The Theoretical And Experimental Study Of Kidney Not Storing Will Type Of Insomnia

Objective:（1）This study is aimed to explore the theoretical basis of kidney not storing will type of insomnia,the etiology,pathogenesis and syndrome differentiation treatment,and the common prescriptions of adjusting will.（2）D-galactose combining with p-chlorophenylalanine（PCPA）was used to establish rat model with kidney not storing will type of insomnia.The changes of aging aspect,sleep aspect and transcriptome in hippocampus of rat model with kidney not storing will type of insomnia were observed.The aspects of aging included memory ability,oxidative stress,apoptotic genes,the sleep aspects included sleep duration,electroencephalogram（EEG）,inflammatory cytokines and neurotransmitters.（3）The effects of Polygala Tenuifolia on sleep,neurotransmitters and inflammatory factors in model rats with kidney not storing will type of insomnia,as well as the transcriptomics effects of Polygala Tenuifolia on hippocampus of model rats were observed.Methods:（1）The related theory and prescriptions of the treatment of kidney not storing will type of insomnia were searched,summarized and discussed.（2）The experimental animals were randomly divided into blank control group,D-galactose group,PCPA group,and kidney not storing will type of insomnia group（model group）.The kidney not storing will type of insomnia group was induced by the subcutaneous injection of D-galactose 120mg/kg/day for 42 days,combining with the intraperitoneal injection of PCPA 300mg/kg/day for 3 days.（3）The learning and memory ability of the model rats was observed by Morris Water Maze（MWM）,the brain contents of superoxide dismutase（SOD）,malonaldehyde（MDA）,catalase（CAT）and glutathione peroxidase（GSH-px）in model rats were detected.The relative expressions of superoxide dismutase 1（SOD1）,superoxide dismutase 2（SOD2）,CAT,B-lymphocyte tumor-associated X protein（Bax）,B-lymphocyte tumor-2（Bcl-2）,and cysteine protease（Caspase-3）in hippocampus of model rats were detected.The ageing characteristics of rat model were evaluated.（4）The pentobarbital test was used to observe the sleep quality of model rats.Wake、SWS₁、SWS₂、REM and TST of the rats in each group were observed by EEG.The plasma levels of interleukin-1β（IL-1β）,interleukin-6（IL-6）,and tumor necrosis factor-α（TNF-α）were observed.The brain contents of 5-hydroxytryptamine（5-HT）,glutamic acid（Glu）,γ-aminobutyric acid（GABA）in model rats were detected.The relative expressions of5-hydroxytryptamine 1A receptor(5-HT_1AR),γ-aminobutyric acid A receptorα1 type(GABA_AR_α1),metabolic glutamate receptor 2（m Glu R2）,IL-6,TNF-α,nuclear transcription factor-B（NF-κB）in hippocampus of model rats were detected.The sleep characteristics of rat model were also evaluated.（5）Total RNA of the hippocampus in blank control group and model group were extracted and analyzed by transcriptome sequencing.（6）The effects of polygala tenuifolia on model rats;the experimental animals were randomly divided into blank control group,kidney not storing will type of insomnia group（model group）,low dose group（0.0875g/kg）,middle dose group（0.175g/kg）,high dose group（0.35g/kg）and diazepam group（0.92mg/kg）.The learning and memory ability of model rats were observed by MWM.The contents of 5-HT、Glu、GABA in hippocampus,and IL-1β、IL-6、TNF-αin plasma were measured.（7）Total RNA of the hippocampus in blank control group,model group,polygala tenuifolia middle dose group（polygala tenuifolia group）and diazepam group were extracted and analyzed by transcriptome sequencing.Results:（1）Kidney will can regulate sleep.The early awakening was the cardinal symptom of kidney not storing will type of insomnia.Kidney deficiency was the main cause.Kidney not storing will was the core pathogenesis.The clinical common type of kidney not storing will insomnia included six types,kidney qi deficiency type,kidney essence deficiency type,kidney yang deficiency type,kidney yin deficiency type,kidney deficiency water overflow type and kidney does not inspire air type.Polygala tenuifolia,Acanthopanax,Ginseng,Poria cocos,Schisandra chinensis,Jiaotai pill,Huanglianejiao soup,Liuweidihuang pill,Ci zhu pill and Kongshengzhenzhong pill were commonly used prescriptions for treating kidney not storing will type of insomnia.（2）The rat model could be established by the subcutaneous injection of D-galactose 120mg/kg/day for 42days,combining with the intraperitoneal injection of PCPA 300mg/kg/day for 3days.（3）The changes of ageing aspects of rat model;compared with the blank control group,the body weight of model rats decreased.The numbers of platform crossing reduced.The brain levels of SOD,CAT and GSH-px of model rats decreased,and MDA by contrast.The plasma levels of IL-1β,IL-6,TNF-αof model rats increased.The relative expressions of SOD1,SOD2,CAT,Bax,Caspase-3 in hippocampus of model rats were up-regulated,Bcl-2 by contrast.（4）The changes of sleep aspects of rat model;compared with blank control group,the sleep latency of model rats was prolonged,and the sleep duration was shortened in pentobarbital experiment.Compared with blank control group,WAKE of model group increased in EEG,SWS₁、SWS₂、REMS,and TST by contrast.The brain contents of 5-HT and GABA of model rats decreased,and Glu increased.The relative expressions of 5-HT_1AR、GABA_AR_α1in hippocampus of model rats were down-regulated,the relative expressions ofmglu R2,IL-6,TNF-αin hippocampus of model rats were up-regulated.Polygala tenuifolia could improve the memory ability of the model rats,increased the contents of 5-HT and GABA,reduced the content of Glu in hippocampus,and decreased the plasma levels of IL-1β、IL-6、TNF-αin the model rats.（5）The transcriptome analysis results of hippocampus of kidney not storing will type of insomnia rats;compared with the blank control group,there were 1628 differentially expressed genes（DEGs）in kidney not storing will type of insomnia group,including 887 up-regulated DEGs and 741 down-regulated DEGs.The functions of the DEGs mainly involved nerve development,cell development,triglyceride metabolism,ribosomes,oxidative stress,inflammatory factors,and were closely related to Parkinson’s disease,Alzheimer’s disease,retrograde nerve signaling pathways and chronic progressive chorea.GO analysis,KEGG Pathway analysis,PPI and connotation of kidney not storing will type of insomnia were analyzed.The significant DEGs of model group were explored and identified.The results turned out that the potentially significant DEGs of hippocampus were Gnb3,Faah,Mmp9 and Twist1 in model group.（6）Polygala tenuifolia could improve the memory ability of the model rats,increase the contents of5-HT and GABA,reduce the content of Glu in hippocampus,and decrease the plasma levels of IL-1β、IL-6、TNF-αin the model rats.（7）The transcriptome analysis results of hippocampus of kidney not storing will type of insomnia rats which were treated by drugs;（1）Compared with the model group,there were 663 DEGs in polygala tenuifolia group,including 240 up-regulated genes and 423 down-regulated genes.The functions of the DEGs mainly involved in neurogenesis,oxidative phosphorylation,apoptosis and proliferation,circadian regulation,energy metabolism,immune regulation,and were closely related to Parkinson’s disease,Alzheimer’s disease,chronic progressive chorea,nonalcoholic fatty liver disease,type I diabetes and asthma;（2）Compared with the model group,there were 1660 DEGs in diazepam group,including 732 up-regulated genes and928 down-regulated genes.The functions of the DEGs mainly involved in nervous system development,oxidative phosphorylation,neuronal apoptosis,oxidative stress,energy metabolism,and were closely related to Parkinson’s disease,chronic progressive chorea,Alzheimer’s disease and nonalcoholic fatty liver disease;（3）There were 235 DEGs in polygala tenuifolia group,and there were 37 DEGs with significant therapeutic effects.The functions of the significant DEGs mainly involved neuronal development,activation of theγ-aminobutyric acid signaling pathway,cell growth regulation,participation in immune response mediated by immunoglobulin,energy metabolism,and were closely related to chronic progressive chorea,Parkinson’s disease,Alzheimer’s disease,type I diabetes,non-alcoholic fatty liver disease and etc;（4）There were 753 DEGs in diazepam group,and there were 49 DEGs with significant therapeutic effects.The functions of the DEGs mainly involved central nervous system development,ageing,synaptic regulation,apoptosis regulation,dopamine,glycogen,fatty acids,purine and pyrimidine nucleotide metabolism,and were closely related to chronic progressive chorea,Parkinson’s disease,Alzheimer’s disease,non-alcoholic fatty liver disease,myocardial contraction,herpes simplex virus infection and etc.（5）Target DEGs analysis of correlation and molecular mechanisms between kidney not storing will type of insomnia and polygala tenuifolia were explored.GO analysis,KEGG Pathway analysis,PPI and connotation of kidney not storing will type of insomnia were analyzed.The significant DEGs of model group,polygala tenuifolia group and diazepam group were identified.The results turned out that the potentially significant DEGs of hippocampus were Gnb3,Faah,Mmp9 and Twist1 in model group.The effective target genes of polygala tenuifolia were Faim,Pcp2 and Elovl6,the effective target genes of diazepam were Npr3 and Trh,the target of the two drugs were BDNF and Gabra6.Conclusion:（1）Kidney will was closely related to insomnia.Kidney not storing will was the core pathogenesis of kidney not storing will type of insomnia.Tonifying the kidney tranquilization and enriching themind was the principle of treatment.Polygala tenuifolia which effects on kidney had the effects of tranquilization,enriching themind and tonifying the kidney effects,and it is the priority drug in the treatment of the abnormal will.（2）D-galactose combined with PCPA could establish the rat model of kidney not storing will type of insomnia.（3）The aging aspects which included learning and memory ability,oxidative stress,inflammatory factors and apoptotic genes were changed in model rats.The sleep aspects which included sleep duration,neurotransmitters and inflammatory factors were also changed in model rats.（4）There were differences in gene expression in the hippocampus of rat model,and the function of significant DEGs were related to nervous system,metabolism and immunity.（5）Polygala tenuifolia could improve sleep,regulates neurotransmitters and reduces the expression of inflammatory factors of the model rats.（6）Both polygala tenuifolia and diazepam had certain intervention effects on hippocampus transcriptome of model rats,especially on genes involved in nervous system and immunity.And Polygala tenuifolia seemed favor the regulation of neuro-associated target genes.