group was significantly reduced on the 3rd， 4th and 5th day compared to that of the model group （

P

＜0.05）. The times of platform crossing the third quadrant of the model group was less than that of the aging group， while those of the GJAF high dose group and the estazolam group were more markedly increased than the model group （

P

＜0.05）. The daytime sleep time and the total sleep time of the model group， as well as the Clock and Bmal1 mRNA expression in the CA1 region of the hippocampus decreased significantly compared to those of the aging group （

P

＜0.05）. Compared to those of the model group， the daytime sleep time and total sleep time of the GJAF high dose group and estazolam group， as well as the Clock and Bmal1 mRNA expression in the CA1 hippocampus region increased significantly （

P

＜0.05）. There was no significant difference in nighttime sleep time among the groups （

P

＞0.05）. The results of Nissl staining and immunohistochemistry showed that there was rich Nissl bodies in the CA1 region of the aging group， while the hippocampus CA1 region was lightly stained in the moel gorup， where certain Nissl bodies were dissolved， and the positive particles of Clock and Bmal1 reduced. The number of Nissl bodies and the expressions of Clock and Bmal1 proteins in the hippocampus CA1 region significantly increased in all medicated groups， and the GJAF high dose group had most obvious effect.

Conclusion

2

GJAF can improve the sleep rhythm disorder， repair hippocampal neurons and improve the ability of learning and memory. The mechanism may be related to regulating the expression of Clock and Bmal1.