Liver Stagnation And Spleen Deficiency Syndrome - Xiaoyao San (Fangzheng) Arcuate Nucleus Genome-wide DNA Methylation And Its Regulation

1 BackgroundLiver depression and spleen deficiency syndrome,also known as liver and spleen deficiency syndrome,is caused by liver uncontrolling conveyance and dispersion and dysfunction of spleen in transportation.It mainly manifested that pain andfullness in chest and hypochondrium,abdominal distention,low food,emotional depression,loose stools,ordiarrhea caused by abdominal pain but pain reduction after diarrhea,stringy pulse.It said that liver depressionis the primary pathogen for many diseases.Liver depression and spleen deficiency syndrome could be found in many common clinical diseases including irritable bowel syndrome,functional dyspepsia,ulcerative colitis,chronic hepatitis B,fatty liver,breast hyperplasia,thyroid dysfunction,depression,sub-health,and during the early stage of stubborn diseases like polycystic ovary syndrome,thyroid malignancy and uterine malignancy.Xiaoyaosan was initially recorded in Taiping Huinin Heji Jufang in Song Dynasty,which was born out of Sinisan and Danggui Shaoyaosan created by Zhang Zhongjing.It is one of therepresentative for the relieving qi stagnation.It has been widely used in clinical departments including internal medicine,gynecology,pediatrics,andrology and five senses.The correlation of Formula and Syndorme refers that there was close correlation between the ingredients of a Formula including medicinal taste,dose,dosage form,and usage and the Syndorme treated by the Formula including the pathogenesis,disease nature,disease location and disease process.Previously,our group have researched the Xiaoyaosan for more than 20 years and found that there were high correlations between compatibility and efficacy of Xiaoyaosan and the main symptoms,pathogenesis and its evolution of liver-qi stagnation and spleen-deficiency syndrome.Therefore,it is of great significance for the diagnosis,syndrome differentiation,and precise treatment using traditional Chinese medicine for many common diseases and some complicated diseases to further research on the biological connotation ofsyndrome of liver stagnation and spleen deficiency and Xiaoyaosan.The animal model of TCM syndromes is the basis for the modernization researches of TCM theories,methods,prescriptions and medicines that were consisted of TCM theory.TCM syndromes,TCM compound formulas,TCM specialty therapies,etc.Previously,many animal models of liver-depression and spleen-deficiency syndrome were developed with simple etiology(emotional stimulation)or the combination of complex etiology and pathology methods that were evaluated by simple assement methods,resulting in the low reliability,repeatability,and clinical applicability.Systems biology is a new discipline that uses holistic and systematic research methods to analyze complex life processes.The charactises including whole,dynamics,level and integration of systems biologyareconsistent with those of TCM syndrome,so it is of great significance for the reseaches of TCM syndrome to introduce the system biology.Whole genome DNA methylation is a hotspot in the epigenetic branch of system biology.DNA methylation can achieve a relatively stable and genetically apparent modification of DNA that can regulate gene transcription and thereby cause gene silencing.However,the study on the genome-wide DNA methylation and its regulation ofliver-depression and spleen-deficiency syndrome&Xiaoyaosan(Fang Zheng)has not yet been found in the previous study.The hypothalamus plays a key role in maintaining the physiological activities of energy metabolism.Hypothalamic nuclei,especially the arcuate nucleus,play an important role in energy intake and expenditure balance.Also,the arcuate nucleus actively participates in cognition,learning and memory,rewards,emotions and other behaviors by extensively receiving nerve afferents from the amygdala,brain stem,cortex,and most of the inner nuclear cores of the hypothalamus.Our group previously found that thepromoting and suppressing appetite neuropeptide disorders in arcuate nucleus may be potential mechanism of feeding disorder of liver depression and spleen deficiency syndrome and one of the targets of Xiaoyaosan.However,recent studies have reported that there were close relationships between depression and sugar,lipid and insulin metabolism,indicating thatenergy metabolism disorders may be deeper mechanism underlying the eating disorders(spleen deficiency)induced by poor mood(liver stagnation).Therefore,this current study is to firstly develop an ideal animal model of liver-depression and spleen-deficiency syndrome.Then,the whole genome-wide DNA methylationspectrumin hypothalamic arcuate nucleus of ratswas researched using whole genome bisulfite sequencec(WGBS),to elucidate thebiological mechanism for the core pathogenesis of liver depression and spleen deficiency syndrome in the eating disorders(spleen deficiency)induced by poor mood(liver stagnation)as well as the therapeutic mechanism of Xiaoyaosan.2 MethodsThis study was consisted of three parts of the experiment.Part ?:(1)100 male SD rats were randomly divided into control group,model group,Fluoxetine group,Guipiwan group and Xiaoyaosan group,with 20 rats in each group.Except for the control group,the rats were daily given the chronic immobilization stressfor 3 hours,consecutively for 21 days.At the same time of model establishment,each drug administration group was respectively treated with Fluoxetine(2.0mg/kg/d),Guipiwan(4.6275g/kg/d)and Xiaoyaosan(2.224g/kg/d)by gavage.Then,the "stagnation of the liver" behavior of the rats in each group was assessed using open field test,forced swimming test,sucrose preference test,and HPA axis(stress axis);the "spleen deficiency(induced by liver qi stagnation)behavior of the rats in each group was assessedusingbody weight,food intake,body fat,intestinal propulsion rate,fecal water content,visceral hypersensitivity,and D-xylose excretion;the model was also assessed through the observation of effects of Xiaoyaosan(corresponds to the syndorme)and Guipiwan(doesn't corresponds to the syndorme).(2)100 male SD rats were randomly divided into control group,3-week model group,4-week group.6-week group and 8-week group,with 20 rats in each group.Except for the control group,the rats were given thechronic unpredictable mild stress(CUMS).The model was evaluated using the method of evaluating the behaviors of "liverqi stagnation" and "spleen deficiency(induced by liver stagnation)in the first experiment.(3)On the basis of previous experiment,100 male SD rats were randomly divided into control group,model group,Fluoxetine group,Guipiwan group and Xiaoyaosan group,with 20 rats in each group.Except for the control group,the rats were daily given the CUMS,consecutively for 6 weeks.At the same time of model establishment,each drug administration group respectively treated with Fluoxetine(2.0mg/kg/d),Guipiwan(4.6275g/kg/d)and Xiaoyaosan(2.224g/kg/d)by gavage.Then,a comprehensive evaluation model was used to assess the behaviors of "liver qi stagnation" and "spleen deficiency(induced by liver stagnation)" in accordrance with the first experiment.Part ?:On the basis ofPart ?,the genomic DNA methylation profile of rats in hypothalamic arcuate nucleus in the control group,model group and Xiaoyaosan was determined using WGBS,and their target genes regulated by the genomic DNA methylation.as well.Part ?:On the basis of Part ?,thecandidatetarget genes regulated by the genomic DNA methylation including Slc2a4,Nmur2,Irx3,Irx5,Bmp4,Nkx2-1 Glp1r,Ankrd26,Fads2.Mlycd,Gpr26,Angptl4,Prkaca,Drd1,Grm5 were verificated using the real-time fluorescence quantitative PCR method.3 ResultsPart ?:(1)There were significant differences in the forced swimming test,body weight and food intake test between the model group and control group,but these changes were not reversed by the treatment of Xiaoyaosan,indicating that when the sample size was enlarged,it became difficult for 21-day chronic restraint stress method to develop a high stability and success rate of rat model of liver stagnation and spleen deficiency syndrome.(2)Different time duration CUMS including 3 weeks.4 weeks,6 weeks,and 8 weeks could successfully establish a rat model of depression.However,the results of assessment including open field test,forced swimming test,sucrose preference test.HPA axis(stress axis),body weight,food intake,body fat,small bowel propulsion rate and visceral hypersensitivity,showed that the 6-week CUMS rat model may be the window of time when depression is in the syndrome stage of liver stagnation and spleen deficiency.(3)The6-week CUMS rats exhibitedsignificant depressive behavior in the open-field test and forced-swimming test,which were significantly improved by the treatment of Fluoxetine,Guipiwan and Xiaoyaosan.The6-week CUMS significantly reduced the body weight of rats,which were significantly reversed by the treatment of Fluoxetine and Xiaoyaosan in two weeks after the experiment.The6-week CUMS significantly reduced the food intake of rats,but the Xiaoyaosan could obviously improve the food intakin the first two weeks after the experiment.The6-week CUMS significantly increased the visceral sensitivity of rats,which could be effectively treated by the administration of Fluoxetineand Xiaoyaosan.In addition,we found that the changes in HPA axis,fecal water content and D-xylose excretion ratefailed to effectively the respondingsymptoms in the rat model of liver stagnation and spleen deficiency syndrome.Part ?:The result of profiling analysis showed that there was a significant difference in the methylation levels of the CG loci before and after the TSS segment.The analysis of differentially methylated regions(DMRs)of CG loci revealed that 30395 DMRs(model group vs control group)and 40164 DMRs(Xiaoyaosan groupvsmodel group)were found,and when these DMRs were annotated,there were a total of 13346 DMRs(model group vs control group),19914 DMRs(Xiaoyaosan groupvsmodel group)and 3055 Share DMRs(model group vs control group&Xiaoyaosan groupvsmodel group).A total of 486 DMRs fall into the Transcription start site(TSS)segment in these Share DMRs.The results of the methylation level of Share DMRs indicated that the hypermethylated DMRs in the model group were significantly more than those in the control group and Xiaoyaosan group.The hypomethylated DMRs in the model group were significantly less than those in the control group and Xiaoyaosan group.The GO analysis of the Share DMRs in the TSS segment showed that these candidate genes for differentially regulated transcription are mainly involved in biological processes including thyroid hormone-stimulated responses,pituitary development,G protein-coupled receptor signaling pathways,and long-term synaptic enhancementpathways,cell development,learning,neuronal differentiation,protease activity,brain development,cell response to insulin stimuli,located in cellular components including chromatin,myelin,mitochondria,neuromuscular junctions,endoplasmic reticulum,transcription factors,Golgi,extracellular matrix,had molecular functions including protein binding,transforming growth factor beta receptor binding,transcription factor activity,ligase activity,oxidoreductase activity,growth factor activity,and microtubule binding.The KEGG analysis results of the Share DMRs in the TSS segment indicated thatthese candidate genes regulatedby differentially methylated sites are mainly involved in signaling pathway and diseases pathway,such as thyroid hormone signaling pathway,apoptosis,AMPK signaling pathway.TCA cycle,insulin signaling pathway,fatty acid synthesis,and Parkinson's Disease,ABC transporters,Notch signaling pathways,glucose metabolism,and type 2 diabetes mellitus.Interestingly,we paid attention to the candidate genes closely related to the signaling pathways involved in food intake,fat.insulin,and emotional behavior,includingSlc2a4,Nmur2,Irx3,Irx5,Bmp4,Nkx2-1,Glp1r,Ankrd26,Fads2,Mlycd,Gpr26,Angptl4,Prkaca,D rd1and Grm5,which may be the potentialDNA methylation markers of core pathogenesis of liver depression and spleen deficiency syndrome in the eating disorders(spleen deficiency)induced by poor mood(liver stagnation)as well as the therapeutic mechanism of Xiaoyaosan.Part ?:The validationresults of real-time fluorescence quantitative PCR showed:the expression of Slc2a4mRNA in the hypothalamic arcuate nucleus of rats in the model group was significantly lower than that in the control group,and the expression of Nmur2 mRNAwas significantly higher than that in the control group,which were significantly reversed by the treatment of Xiaoyaosan.These results were consistent with their methylation-regulated transcription;The expression of Irx3 and Irx5 mRNA in the hypothalamic arcuate nucleus of rats in the model group were significantly reduced when compared with the control group,which were significantly increased by the treatment of Xiaoyaosan.These results werewas contrary to their methylation-regulated transcription;There were no differences in the expressions ofBmp4,Nkx2-1,Glp1r,Ankrd26,Fads2,Mlycd,Gpr26,Angptl4,Prkaca,Drd1and Grm5 mRNA in the hypothalamic arcuate nucleus of rats in each group.4 ConclusionThe rat model of depression induced by CUMShas a time window of 6 weeks for syndrome stage of liver depression and spleen deficiency syndrome.The6-week CUMS could successfully establish a combination animal model of depression and liver depression and spleen deficiency syndrome with high similarity,reproducibility and utility,which could be widely applied to theresearchof theory,modern biological basic and clinical diagnosis and treatment for liver depression and spleen deficiency syndrome.On this basis,we found that the candidate target genesregulated by differentially methylatedregions that Slc2a4 and Nmur2 in the hypothalamic arcuate nucleus may be the bio markers of core pathogenesis of liver depression and spleen deficiency syndrome in the eating disorders(spleen deficiency)induced by poor mood(liver stagnation)as well as the therapeutic targets of Xiaoyaosan,which will provide experimental evidence for deepening the theory understanding of liver depression and spleen deficiency syndrome&Xiaoyaosan(Fang Zheng)and treating individualized diagnosis and treatment based on syndrome differentiation.