| Objective:Based on the previous research,this paper intends to study the theoretical connotation of life cycle and the effect and mechanism of anmei Dan on sleep deprivation in rats with different ages.The theoretical part will mainly discuss the origin,manifestation and internal mechanism of life cycle formation from huangdi neijing(hereinafter referred to as neijing),and study its guiding significance for modern clinical practice on this basis.Experiment part based on the early age sleep deprivation model rats,classic square An Mei Dan intervention,by observing the general situation,the learning and memory,Orexin and mediated gene sleep rhythm signal pathway OXA/CREB/PER1,to study An Mei Dan's function and mechanism on sleep deprivation rats,in the hope of preliminary to clarify the treatment's mechanism of insomnia and provide new theoretical and experimental basis for clinical treatment of insomnia.Methods:Theoretical discussion: Collecting and sort out literatures related to the life cycle of neijing systematically,exploring the elaboration of this theory by other doctors,so as to enrich and develop the life cycle theory of traditional Chinese medicine.To search the records of An Mei Dan in ancient literature,construction,intragastric administration respectively for a week,then compared the different months blank group,model group,estazolam,An Mei Dan of sleep deprivation model general situation,the learning and memory,gene protein expression sleep rhythm and its Orexin system?OXA/ CREB PER1 signaling pathways,to evaluate An Mei Dan's effect of sleep deprivation model and its mechanism preliminarily.Results:1.Before the intraperitoneal injection of PCPA,the hair of each group was shiny,the skin was clean,the lips and nails were pale pink,and the daytime spirit was weaker than the nighttime.During the day,it was easy to sleep,and it was good at night.After intraperitoneal injection of PCPA,the rats showed excitement and hyperactivity.Some rats had rough hair,dull skin,and white or yellowish claws.On the third day of intraperitoneal injection,the circadian rhythm was obviously destroyed,and anxiety and agitation occurred during the day and night.Loss of appetite and apathy;most rats have no eyes,no sorrow,lips,nails are white,hair is fluffy and messy,slow response,a few rats are easy to irritate,and the tail lips see blood stains.The blank group was in good condition,free from activities,and had normal sleep and diet.There was no significant difference in body weight between the groups before and after model establishment(P>0.05),but the body weight of each group was reduced after modeling,and there was no difference between the model group and the blank group(P>0.05).The 24-hour spontaneous activity time and Experimental research:Choosing 3/6/9 months male SD rats respectively,using the random number table to divided them into blank group?model group?estazolam?An Mei Dan group,except the blank group,all the rats were using the PCPA abdominal cavity injection and superposition multi-platform deprived of water environment sleep deprivation model analyzing its prescription analysis and study its modern clinical applicationvalue.difference in the activity distance between the 9-month-old rats and the model group(p<0.01).),there was a difference in activity time(P<0.05).2.The 3/6/9-month-old rat Morris water maze-positioning navigation experiment showed that the platform latency of the 3-month-old rats in the blank group <6 month-old rats on the platform latency>9-month-old rats on the platform latency,3 The total swimming distance of the rats of the age of the moon>the total swimming distance of the 6-month-old rats>the total swimming distance of the 9-month-old rats.In the 3 month old rats and the blank group,the platform latency and total swimming distance were significantly prolonged in each group(P<0.01).Compared with the model group,the platform latency was significantly different in the blank group(P<0.01),estazolam There was no significant difference in the latency of the platform between the group and the Anzhendan group(P>0.05),but the total distance of swimming was significantly shorter(P<0.01),but it was longer than the blank group.Compared with the blank group,the platform latency and total swimming distance of the 6-month-old rats were significantly longer(P<0.01).Compared with the model group,the platform latency was significantly shortened in the blank group(P<0.01),estazolam group There was no significant difference(P>0.05),and the latency of the platform in the Anzhendan group was shortened(P<0.05).The total distance of swimming in each group was significantly shorter(P<0.01),but it was still longer than the blank group.In the 9-month rat and the blank group,the platform latency was significantly reduced in the model group and the estazolam group(P<0.01),and the total swimming distance in the model group,the had different activity distance and activity time(P<0.05).There was a significant difference in the distance between the two groups in the 6-month-old rats and the model group(p<0.01).There was a significant activity distance of the 3/6/9-month-old rat model group were higher than those of the blank group;the 3-month-old rat blank group and the model group distance of the swimming group in the blank group,estazolam group and Anzhendan group was significantly decreased(P<0.05).<0.01).In addition,in terms of the latency of the upper platform,there was no significant difference between the 3/6/9 month old rat estazolam group and the Anzhendan group(P>0.05);in terms of total swimming distance,6 months old There was significant difference between the rats in the Anzhendan group and the estazolam group(P<0.01).However,the latency of the upper platform and the total swimming distance were the shortest after the rats in the 9-month-old rats.The Morris water maze-space exploration experiment of 3/6/9 month old rats showed that the number of rats crossing the platform in the blank group of 3 months old > 6 months old rats crossing the platform times > 9 months old rats crossing the platform times,3 months old Rat quadrant activity time > 6-month-old rat quadrant activity time > 9-month-old rat quadrant activity time.Compared with the blank group,the age of the three-month-old rats in the model group decreased and the station quadrant time decreased significantly(P<0.01),the estazolam group was meaningful(P<0.05),and the Anzhendan group crossed the platform.There was no significant difference in the number of times(P>0.05),and the time of quadrant activity was significant(P<0.01).Compared with the model group,the number of crossing stations and the station quadrant time of the blank group and the estazolam group increased significantly(P<0.01),and the number of crossing stations of the Anzhendan group was significant(P<0.01).There was a difference in activity time(P<0.05).Compared with the blank group,the number of crossings and the platform activity time of the model group decreased and significantly decreased(P<0.01).There was no significant estazolam group,and the Anzhendan group was significantly shortened.(P<0.01),but still longer than the blank group.Compared with the model group,the latency of the platform was significantly reduced in the blank group(P<0.01),and decreased in the Anzhendan group(P<0.05).The total significant difference(P<0.01).Compared with the model group,the number of crossing stations and the station activity time of the blank group were significantly increased(P<0.01),and the number of crossing the platform of the western medicine group was decreased(P<0.05).There was no significant difference in the Chinese medicine group(P>0.05).Compared with the blank group,the number of crossings and the platform activity time of the model group decreased and significantly decreased(P<0.01).There was no significant difference between the western medicine group and the Chinese medicine group crossing the platform(P>0.05).Activity time decreased and there was a significant difference(P<0.01).Compared with the model group,the number of crossings of the blank group and the activity time of the platform were significantly increased(P<0.01),the number of crossings of the western medicine group and the activity time of the quadrant decreased(P<0.05),and the number of crossing the platform of the Chinese medicine group decreased significantly(P< 0.01),there was no significant difference in quadrant activity time(P>0.05).During the whole process,the model group was rarely able to cross the platform.A small number of rats in the Chinese medicine group and the Anzhendan group could pass through the platform.The data of each group were statistically significant.In addition,there was no significant difference between the 3/6/9 month old rat estazolam group and the Anzhendan group in terms of the number of crossing stations(P>0.05);in terms of quadrant activity time,3/6 months There was no significant difference between the estazolam group and the Anzhendan group(P>0.05),but there was a difference between the 9-month-old rat estazolam group and the Anzhendan group(P<0.05).The number of crossing stations and the quadrant activity time of the Anzhendan group of the month-old rats were higher than that of the 6th and 9th month.difference between the western medicine group and the Chinese medicine group crossing the platform(P>0.05).Activity time decreased and there was a3.3/6/9 rats hypothalamus area OX Immuneofluorescence results showed that content of OX blank group were lower,the model group compared with blank group was significantly increased,especially in the 9-month old rat,whose increase was most pronounced in the rat OX content,each OX content also decreased to different degrees after the treatment,macroscopic observation 3 months estazolam and An Mei Dan group,no significant differences of 6 rats estazolam group improved significantly in the An Mei Dan group,group of 9 rats An Mei Dan improve the estazolam group obviously.4.OXA and OXB ELISA results in the hypothalamus of rats aged 3/6/9 months showed that OXA and OXB in the 3/6/9 months old SD rat model group were higher than those in the blank group(P < 0.01),consistent with the circadian rhythm and immunofluorescence results described above,indicating that PCPA intraperitoneal injection combined with multi-platform water environment deprivation can cause sleep rhythm disorder and insomnia in rats.Compared with the model group,the content of the two groups can be reduced to different degrees in the anmei group and eszolam group(P < 0.05),and the improvement effect of the anmei group and eszolam group in 3/9 months old rats is significantly different(P < 0.01),the content of OXA and OXB in the anmei group is significantly lower than that in the eszolam group.However,compared with the blank group,the content of An Mei Dan group and eszolam group was higher than that of the normal group(P > 0.05).According to the month age comparison,the OXA and OXB content of 3-month old rats decreased the most significantly after administration,but only the OXA content of 6-month old SD rats in the An Mei Dan group and eszolam group showed significant difference(P < 0.05),and the OXA and OXB expression between the other groups showed no significant difference(P > 0.05).5.RT-PCR results of OXA/CREB/PER1 signaling pathway in the hypothalamic region of 3/6/9 month old rats showed:Compared with the blank group: OXA m RNA expression was up-regulated in the model group,OXA m RNA was significantly increased in 3 months old SD rats(P<0.01),OXA m RNA was increased in 6-month-old SD rats(P>0.05),9-month-old SD OXA m RNA was increased in rats(P<0.05);OXA m RNA was increased in ezetren group 3/6 months old SD rats(P<0.05),and OXA m RNA was significantly increased in 9-month-old SD rats(P<0.01).OXA m RNA was increased in SD rats of 3 months old in Anzhendan group(P<0.05),and OXA m RNA was significantly increased in SD rats aged 6/9 months(P<0.01).Compared with the blank group: CREB m RNA expression was up-regulated in the model group,CREB m RNA was increased in SD rats at 3 months of age(P<0.05),and CREB m RNA was significantly increased in SD rats at 6/9 months(P<0.01).The content of CREB m RNA in SD rats of 3/6/9 months old was significantly decreased by azolam group(P<0.01).The content of CREB m RNA in SD rats of 3/6/9 months old was decreased in Andandan group(P<0.05).Compared with the blank group,the expression of PER1 m RNA was down-regulated in the model group,and PER1 m RNA was significantly down-regulated in 3/6-month-old SD rats(P<0.01),and PER1 m RNA was down-regulated in 9-month-old SD rats(P<0.05);estazolam PER1 m RNA was significantly down-regulated in SD rats at 3 months of age(P<0.01),PER1 m RNA was down-regulated in 6/9-month-old SD rats(P<0.05).PER1 m RNA in SD rats aged 3/6/9 months in Anzhendan group Significantly down-regulated(P <0.01).Compared with the model group,the expressions of OXA m RNA and CREB m RNA in the 3/6/9 month blank group were significantly decreased(P<0.01),and the expression of PER1 m RNA was significantly increased(P<0.01).Both estazolam and ampoule were able to reduce OXA m RNA levels in SD rats at 3 months of age(P<0.05),and significantly reduce OXA m RNA levels in SD rats aged 6/9 months(P<0.01).Compared with the model group,the estazolam group can reduce the expression of CREB m RNA in SD rats of 3 months old(P<0.05),which can significantly reduce the expression of CREB m RNA in SD rats of 6 months old(P<0.01),which can reduce the age of 9 months.CREB m RNA expression in SD rats(P>0.05).The Anzhendan group can reduce the expression of CREB m RNA in SD rats aged 3/6 months(P<0.05),and decrease the expression of CREB m RNA in SD rats of 9 months old(P>0.05).Compared with the model group,the expression of PER1 m RNA was up-regulated in the blank group(P<0.01).The estrazole group and the sedative group could significantly improve the expression of PER1 m RNA in SD rats of 3 months old(P<0.01),which could improve 6/9 PER1 m RNA expression in SD rats of the age of age(P<0.05).6.Western Blot results of OXA/CREB/PER1 signaling pathway in hypothalamus showed that OXA and CREB protein expression were up-regulated and PER1 protein expression was down-regulated in hypothalamus tissues of the model group compared with the blank group(P < 0.01).Eszolam and ammetan can reduce OXA and CREB content in 3-month old SD rats(P > 0.05)and increase PER1 content(P < 0.05).Estazolam decreased OXA content in 6-month old SD rats(P < 0.05).OXA content in 9-month-old SD rats was also decreased(P < 0.01).However,there was no statistically significant change in the expression of CREB and PER1 in SD rats aged 6/9 months(P > 0.05).The OXA content(P < 0.01),PER1 content(P < 0.01)and CREB content(P > 0.05)in 6-9 month old SD rats could be decreased in the An Mei Dan group.Compared with the model group,OXA and CREB protein expressions were down-regulated(P < 0.01)and up-regulated(P < 0.01)in eszolam group and omidan group of SD rats at the age of 3 months,and PER1 protein expression was up-regulated(P < 0.01).Estazolam decreased OXA and CREB content in 6-month-old SD rats(P > 0.05,P < 0.01),and increased PER1 content(P < 0.01).The OXA and CREB contents in 9-month-old SD rats were decreased(P < 0.01,P > 0.05),and the PER1 contents were increased(P < 0.01).Methadine can reduce OXA and CREB content(P > 0.05,P < 0.01)and increase PER1 content(P < 0.01)in 6-month-old SD rats.OXA and CREB contents in 9-month old SD rats were decreased(P < 0.01,P > 0.05)and PER1 contents were increased(P < 0.01).Conclusion:1.Nei Jing has a rich and systematic understanding of life cycle from the aspects of origin,manifestation,formation mechanism and clinical application.Which calls Life cycle as "birth,long,strong,old,have been",and the law is divided into "small young old" general rule,the general rule of "10 years of stage" and "female seven male eight" special laws,especially in such aspects as insomnia,forgetfulness,constitution has the thorough elaboration,and that its formation mechanism and elements,the gods,qi and blood,organs,modern,gender,etc.,and for clinical knowledge often change with syndrome differentiation,outside the department within the chuai with treatment,not disease prevention first in order to preserve one's health has important theory instruction value.Later generations of doctors further supplemented and innovated the life cycle theory on the basis of the rich understanding of "neijing",and formed the strategic thought of "full life cycle health" today,which fully reflected the high combination of original theoretical innovation of traditional Chinese medicine and modern inheritance of traditional Chinese medicine.2.The life cycle is an important influencing factor of insomnia,and age-related insomnia research has important clinical application value.PCPA intraperitoneal injection combined with multi-platform water environment deprivation is a relatively mature SD technology,which can be widely used in basic research on insomnia.Orexin is closely related to insomnia,SD can lead to circadian rhythm disorder,increased Orexin content and weight loss,and improving abnormal Orexin content may become a new target for insomnia prevention.3.An Mei Dan can improve 3/6/9 months SD rat model of learning and memory ability,reduce the 3/6/9 months SD rat model of Orexin levels,improve its 3/6/9 months SD model rat circadian rhythm disorders and the ability of learning and memory mechanism may be related to the regulation of Orexin and its mediated OXA/ CREB/PER1 signaling pathways,and preliminary point out that An Mei Dan effected for 9 months SD model. |
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