| Objective: Based on the previous research,this paper intends to start from the theoretical and experimental aspects.In the theoretical part,we will review the understanding,physiological basis and regulatory mechanism of light effects on circadian rhythm in traditional Chinese and Western medicine,as well as the important role of eyes in the generation and maintenance of circadian rhythm,so as to lay the foundation for later experimental research.In the experimental part,the light-mediated circadian rhythm was used as the starting point to observe the correlation between light and zebrafish growth,establish the light sleep deprivation model of zebrafish,and select Anmeidan(AMD),the classical representative of restoring circadian rhythm,to analyze the effect of MAPK / MNK / e IF4 E pathway related genes and proteins at different time points of zebrafish,so as to explore the mechanism of intervention treatment of insomnia and recovery of circadian rhythm disorder,so as to provide reference for the development of sleep and circadian rhythm research of traditional Chinese medicine and the prevention and treatment of clinical insomnia.Methods:Theoretical discussion: using the methods of classical combing,literature retrieval and theoretical research,systematically collect and sort out the literature on circadian rhythm for analysis and induction,condense its theoretical connotation,physiological basis,manifestation and clinical application,and take light as the starting point,analyze the influence of light on circadian rhythm in the external environment from the eye,and excavate the thinking of this theory and its modernity.On this basis,we select AMD,the famous prescription of Qing Dynasty,to provide theoretical guidance for the development of experimental research,by searching and analyzing its applicable syndromes,compatibility,clinical application.Experimental study: 1-9dpf wild-type AB strain zebrafish was selected.The first experiment was the effect of light on the circadian rhythm of zebrafish and the establishment of sleep deprivation model.The morphological differences and MAPK / MNK / e IF4 E pathway m RNA levels of 1-5dpf in different light and dark environments were observed,and the circadian rhythm of 3-9dpf zebrafish under continuous light stimulation was analyzed to establish the zebrafish sleep deprivation model.The second experiment was the effect of amiodan on the circadian rhythm of zebrafish Behavioral effects of zebrafish sleep deprivation model: behavioral analysis of the effects of light-mediated 5dpf zebrafish SD model,as well as the behavioral effects of AMD and its components and single drugs on zebrafish sleep deprivation model.The third experiment is the effect of AMD on the MAPK / MNK / e IF4 E pathway of zebrafish SD model: the effect of AMD on the m RNA level of MAPK/MNK/eIF4 E pathway of zebrafish SD model was studied by PT-PCR,ISH and if methods,and the expression of related genes and proteins in brain was observed.Results: Theoretical discussion: circadian rhythm is a cycle of biology,physiology and behavior regulated by suprachiasmatic nucleus in anterior hypothalamus,and it is a manifestation of organism adapting to nature.Light,as an important timing factor,can activate the rhythmic oscillator and regulate the changes of systemic rhythm.The mechanism of this action is studied in western medicine from the micro perspective of the biological clock receiving and input pathway,and in traditional Chinese medicine from the corresponding macro perspective of heaven and man.Although the starting point of the understanding of Chinese and Western medicine is different,both of them can not be separated from the eye,which is an important organ for receiving light signals.In traditional Chinese medicine,the eye and the life gate are white and black,transmitting the brain and the heart,and then the channels and veins headed by the foot sun act on them The theory that the whole body receives external signals from the retina to the suprachiasmatic nucleus and outputs circadian rhythms through the output pathway coincides with that of modern medicine.This point of view can provide inspiration and ideas for further research on the correlation between light and rhythm,and guide the prevention and treatment of clinical circadian disorders.Experimental study: Experiment 1:(1)the development of normal 14:10h light dark cycle zebrafish was significantly faster than that of DD group in 24 h full dark environment,while that of DD group was significantly later than that of LD group from the beginning of cell cycle,followed by primordial organogenesis,body axis extension,pigmentation,embryo hatching,movement behavior,yolk consumption and foraging.The results showed that light had an effect on the growth and development of zebrafish.(2)With the increase of time,mapk3,mknk1,mknk2 a,per1a and per2 increased gradually,while mapk1,eif4 ea and eif4e1 b decreased gradually.Compared with DD group,the m RNA levels of MAPK / MNK / e IF4 E pathway genes in LD group were generally increased,while the m RNA levels of DD increased gradually from the third day.It was proved that the light environment can promote the development of zebrafish,and the light can affect the related genes of MAPK / MNK / e IF4 E pathway.(3)The zeitgeber time(ZT)began to appear circadian rhythm disorder at about 30 h,the resting state was reduced,and the rhythm cycle was fair,which was manifested as the fragmented movement with the increase of small movement distance and the decrease of time length.This situation lasted for about 20 h,then the resting state was increased,the small movement was decreased,the large movement was increased,and gradually adapted to the environment.The rhythm recovered at about zt100,but the time phase shifted backward,and the time of rhythm cycle was prolonged until the next resting state appeared(ZT150).Experiment 2:(1)according to the tracking map of zebrafish,the movement track of juveniles of zebrafish is mainly middle motion.Compared with the blank group,the other groups increased their distance and coverage.After the treatment,compared with the model group,the activity increased and the large exercise increased significantly.The quantitative results showed that compared with the blank group,the total rest time,continuous long-time rest and the number of rest rounds in the zt12-24 time period of the model group decreased;the total activity and the activity in the zt6-18 time period increased.After administration,ADMM and ADMH increased the dormancy of zt12-24,but ADML decreased gradually.(2)In the low dose group of AMD,Zaoren has the excitatory effect,Maidong has the characteristics of daytime excitatory and night sedative effect,and the rest groups have the sedative effect.In the middle dose group,the resting state increased during zt0-12,while the small movement and large movement decreased,while in the zt12-24 period,the resting state decreased and the movement increased,especially in the group of removing Fu Shen.In the high-dose group,the rest decreased and the movement increased,and the small movement increased,especially in the zt12-24 interval.(3)In the single drug experiment,there were intolerances in ginseng group,angelica group,Danshen group,Shichangpu group,Schisandra group and liquorice group.Compared with SD model,the total distance and duration of static state of Fuzhen,middle dose of ginseng,high dose of Ophiopogon japonicus and high dose of Angelica zt0-18 increased,while the distance and duration of exercise decreased;those of Glycyrrhiza low dose group also had the same phenomenon,but appeared in zt12-24 period;the distance and duration of rest state of other groups decreased,and the distance and duration of exercise state increased mainly.Experiment 3:(1)the related genes of MAPK / MNK / e IF4 E pathway in normal zebrafish showed rhythmic changes.The expression of MAPK gene,per gene and eif4e1 b in SD model was down regulated,but there was no statistical difference.Compared with the model group,the Chinese medicine group can up regulate the MAPK / MNK / e IF4 E pathway related gene expression in 10 points and 16 points,especially mapk3,mapk1,mknk2 a,eif4e1b and per2,which is similar to the up regulated gene and time in the control group.(2)Ish experiment showed that the expression of pathway related genes in the brain also had obvious circadian characteristics,mainly covering the eyes,telencephalon,diencephalon and midbrain.The expression of each gene was higher than that in the morning,and then decreased gradually.The expression of each gene was slightly up-regulated after light exposure,and the difference in each time period was reduced.After drug intervention,it can reduce the level of up regulation and restore the phenomenon of disordered expression.(3)If indicated that mapk3,e IF4 E and per2 were mainly expressed in eyes,telencephalon and diencephalon.Compared with the control group,the expression of mapk3,p-ERK1 and e IF4 E decreased in the model group,while the expression of per2 increased.Compared with the model group,MT decreased mapk3 and e IF4 E,and increased the expression of per2.The expression of four pathway related proteins could be up-regulated in the low-dose and high-dose groups of AMD,but there was no significant difference in statistical fluorescence intensity.Conclusion: 1.Light is the most important timing factor of circadian rhythm.A large number of circadian rhythms related to life and health are recorded in traditional Chinese medicine represented by Inner Canon of Huangdi.Based on the corresponding premise of heaven and man,the material basis is Yuanqi-Yin Yang-Five zang organs sprit,and the eye is the opening and closing hub,observing the change of sun and moon,day and night,feeling the call of heaven and earth,and relying on Yuanqi-Yin Yang-Five zang organs sprit,to coordinate and use each other to ensure the normal operation of human body rhythm.So as to adapt to the constant changes of natural and social development laws.However,with the change of modern lighting conditions and the development of science and technology,the lighting time in people's living environment is no longer only dependent on the rise and fall of the sun.The widespread light pollution caused by artificial light sources at night has led to a series of diseases such as circadian rhythm disorder and sleep disorder.Therefore,it is still an important subject in time medicine and time biology in the future to deeply understand the physiological and pathological significance of circadian rhythm to human body and to explore the influence and mechanism of exogenous factors such as light on circadian rhythm in all aspects of human body.2.As an important time factor,light is one of the bases of circadian rhythm,and it is also an important factor of insomnia.As shown in Experiment 1,the development of normal 14:10h LD light dark cycle zebrafish was significantly faster than that of DD group(Fig.1),and the m RNA level of MAPK / MNK / e IF4 E pathway mediated by light also changed accordingly(Fig.2).Under the continuous light interference,the circadian rhythm changed from disorder to recovery,but the time phase shifted backward,and the rhythm cycle prolonged.In Experiment 2 and Experiment 3,it was found that the model group had more rest in the daytime and less rest at night,and its activity rhythm was lower than that of the normal zebrafish.Amd could recover the circadian rhythm,which was close to the blank group.The excitatory effect of low dose group was relatively obvious,the sedative effect of high dose group was more obvious,and the middle dose group was between them,which increased the activity in the daytime and rest at night The rest time is prolonged,and the key is to restore the rhythm(Figure 6-8).The expression of MAPK / MNK / e IF4 E pathway related genes in the head mainly covers the eyes,telencephalon,diencephalon and midbrain.Luciferase is highly expressed in the retina,pineal gland,thalamus,preoptic area,retrooptic area and most of the midbrain.The inhibition effect of continuous light stimulation is more obvious.After drug intervention,the gene and protein levels of the pathway can be up-regulated,and the results are consistent with the behavior(Figure 9-11).It is suggested that the effect of light on the circadian rhythm of zebrafish is related to the intensity,duration and cycle of light.AMD can restore the circadian rhythm of SD model of zebrafish mediated by light,and the effect of improving sleep is in direct proportion to the dose.The mechanism may be related to the regulation of MAPK / MNK / e IF4 E pathway.3.The juveniles in 9dpf of zebrafish,a model organism,were selected as the research objects,and the effects of the drugs on them were observed by soaking.On the third day,the viscera of juveniles began to develop and mature gradually,5dpf began to look for food,and 9dpf could obtain nutr ients from their yolk sac.Therefore,it can be inferred that the penetration pathway of drugs in juveniles should be skin,mucous membrane,gill and digestive system mainly composed of mouth,esophagus and gastrointestinal tract,which is in line with the administration method of traditional Chinese medicine and the theory of viscera centered action.Light stimulation as a disease model is also more in line with the clinical factors of insomnia.Therefore,the hypothesis is put forward that whether the function of traditional Chinese medicine can play a role in treating diseases by changing the internal and external environment of human body and adjusting the homeostasis of internal and external environment.The primary screening method of drug MTC in the experiment is to take the drug in the proportion range of body weight and body fluid,and the body fluid accounts for about 1 / 10 of the body weight,which can be regarded as the proportion of body weight is that the body is immersed in the drug solution,similar to the method of external application of traditional Chinese medicine;the proportion of body fluid is that the viscera are immersed in the drug solution,similar to the method of oral internal application of traditional Chinese medicine.It was fo und that the MTC of the formula was not suitable for all the single drugs in the formula.When the concentration of Panax ginseng,angelica,Salvia miltiorrhiza,Acorus tatarinowii,Schisandra,liquorice and other drugs reached amd MTC(4.00 mg · m L-1),zebrafish died,and even some 1 / 3 MTC of some drugs also died(Table 2).In the previous experiment,it was found that the MTC of fried jujube kernel reached 35 mg · m L-1.It was found that the sedative effect of low dose group increased,but the sedative effect of increased dose decreased,and the effect of recovery of circadian rhythm was obvious.The above results suggest that the drug compatibility can reduce toxicity and increase efficiency,and also give us enlightenment on whether the way of drug action can change the external environment and humoral environment of zebrafish through adjusting p H value,so as to correct the disorder and restore the balance of yin and Yang.At the same time,when the dosage is not appropriate or the compatibility is not appropriate,it will also have an impact on the body,even the toxic reaction of the drug.If the light affects the body by changing the external environment,then whether the traditional Chinese medicine affects the body by changing the internal environment.This problem is worthy of further study. |
PDF Full Text Request