| 1 BackgroundThe syndrome of liver stagnation and spleen deficiency is a traditional Chinese medicine syndrome.This syndrome is mostly due to extreme emotional damage to the liver,stagnation of liver qi,and spleen soil insult,which leads to liver and spleen disease.The most common clinical symptoms are:pain in the ribs,emotional depression,poor appetite,diarrhea,slow pulse strings,fat tongue and greasy tongue fur.Liver depression and spleen deficiency syndrome is related to nervous system,digestive system,immune system,endocrine system and many other system dysfunctions.It is common in depression,anxiety,non-alcoholic fatty liver disease,irritable bowel syndrome,functional dyspepsia,and chronic atrophy.Gastritis,chronic fatigue syndrome,sub-health,polycystic ovary syndrome and many other diseases.Therefore,the modern biological basic research of liver stagnation and spleen deficiency syndrome has practical significance for the diagnosis and treatment of clinical common and difficult diseases.Xiaoyaosan is from Taiping Huimin Hejijufang in Song dynasty.The whole prescription consists of Bupleurum,radix paeoniae alba,Angelica,Poria,Atractylodes,Licorice,Mint and Ginger.It has the functions of relieving liver and stagnation,strengthening the spleen and nourishing blood.Modern clinical practice has further broadened the application field of this prescription,and it can be widely used in various diseases of internal medicine,women,children,men,and facial diseases.The research team carried out a large number of experimental researches on Xiaoyaosan in the early stage,and found that there is a high degree of correlation and correspondence between the medicine taste,compatibility of Xiaoyaosan and the pathogenesis,etiology,location of liver stagnation and spleen deficiency syndrome.Therefore,Xiaoyaosan can be used as an effective treatment for liver stagnation and spleen deficiency syndrome.At present,there are mainly three animal models in experimental studies of traditional Chinese medicine:traditional Chinese medicine disease model,traditional Chinese medicine syndrome model,and animal model combining disease and syndrome.Among them,animal model combining disease and syndrome is under the guidance of the traditional Chinese medicine theory of overall concept and dialectical treatment.Through the etiology and pathogenesis theory,it is achieved the simulated replication of human clinical disease syndrome characteristics on animals,which is an indispensable link in modern research of traditional Chinese medicineSystems biology is a new discipline that deals with the interrelationships between the constituent components(DNA,mRNA,protein,metabolites,etc.)from the system level.The main technical means is high-throughput omics research.Systems biology has the characteristics of complexity,integration and informatization,which coincides with the characteristics of integrity,timeliness and complexity in traditional Chinese medicine syndrome.Therefore,in the basic research of traditional Chinese medicine syndrome,system biology will play an increasingly important role.Among them,transcriptomics research can reflect the expression of genes in different physiological states and different external stresses.Promoter DNA methylation regulates gene transcription and inhibits gene expression.At present,there are few high-throughput omics studies on liver stagnation and spleen deficiency syndrome.The combined sequencing analysis of the two omics is of great significance to reveal the modern biological basis of liver stagnation and spleen deficiency syndromeThe hypothalamus is one of the important centers of physiological regulation,regulating various physiological processes such as food intake,body temperature,endocrine,emotional response,cognition,learning and memory.Previous research by the research group found that the hypermethylation status of the hypothalamus arcuate nucleus in rats with liver stagnation and spleen deficiency syndrome suggests that hypothalamus is important in liver stagnation and spleen deficiency syndrome.In the early stage of this experiment,Lmna,a target gene for hypothalamic arcuate nucleus in liver stagnation and spleen deficiency syndrome,was screened using transcriptome and methylation sequencing technology.Lmna-mediated Leptin signaling pathway is involved in the development of depression-like behaviors in liver stagnation and spleen deficiency syndrome.Therefore,it is inferred that Leptin signal may be a key regulatory node of Lmna's participation in liver stagnation and spleen deficiency syndrome.In summary,based on previous experimental research,the hypothesis that the Lmna-mediated Leptin signaling pathway in the hypothalamic arcuate nucleus is an internal mechanism of liver stagnation and spleen deficiency syndrome and a key pathway for Xiaoyaosan regulation.In this experiment,a stable animal model of liver stagnation and spleen deficiency syndrome is first copied.Based on this,the hypothalamic arcuate nucleus is selected as the subject area.The high-throughput sequencing(transcriptome and methylation sequencing)technology is used to explore the biological basis of liver stagnation and spleen deficiency syndrome and the treatment mechanism of Xiaoyaosan.2 MethodsThis experimental study consists of three parts.Part 1:A total of 80 male SD rats were randomly divided into normal group,model group,Xiaoyaosan group and fluoxetine group,with 20 rats in each group.Except the normal group,the other groups were received 6 weeks of chronic unpredictable mild stress(CUMS)modeling.At the same time,Xiaoyaosan group and fluoxetine group were treated with Xiaoyaosan(2.224 g/kg/d)and fluoxetine(2.0 mg/kg/d).Six weeks after modeling and administration,the model was evaluated using sugar preference test and open field test Part 2:On the basis of experiment part 1,the transcriptome and methylation sequencing technologies were used to detect mRNA and methylation differentially expressed genes in the hypothalamus arcuate nucleus of the normal group,model group and Xiaoyaosan group.Part 3:On the basis of experiment part 2,the screening gene LMNA and its mediated Leptin signaling pathway were verified.Western blot(WB)and quantitative real-time polymerase chain reaction(qRT-PCR)techniques were used to detect the gene and protein expressions of Lmna,LepR,Jak2,STAT3,IRS-1,PI3K,AKT,POMC,AGRP and NPY.Enzyme linked immunosorbent assay(ELISA)was used to detect the Leptin level in rat serum3 ResultsPart 1:After 6 weeks of CUMS modeling,the rats in the model group behaved lost weight and reduced food intake,and showed significant depressive behaviors in the sugar preference test and open field test.There was no significant difference in body weight and food intake after Xiaoyaosan and fluoxetine treated,and there was no statistical difference in the sugar preference test.While Xiaoyaosan and fluoxetine played a significant improvement role in open field testPart 2:Differential mRNAs were obtained from the hypothalamic arcuate nucleus of the liver stagnation and spleen deficiency syndrome by transcriptomics.There were 439 differential mRNAs in the model group compared with the normal group,and there were 328 differential mRNAs in the Xiaoyaosan group compared with the model group.Combined with the analysis of the whole genome DNA methylation profile of the hypothalamic arcuate nucleus of the liver stagnation and spleen deficiency syndrome obtained from the previous WGBS screening,Fam111a,Myo5c,Dtx2,Abcc5,Cfb,Rnaset2,and Lmna were screened out as the target genes between the model group and the normal group,and 11 target genes Abcc5,Lmna,Cfb,Clic5,Dnah6,Eln,Fam111a,Mtr,Slc44a4,Tmem51,Vsig10 were screened out between the Xiaoyaosan group and the model group.These genes are related to DNA replication,ATP binding,Notch signaling pathway,ATPase activity,G protein coupled receptor signaling pathway,leptin receptor signaling pathway,insulin resistance,etc.,and may be the pathogenesis of liver depression and spleen deficiency syndrome and Xiaoyaosan regulation target genes.Part 3:WB and Real-time PCR showed that the expression of Lmna in the model group decreased,and Xiaoyaosan could reverse this change.ELISA result showed that the serum leptin level in the model group was significantly increased.WB and Real-time PCR showed that the LepR-STAT3 signaling pathway was activated in the hypothalamus arcuate nucleus of the model group,in which the expressions of JAK2 and STAT3 were up-regulated.In addition,CUMS had an activating effect on PI3K signaling pathway.Real-time PCR results showed that the mRNA expressions of POMC,NPY and AgRP that control appetite changed to varying degrees,showing an appetite-suppressing state.After Xiaoyaosan treatment,the LepR-STAT3 signaling pathway and some nodes of the PI3K pathway were reversed,which meant Xiaoyaosan co?uld inhibit the LepR-STAT3 and PI3K signaling pathways.In addition,Xiaoyaosan could also reverse the changes of POMC,NPY and AgRP.4 ConclusionA rat model of liver stagnation and spleen deficiency syndrome was established by 6 weeks CUMS modeling,and the model was evaluated by weight,food intake,behavioral methods,etc.It proved that the rat model of liver stagnation and spleen deficiency syndrome established by 6 weeks CUMS could be very stable,which could be used for modern biological basic research of liver stagnation and spleen deficiency syndrome.Based on the successful establishment of a rat model of liver depression and spleen deficiency syndrome,combined with transcriptomics and methylation analysis,Fam111a,Myo5c,Dtx2,Abcc5,Cfb,Rnaset2,Lmna,Clic5,Dnah6,Eln,Mtr,Slc44a4,Tmem51,Vsig10,a total of 14 target genes were identified in the hypothalamic arcuate nucleus region These genes were related to DNA replication,ATP binding,Notch signaling pathway,ATPase activity,G protein-coupled receptor signaling pathway,leptin receptor signaling pathway,insulin resistance,etc.,which may be the biological basis of liver stagnation and spleen deficiency syndrome and the target genes regulated by XiaoyaosanFurther research results indicated that Lmna-mediated Leptin signaling pathway in the hypothalamic arcuate nucleus may be an internal mechanism of liver stagnation and spleen deficiency syndrome and a key pathway for Xiaoyaosan regulation. |
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