| Objective:The theoretical part systematically sorts out the historical origin,etiology,pathogenesis and treatment methods of insomnia,and focuses on the theoretical basis of the treatment of insomnia by cultivating the heart and calming the nerves.The experimental part takes sleep and circadian rhythm as the breakthrough point,and takes the representative of Peiyuan Yangxin Anshen as the intervention drug,and establishes a light sleep deprivation zebrafish model,mainly focusing on the effect of light on sleep,behavior,learning and memory ability,biological clock The effect of gene and tissue cell structure and the intervention effect of Anmedan on sleep deprived zebrafish were studied,in order to explain the effect of sleep deprivation on zebrafish and the mechanism of Anmeidan’s regulation on it,and to provide advices and methods for the prevention and treatment of insomnia by traditional Chinese medicine.Methods:Theoretical discussion: Mainly adopts literature retrieval and the methods of ancient books sorting,collecting literatures,insomnia,integrated ancient and modern subject,system combing wakefulness,analysis of disease,pathogenesis and treatment methods of analysis of traditional Chinese medicine’s understanding of "qi",from the concept of "qi",the source,distribution,function and the close relationship of the zang-fu organs,in-depth,To explore the relationship between "qi" and sleep,and further put forward that "qi deficiency,the heart does not contain the spirit" is the key pathogenesis of insomnia,analyze its rationality and scientific,to provide theoretical support for the choice of Anmei Dan treatment of insomnia,and enrich the theoretical basis of TCM prevention and treatment of insomnia.Focusing on the relationship between light and sleep,the influence of light on circadian rhythm was analyzed to provide theoretical guidance and access selection basis for subsequent experimental research.Experimental study: Healthy 4-month-old male zebrafish of wild type AB line were selected as experimental animals,and feeding conditions were carried out according to zebrafish feeding specifications.Experiment 1 was the establishment and evaluation of zebrafish sleep deprivation model,observing the effects of light on zebrafish motor behavior,learning and memory ability,clock genes and brain tissue structure,exploring the best conditions for light to simulate sleep deprivation in zebrafish,and establishing zebrafish sleep deprivation model.The second experiment was to determine the maximum tolerance concentration of zebrafish drugs,mainly to determine the safe dose range of Anmeidan and melatonin.In experiment 3,the effect of Anmeidan on behavior and learning and memory ability of sleep-deprived zebrafish was studied.Zebrafish behavior analysis equipment was used to record and analyze the motor behavior and learning and memory ability of zebrafish in each group,and the intervention effect of Anmeidan was observed.In experiment 4,the effect of Anmeidan on brain structure changes of sleep-deprived zebrafish was studied.The brain tissue sections of zebrafish were photographed by using ultraelectron microscopy to observe the intervention effect of Anmeidan.Experiment 5 was about the effect of Anmeidan on clock genes of sleep-deprived zebrafish.RT-PCR was used to detect clock genes in brain and liver tissues of zebrafish in each group,compare their differences,analyze their expression level and trend change in a day,and explore the intervention effect of Anmeidan on them.In experiment 6,the effect of Anmeidan on Alk-Smad3-Dec1 clock signaling pathway of sleep-deprived zebrafish was studied.The transcription level of pathway genes was detected by RT-PCR,the protein translation level of pathway was detected by Western Blot,and the expression of pathway proteins was detected by immunofluorescence method.Results:Theoretical discussion: deficiency of qi and absence of mind are the key pathogenesis of insomnia,and the method of cultivating and calming the mind is an important method to prevent and cure insomnia.Experimental study:Experiment 1:（1）Compared with Control group（CK group）,the resting time of zebrafish in Sleep deprivation for 1 day（SD1）,SD2 and SD3 groups increased,while the resting time of zebrafish in SD4-SD7 groups did not change significantly.（2）The learning and memory ability of zebrafish in CK group was better than that in SD group.Compared with SD group,the learning and memory ability of zebrafish in SD1 group was better than that in SD2 and SD3 groups,and the learning and memory ability of zebrafish in SD2 group was better than that in SD3 group.（3）The m RNA expression levels of per1 a,per2,bmal1 and cry1 b in the brain of ZEbrafish in SD3 group were significantly different from those in CK group.（4）According to transmission electron microscopy,CK group of zebrafish brain tissue structure is complete,the nucleus structure integrity,mitochondrial rich and complete structure,morphological rules,plate laminated crest,did not see obvious fracture,SD2 and SD3 is recommended for zebrafish brain tissue necrosis occurs,microscopically the nucleus collapse,border crack even dissolve,chromatin pyknosis,Mitochondria swelling,even a large number of vacuoles,cristae fracture or most of the disappearance.Experiment 2: The MTC of Anmeidan was 0.08 mg/ ml,and The MTC of melatonin was 0.4 mg/ ml.Experiment 3 :（1）the total resting time of zebrafish in SD group was decreased compared with CK group,and the total resting time of zebrafish in AMD-H group was increased compared with SD group.（2）The resting time of zebrafish in SD group was significantly reduced compared with that in CK group.We divided one day and night into four time periods,and the results showed that zebrafish in each group had different movement at different time periods,and the effectiveness of intervention drugs also had different time periods.（3）The latent time of zebrafish finding TA region in CK group was continuously reduced,and the continuous illumination SD significantly affected the learning and memory ability of zebrafish,and this injury was recovered to varying degrees after the intervention of Anmeidan.（4）Zebrafish in all groups swam at the bottom of the fish tank when they were put into the new fish tank.The swimming time of zebrafish in SD group was longer than that in CK,and the anxiety behavior of zebrafish was improved after the intervention of Anmeidan.（5）Zebrafish in all groups showed obvious aggressive behavior in mirror mirror experiment,but zebrafish in SD group hit mirror more frequently than that in CK group.After drug intervention,zebrafish in SD group did not improve this symptom.Experiment 4: Compared with CK group,zebrafish in SD group had abnormal brain tissue cell structure,such as collapse of nucleus,rupture or even dissolution of nuclear membrane,chromatin shrinkage,swelling of mitochondria,and even a large number of vacuoles and water like changes,crest fracture or most disappearance,and forming dense lamellar myelin like structure.Give Anmeidan and melatonin after the intervention,zebrafish brain cells recovered structure,nuclear membrane is complete,the homogenization of chromatin,mitochondria vacuoles or swelling,alleviate some crest rendering performance,complete with AMD-H group zebrafish cellular structure improvement is most obvious,almost close to CK group status,and AMD-L and MT group is relatively poor.Experiment 5 :（1）in the brain tissues of zebrafish in CK group,the m RNA expressions of clock1 a,cry1a and per2 showed a certain rhythm,they showed an upward trend in the light period and a downward trend in the dark period;The m RNA expression of per1 a started from time point ZT06 and showed a trend of decline-upgrade-decline,and its peak appeared at time point ZT24.In zebrafish liver tissue of CK group,clock1 a m RNA expression showed a downward trend in light period and an upward trend in dark period.The expression of cry1 a m RNA decreased in the light phase,but did not change significantly in the dark phase.The m RNA expressions of per1 a and per2 both started from the time point of ZT06 and showed a down-up-down,and their peak appeared at the time point of ZT18.The expressions of per1 a,per2,clock1 a and cry1 a m RNA in the brain tissue and liver of zebrafish in SD group were up-regulated at each time node,but the expression trend was consistent with that of the CK group.However,the relative expression levels of per1 a m RNA in both the brain tissue and liver of zebrafish not only changed.The phase also changed in advance,and the above changes were improved in different degrees after Anmeidan intervention.（2）The relative expression level of biological clock genes in liver was higher than that in brain tissue,and some biological clock genes showed rhythm disorder.Experiment 6 :（1）the m RNA expressions of alk5,smad3,smad4,dec1 and dbp in CK group showed a certain rhythm.The m RNA expressions of the five genes showed a gradually increasing trend in the dark period,while the m RNA expression of cry1 a showed a gradually decreasing trend in the dark period.The m RNA expression of alk5,smad4,dec1,dbp and cry1 a increased gradually during photic phase,while the m RNA expression of smad3 decreased gradually during photic phase.The m RNA expressions of alk5,smad3,smad4,dec1,and dbp all showed a downward trend from light stage to dark stage.The rhythm of SD group was relatively chaotic,and the trend of each gene changed during the light and dark period.The m RNA expression levels of alk5,smad3,smad4,dec1,dbp and cry1 a at each time point were higher than those of CK group,and the rhythm of zebrafish was improved after the intervention of Anmeidan and melatonin.（2）The protein expressions of Alk5,p-Smad3 and Dec1 in CK group decreased gradually in light phase and increased gradually in dark phase;The rhythm of Alk5,p-Smad3 and Dec1 protein expression was disturbed in SD group,and the overall expression level of Alk5,p-Smad3 and Dec1 protein increased at each time point,and the expression of each indicator was also different at our fixed 4time points.The above situation was recovered without any degree after the intervention of Anmeidan and melatonin.（3）Compared with CK group,the fluorescence intensity of Alk5 and p-Smad3 in SD group was significantly enhanced at each time point.The fluorescence intensity of Dec1 was significantly increased at ZT12 and ZT18 day time points in SD group,and decreased at ZT6 and ZT24 night time points.Compared with SD group,Alk5 and p-Smad3 fluorescence intensity of zebrafish treated with Anmeidan decreased at different time points,and there was no dose dependence,indicating that Anmeidan might inhibit the expression of Alk5,but it had nothing to do with dose.In the same brain tissue section,p-Smad3 and Dec1 were labeled with two-color,Dec1 protein was labeled with red fluorescence,and p-Smad3 protein was labeled with green fluorescence.Compared with CK group,p-Smad3 and Dec1 significantly enhanced fluorescence intensity at each time point in SD group.Compared with SD group,p-Smad3 and Dec1 of Anmeidan and melatonin administration groups showed varying degrees of fluorescence intensity reduction at each time point,and was not dose-dependent on Anmeidan.Conclusion:（1）the huangdi neijing,Yin and Yang can shift as a starting point,on the basis of business health run,who established in camp as the main theory of Yin and Yang theory of sleep,the later development and inheritance of sleep theory,summed up the imbalance of Yin and Yang,the camp who disorder,the main pathogenesis of zang-fu organs and to wakefulness,dig its pathogenesis,all have a lack of strength,the heart is not hidden god mechanism in it,Yin and Yang are coordinated to a certain degree,ying and Wei operate in a proper way,and zang-fu activities are orderly,all of which need to be promoted and regulated by the vitality.Therefore,we further analyze the pathogenesis of insomnia from the whole point of view,and conclude that the deficiency of qi qi and the absence of god in the heart are the key pathogenesis of insomnia.Based on this,we establish peiyuan nourishing the mind and calming the spirit therapy as an important method for the treatment of insomnia,and its representative is Anmeidan.（2）The 4-month-old male zebrafish were exposed to light for 3 days.The results showed that the zebrafish showed obvious changes in movement time,behavior,learning and memory ability,biological clock gene and brain structure after light exposure.（3）Anmeidan can correct the reduction of resting time caused by continuous light exposure,and improve the motor behavior pattern,learning and memory ability,anxious behavior and aggressive behavior of sleep-deprived zebrafish.（4）Anmeidan has a recovery effect on the damaged brain tissue and cell structure of SD zebrafish,and this recovery effect is positively correlated with the dosage scale of Anmeidan.（5）The expression of the same clock gene was significantly different in the brain tissue and liver of zebrafish.After continuous illumination for 3 days,the expression of per1 a,per2,clock1 a and cry1 a m RNA in the brain tissue and liver of zebrafish were up-regulated at each time node,and the rhythm appeared disorder.The intervention of Anmeidan can be down-regulated and its rhythm can be restored.（6）Anmeidan can improve the protein expression level of Alk-Smad3-Dec1 pathway,which may improve zebrafish sleep and circadian rhythm through this pathway. |
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