Study On The Effect And Mechanism Of The An Mei Dan On Energy Metabolism In Sleep Deprived Rats From The Perspective Of "The Brain Is The House Of The Vital Energy"

Objective:Based on the team's previous research,this paper will focus on the mechanism of the effect of An Mei Dan(AMD)on sleep deprived(SD)rats from two levels:theoretical and experimental aspects.The theoretical part will be based on the view that"brain is the house of Yuan Shen"in Chinese medicine,from the understanding of brain,Yuan Shen,and the relationship between brain,Yuan Shen and sleep,and then propose our"Yuan Qi-Yin Yang-Wu Zang Shen"theoretical system of insomnia prevention and treatment.We will then propose our theoretical system of insomnia prevention and treatment of"Yuan Qi-Yin Yang-Wu Zang Shen"and elaborate its Chinese medical connotation.In the experimental part,we established the SD rat model and carried out the AMD on the mechanism on energy metabolism,including metabolic level,activity intensity,mitochondrial biological function,energy metabolic pathway proteins and the relationship between sleep and energy,to provide new ideas and methods for the treatment of sleep disorders in Chinese medicine.Methods:Theoretical discussion:Through literature search,collation of ancient texts,and systematic summary,we explored the connotation of the theory of"brain as the house of Yuan Shen",analyzed the understanding of"brain"and"Yuan Shen"in Chinese medicine,deeply analyzed their concepts,development and evolution,physiological functions,and derived connotations,explored the Chinese and Western medical understanding of the relationship between brain,Yuan Shen and sleep,and further proposed the insomnia prevention and treatment system of"Yuan Qi-Yin Yang-Wu Zang Shen",analyzed its rationality and scientificity,to establish the treatment method and prescriptions for cultivating Yuan Shen,and also provided support for enriching the theory of insomnia prevention in Chinese medicine and the application of the treatment method for cultivating Yuan Shen.Experimental study:Healthy Sprague Dawley male rats,body mass 220±10g,controlled temperature,humidity and light,sterilized food and bedding,etc.After 1 week of environmental adaptation,the rats were randomly divided into Ctrl,SD,AMD and MT groups.Gavage was administered once daily for 4 weeks.Modified multi-platform water environment method was used to prepare SD model.Body mass,food intake and exercise tolerance,blood glucose and lipid levels,glucose and lipid genes,ATP,Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase were detected in Experiment 1.In Experiment 2,the morphology and mitochondrial structure of hypothalamus were observed by HE staining and TEM,and the expression of TFAM,Nrf1 Drp1 and Mfn2 were detected by Western blotting(WB)and the targeting of the four proteins by immunofluorescence;the levels of SOD,MDA and GSH-Px were detected by ROS staining and ELISA.The expression and co-expression of adenosine receptors A1R and A2AR and neurotransmitter receptors mGluR5,PTGDR,and GABAA1?were detected by immunofluorescence in experiment 4.Experiment 3 was divided into the same groups as experiment 1,and each group was further divided into 4 subgroups(n=5):ZT0,ZT6,ZT12 and ZT18groups.The model establishment and drug administration were the same as in Experiment 1.Spontaneous activity was monitored by the Autonomous Activity Video Analysis System,AMPK/PGC-1?/Nrf-1 pathway and Per1,Cry1,Bmal1,Clock were detected by Real Time-q PCR,p-AMPK,PGC-1?,Nrf-1 were detected by WB,and rhythm was detected by immunohistochemistry gene expression.Results:Theoretical discussion:Chinese medicine has a very rich understanding of"the brain as the house of the vital gods",and has a more comprehensive understanding of the brain and the Yuan Shen,which is reflected in the spiritual,emotional and thinking activities,of which sleep belongs to the category.The connection between sleep and the brain occurs through the coordination of the five visceral yin and yang.Therefore,the insomnia prevention and treatment system of"Yuan Qi-Yin and Yang–Wu Zang Shen"is in line with the positioning of the relationship between sleep,brain and spirit in Chinese medicine,where Yuan Qi is the foundation,Yin and Yang is the platform,and Wu Zang Shen is the core.The treatment method of Peiyuan Anshen is an important method to prevent and treat insomnia.Therefore,our group has established the Peiyuan Anshen formula AMD and carried out the mechanism of AMD on SD rats to provide new ideas and methods to guide the clinic.Experimental studies:Experiment 1,(1)Body mass increased over time in all groups,significantly in the Ctrl group and slowly in the SD.Body mass decreased from day 7 in the SD group compared to the Ctrl group,and decreased significantly compared to the Ctrl group;body weight increased from day 14 in the AMD group compared to the SD group,and continued to increase at subsequent times.Ingestion showed an increase in total food intake in the SD group within 4 h.The amount of food eaten by SD rats in the first 2 h and the last 2 h was significantly increased compared with the Ctrl group;no statistical difference was seen in the amount of food eaten from 0-2 h and 2-4 h after the AMD intervention compared with the SD group.(2)The results of glycolipid metabolism showed that FBS,TG,TC and LDL-C levels were increased and HDL-C content was significantly decreased in the SD group compared with the Ctrl group;AMD decreased FBS,TG,TC and LDL-C levels and increased HDL-C concentration compared with the SD group.The mRNA results of glucose and lipid metabolism genes in liver showed that FAS and ACC1 expression was upregulated and PPAR?and GLUT4 expression was downregulated in SD rats compared with Ctrl group;AMD was able to downregulate FAS and ACC1 and upregulate PPAR?and GLUT4,indicating that AMD improved glucose and lipid metabolism was associated with the regulation of FAS,ACC1,PPAR?and GLUT4 expression.(3)Exertional swimming showed a significant decrease in swimming time,swimming speed and total distance swum in SD rats,and the AMD intervention significantly improved all the above three indexes,suggesting that AMD can improve the activity decline caused by SD and improve motor ability.(4)The results of ATP level and ATPase showed that ATP level,Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase were significantly decreased in SD group,while ATP,Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase were significantly increased in AMD group,indicating that AMD can improve the hypermetabolic state caused by SD and correct the metabolic disorder.Experiment 2:(1)HE staining showed that the cells in the Ctrl group were structurally intact and morphologically normal;the SD group had significantly reduced cell numbers and irregular morphology;the cell morphology improved after AMD intervention,with a small amount of nuclear consolidation and cell necrosis.The ultrasonic electron microscopy showed that the cell morphology of Ctrl group was basically normal and the mitochondrial morphology was regular;the nucleus of SD group was irregular,the mitochondria were swollen and deformed;the cell morphology improved after AMD intervention,and some mitochondria were swollen and vacuolated.(2)Western blotting results showed that the relative expressions of TFAM,Nrf1,Drp1and Mfn2 proteins in hypothalamus of SD rats were significantly reduced;compared with the SD group,AMD increased the expressions of TFAM,Nrf1,Drp1 and Mfn2.(3)Immunofluorescence showed co-expression between TFAM,Nrf1,Drp1 and Mfn2 and VDAC1,with more co-expression in the Ctrl group;decreased co-expression in the SD group.increased co-expression of TFAM,Nrf1,Drp1,Mfn2 and VDAC1 in AMD intervention,results consistent with the previous WB.(4)ROS staining showed lower mean optical density values in the Ctrl group and significantly higher mean optical density values in the SD rats;IOD values decreased after AMD intervention;SOD and GSH-Px levels decreased and MDA increased in the SD group;whereas AMD increased SOD and GSH-Px levels and decreased MDA expression.Experiment 3:(1)The mRNAs of CAMKK?,AMPK,PGC-1?and NRF-1 in the Ctrl group showed certain rhythm characteristics,with a trend of decreasing in the light phase and increasing in the dark phase.Compared with the Ctrl group,the SD group showed rhythm disorder,with CAMKK?at ZT18 and PGC-1?at ZT0 with no statistical significance with Ctrl;CAMKK?mRNA was down-regulated at ZT0 and up-regulated at ZT6 and ZT12;AMPK mRNA was up-regulated at ZT0,ZT6 and ZT12 and down-regulated at ZT18;PGC-1?mRNA was up-regulated at ZT6 and ZT12 and down-regulated at ZT18;Nrf-1mRNA was up-regulated at ZT6 and down-regulated at ZT0,ZT12,and ZT18.Compared with the SD group,the rhythm of rats in the AMD group improved,with a trend of decreased expression in the light phase and increased expression in the dark phase.CAMKK?mRNA at ZT0,ZT18 and PGC-1?mRNA at ZT0 were not statistically significant compared with the SD group,while AMPKmRNA and Nrf-1mRNA were significantly improved at all four time points.(2)The results of western blotting showed that compared with the Ctrl group,p-AMPK,PGC-1?and Nrf-1 protein expression was irregular in the SD group,and the overall expression was decreased.p-AMPK was increased at ZT12,and PGC-1?and Nrf-1 were decreased at all four nodes;compared with the SD group,the overall expression of p-AMPK,PGC-1?and Nrf-1 was upregulated in the AMD intervention,in which p-AMPK was not statistically different at ZT12,PGC-1?was decreased at ZT12,Nrf-1 was decreased at ZT18,and the expression of the remaining nodes was increased.(3)The Bmal1,Cry1 and Per1 genes in Ctrl rats had rhythm characteristics,and no significant rhythm characteristics were found in Clock rats.Compared with the Ctrl group,the SD group showed no rhythm,Bmal1mRNA was down-regulated at ZT0,ZT12 and ZT18,ClockmRNA was down-regulated at ZT6 and ZT12,Cry1mRNA was up-regulated at ZT0 and down-regulated at ZT6,ZT12 and ZT18;Per1mRNA was down-regulated at all 4 time points.Compared with SD,Bmal1,Cry1 and Per1genes improved in different nodes in rats after AMD intervention,Bmal1 and Cry1 were statistically significant in 4 nodes compared with SD group,Bmal1 mRNA was upregulated in all nodes compared with SD group except in ZT6 expression was downregulated;Clock,Per1 was only upregulated in ZT6,ZT12 by Clock,Per1 Per1.(4)The most significant differences ZT6 and ZT12 were selected to detect the protein expression of four genes.Compared with the Ctrl group,Bmal1,Clock,Cry1,Per1 protein expression was significantly decreased in ZT6 and ZT12 in the SD group;compared with the SD group,Bmal1,Clock,Cry1,Per1 protein expression was increased in ZT6 and ZT12 and IOD was significantly increased after AMD intervention.(5)The Ctrl group had better circadian rhythm characteristics,while the SD group had relatively disordered circadian rhythms,with TT,LT and DT significantly prolonged.SD had increased activity time at 6 nodes compared with the Ctrl group except for 20:00 and 8:00;AMD shortened TT,LT and DT;there was statistical significance in the 4 nodes from 11:00 to 20:00 with the SD group.The activity speed and number of activities of rats in each group under light and dark conditions were significantly reduced during daytime compared with nighttime;compared with Ctrl group,SD group increased the activity speed and number of activities during daytime and nighttime;compared with SD group,AMD reduced the activity speed,number of daytime and nighttime activities of SD rats.Experiment 4:(1)Compared with the Ctrl group,A1R and mGluR5 fluorescence intensity increased and A2AR,PTGDR and GABA1?fluorescence intensity decreased in the SD group;compared with the SD group,A1R and mGluR5 fluorescence intensity decreased,A2AR,PTGDR and GABA1?fluorescence intensity increased and protein expression increased in the AMD intervention.(2)Immunofluorescence showed that A1R was significantly co-expressed with mGluR5,more obviously in the SD group,with yellow or orange co-expression.However,the co-expression of A2AR and mGluR5 was low or even absent in SD and AMD groups,suggesting that A1R,but not A2AR,may play a sleep regulatory role by binding more closely to mGluR5.The co-expression of A1R,A2AR and GABAA1?was evident in the Ctrl group and relatively low in the SD group,and the co-expression of the protein increased after AMD intervention.Conclusion:(1)Insomnia is called"sleeplessness"in Chinese medicine,and is caused by a decrease in the quality of sleep due to mental disturbance.Both Chinese and Western medicine consider the brain as a very important internal organ,which plays a key role in the mental and emotional activities of higher life.Based on the theory of"brain is the house of the mind"and the"three major theories"of sleep in"Nei Jing",we have concluded that the system of insomnia prevention and treatment is based on the core of"Yuan Qi-Yin and Yang-Wu Zang Shen",and we believe that Yuan Qi is the root of sleep,Yin and Yang is the platform of sleep,and Wu Zang Shen is the core of sleep,and we established the Pei Yuan An Shen treatment method as an important method of insomnia treatment.The formula AMD is the representative formula.(2)SD and energy metabolism disorders interact with each other and are causal.energy metabolism is obviously abnormal after SD,so energy metabolism disorders are involved in the process of SD,and it may become a new idea to prevent and treat sleep disorders from the perspective of energy metabolism.(3)AMD can improve metabolic disorders in SD rats,correct abnormalities of glucolipid metabolism,and increase activity and ATP levels.(4)AMD alleviate energy metabolism disorder by improving the structural damage of mitochondria,improving the functions of mitochondrial biosynthesis,division and fusion.(5)AMD improves the rhythmic expression of AMPK/PGC-1?/Nrf-1 pathway proteins and regulates mitochondrial biological functions and affects the rhythmic characteristics of clock genes,so that AMPK may participate in the metabolic process from both energy and rhythm levels in SD rats.(6)AMD can improve A1R and A2AR expression and may improve sleep in SD rats by regulating with MGluR5,PTGDR,GABAA1?receptor expression.