The TCM Theoretical Discussion Of Circadian Rhythm And The Effects Of Shenghuitang To APP/PS1 Transgenic Mice

Objective:Collate the pertinent literature about Alzheimer’s disease and circadian rhythm, unscramble the meaning and relationship of Alzheimer’s disease and circadian rhythm from the point of view of TCM. Based on our earlier research foundation, discuss the relationship between Alzheimer’s disease and circadian rhythm, study the mechanism of improvement of circadian rhythm on prevent and treatment of AD. AD animal model was been made with APP/PS1 double transgenic mice to observe the effect of Shenghuitang to AD,and the influence of circadian rhythm. And provide some theoretical basis and experimental evidence for further theoretical and clinical research of TCM therapeutic principle and method theory system as well as prevention and treatment of senile diseases.Method:1. Theoretical discussion:Collate the ancient and modern pertinent literature about Alzheimer’s disease and circadian rhythm,analyze, conclude and summarize the basic theory of circadian rhythm, discuss the adhibition of circadian rhythm in disease diagnosis, treatment, prognosis, etc.Analyze the relationship of Alzheimer’s disease and circadian rhythm.2. Experimental study:AD animal model was been made with APP/PS1 double transgenic mice to observe the effect of Shenghuitang to AD, and the influence of circadian rhythm. Spontaneous activity of mice in 24 h was been observed with spontaneous activity experimentvideo analysis system. Cosinor Matlabsoftware was used to analyze the biological characteristics of spontaneous activity of mice with cosine method.Morris water maze test was used to detect the ability of learning and memory of mice. Urine volume of mice in 24 h was observed in different time period, well the content of CORT in urine and s APPα in hippocampus of mice was detect with Enzyme-linked immunosorbent assay. The expression of m RNA in hypothalamusinternal clock gene and melatonin receptor was detected with Real Time-PCR. Western Blot was used to detect protein level of internal clock gene, melatonin receptor,as well as Aβ1-40 and Aβ1-42 in hippocampus of mice. At the same time, SABC was used to detect the content of the main protein of internal clock gene, as well as Aβ1-40 and Aβ1-42 in mice’s hippocampus CA1 region. To observe the effect of Shenghuitang to AD, and the relationship between the influence of circadian rhythm and APP metabolism, and make a comparison with melatonin.Results:1. The typical circadian rhythm of spontaneous activity was observed in 3mooths old APP/PS1 double transgenic mice(P<0.05), but it was not observed in 4 mooths and 5 mooths old mice,even than tall 、 t Lof mice increased(P>0.05).2. The typical circadian rhythm of spontaneous activity was observed in the control group, melatonin group and Shenghuitang group(P<0.05), the sequence of amplitude is: high dose group>control group>low dose group>melatonin group. Morris water maze test showed that compared withthe model group, the platform latency of each group shortened significantly(P<0.01,P<0.05), total swimming distance of each group reduced(P<0.01, P< 0.05); compared with the melatonin group, total swimming distance increased significantly in low dose group(P < 0.05); each group’s through platform number and the target quadrant time increased compared with the model group(P<0.01,P<0.05), and the time of first reach for the original platform reduced(P<0.01,P<0.05); compared with the melatonin group,through platform number and the target quadrant time reduced(P<0.05).3. Urine volumedetection result showed that, compared with the control group, urine volume of the model group increased in daytime and all day long( P<0.05, P<0.01).Compared with the model group, urine volume of themelatonin group and high dose group reduced in daytime(P<0.05, P<0.01),and melatonin group increased in night time(P<0.05); compared with the melatoningroup, urine volume of the low dose group increased significantly(P< 0.05). The typical circadian rhythm of urine volume was observed inthe control group, melatonin group and Shenghuitanghigh or low group(P<0.05),well the typical circadian rhythm of the content of CORT in urine was observed in each group except melatoningroup(P<0.05).4. The expression of m RNA in hypothalamusinternal clock gene in Real Time-PCR test showed that, compared with the control group, Per1 m RNA、Cry1 m RNA、Clockm RNA、Bmall m RNA and Rev-erbam RNA showed a down-regulated expression in modle group. Compared with the modle group,Per1 m RNA、Cry1 m RNA、Clockm RNA、Bmall m RNA and Rev-erbam RNA showed a up-regulated expression in melatonin group; Per1 m RNA 、 Cry1 m RNA、Clockm RNA、Bmall m RNA、Rev-erba m RNA showed a up-regulated expression in high dose group; Per1 m RNA 、 Cry1 m RNA 、 Clockm RNA showed a up-regulated expression in low dose group.5. The expression of melatonin receptor of hypothalamus in RealTime-PCR test showed that,compared with the control group, MT1 m RNA、MT2m RNA showed a down-regulated expression in modle group.Comparedwith the modle group, MT1 m RNA showed a down-regulated expression in melatonin group, while MT2 m RNA showed a up-regulated expression; MT1 m RNA 、 MT2 m RNA showed a up-regulated expression in high dose group; MT1 m RNA showed a up-regulated expression in melatonin group,while MT2 m RNA showed a down-regulated expression.Western Blot test result showed that, compared with the control group, Per1、Bmall、Clock、MT1and MT2 protein level of hippocampus decreased in model group(P<0.05,P<0.01). Compared with the model group, Per1、Bmall、Clock protein level of hippocampus increased significantly in melatonin group(P < 0.01,P < 0.05); Per1 、 Bmall 、 Clock 、 MT1 and MT2 protein level ofhypothalamus increased in high dose group(P < 0.05); Per1 protein level ofhypothalamus increased in low dose group(P < 0.05). Compared with the melatonin group, MT1 and MT2 protein level increased significantly in high dose group(P<0.01); Per1、Bmall、Clock protein level decreased significantly in low dose group(P<0.05), while MT2 protein level increased(P<0.05).6. Compared with the control group, the s APPαcontent of hippocampus reduced in model group all day long(P < 0.01). Compared with the model group, the s APPαcontent of hippocampus increased significantly in melatonin group and high dose group in daytime(P<0.01), increased significantly inlow dose group at night(P < 0.05), while increased significantly in melatonin group 、 high dose group and low dose group all day long( P<0.05,P<0.01).Compared with the melatonin group, the s APPα content of hippocampus increased significantly inhigh dose group in daytime(P<0.05),while increased significantly in low dose group at night(P<0.05). The s APPαcontent of hippocampus showed significant difference indaytime and nightime in both melatonin group and high dose group(P<0.05, P<0.01). Comparedwith the control group, Aβ1-40 and Aβ1-42 protein level of hippocampus increased significantly in model group(P<0.05, P<0.01). Aβ1-40 and Aβ1-42 protein level of hippocampus reduced in melatonin group、high dose group and low dose group compared with the model group( P<0.05, P<0.01).Compared with the melatonin group, Aβ1-40 and Aβ1-42 protein level of hippocampus reduced in high dose group and low dose group( P<0.05,P<0.01).Conclusion1. The theory of TCM contains in-depth understanding of circadian rhythm based on the opinion such as culturecorrespondence between man and universe,change of Yin-yang,supersedure of Wuxing, conversion of Yingwei.The theory of circadian rhythm is widely used indiagnose, treetment and prognosis in TCM.2. AD and circadian rhythm are closely related, they are reciprocal causation and promote each other.Circadian rhythm disordersmay show in patients with AD, while circadian rhythm disordersmay be lead to a risk factor for Alzheimer disease, and can accelerate the pathological process of AD.3. Shenghuitang can improve the learning and memory ability of APP/PS1 double transgenic mice.4. Shenghuitang can redress circadian rhythm disorders of APP/PS1 double transgenic mice.5. The effect of improving the APP/PS1 double transgenic mice learning memory ability and redressing circadian rhythm disordersof Shenghuitang may be related to regulate circadian clock genes, melatonin receptor expression and APP metabolic pathway and so on.