Based On NEI Network And Transcriptomics Technology, Explore The Biological Basis Of The Association Between Chaihu Shugan San And Liver Depression Syndrome

Objective:Syndrome differentiation is the characteristic of diagnosis and treatment of traditional Chinese medicine."Correlation between prescriptions and syndromes"is not only an important part of syndrome differentiation,but also the academic basis of prescription.The modern biological basis of prescription-certification is becoming a hotspot of modern Chinese medicine research.Studies on liver-qi stagnation syndrome show that the modern connotation of liver-qi stagnation syndrome involves multiple phylogenetic changes in the system,and the relationship with the neuro-endocrine-immune(NEI)network is very close,but the current liver-qi stagnation syndrome is related to Shuganfang.The biological connotation is still unclear.In this study,the neuro-endocrine-immune network and its information carrier-gene transcriptomics in peripheral blood leukocytes were used to observe the changes of NEI network under the condition of liver-qi stagnation syndrome and to explore the differential gene expression of peripheral blood leukocytes by molecular biological techniques.At the same time,combined with the effect spectrum of the role of Chaihu-Shugan-San.The bioinformatics method was used to deeply analyze the data,in order to obtain the biological connotation and molecular target of liver-qi stagnation syndrome and Chaihu-Shugan-San at the ensemble-system-molecular level.Methods:Male Wistar rats were randomly divided into normal group(n=10)and model group(n=40).Model rats were used to prepare liver-qi stagnation syndrome rat model by chronic restraint stress method.After modeling two weeks,30 rats in the model group were randomly divided into the high-dose group of Chaihu-Shugan-San group(HC),the low-dose group of Chaihu-Shugan-San group(LC)and the fluoxetine group(Flu).High-dose(1.2g/kg·d),small dose(0.6g/kg-d)and fluoxetine(1.8 mg/kg·d)were administered respectively for 2 weeks.The normal group and the model group were each given an equal amount of distilled water.The body weight of each group was weighed every week,the appearance behavior score was recorded,and the sugar water test and the open field experiment were performed at the end of 2w and 4w.On the 29th day of the experiment,blood was taken from the abdominal aorta of each group,and the leukocytes and plasma were separated,the hypothalamus,pituitary and hippocampus tissues were taken.The levels of CORT,IL-1?,IL-6,IL-4,IL-10,TNF-? and IFN-a in plasma,contents of CRH,5-HT,NE,DA,IDO and GR ? in hypothalamus,ACTH content in pituitary and SERT and BDNF contents in hippocampus were detected by enzyme-linked immunosorbent assay(ELISA),leukocytes were extracted by total RNA for transcriptome sequencing and analysis.Results:1 Changes in appearance behavior and NEI indicators of rats with liver-qi stagnation syndrome and the effect of interventional drugs.(1)Changes in appearance behavior of rats with liver-qi stagnation syndrome:compared with the normal group,the weight of the rats in the model group decreased at different time points during the modeling pernod.The scores of activity,emotion and sleep,diet and stool,skin and hair were significantily increased during 2-4 weeks(P<0.01).The time of central residence was decreased in open field test at the end of 2 weeks(P<0.05),while at the end of 4 weeks,it was increased and the number of crossings were decreased,the sugar water preference was also decreased in the syrup experiment(P<0.05).(2)Changes of NEI-related indicators in rats with liver-qi stagnation syndrome:compared with the normal group,the levels of 5-HT and CRH in the hypothalamus of rats in the model group were increased,the contents of NE,IDO and GR a were decreased,and the content of SERT was decreased,BDNF content was increased in the hippocampus,CORT in plasma was increased(P<0.01 or P<0.05),there was no significant difference on the content of ACTH in pituitary(P>0.05);IL-1p,IL-4,IL-6,IL-10,TNF-?,IFN-? contents of plasma were significantly reduced(P<0.01 or P<0.05).(3)The effect of intervention drugs on rats with liver-qi stagnation syndrome:1)Chaihu-Shugan-San:compared with the model group,the scores of activity,emotion and sleep,diet and stool in HC group and LC group were significantly lower(P<0.01 or P<0.05).The HC increased the saccharide preference and the number of crossings(P<0.05),the LC also increased the number of crossings(P<0.01).In the two dose groups,the contents of IDO and GR a in the hypothalamus were increased,and the content of pituitary ACTH was decreased(P<0.01 or P<0.05),IL-1?,IL-6,TNF-a levels of plasma were increased(P<0.01 or P<0.05),while hypothalamic 5-HT and CRH levels were decreased,DA and NE contents were increased,CORT in plasma was reduced in the HC(P<0.01 or P<0.05),IL-10 level of plasma in the LC was significantly increased(P<0.01).2)Fluoxetine:compared with the model group,the scores of activity,emotion and sleep,diet and stool in the Flu were significantly lower(P<0.01),the saccharification preference and number of crossings were increased(P<0.05).The contents of NE,IDO and GR a in hypothalamus were increased(P<0.01 or P<0.05),the contents of ACTH in pituitary and SERT in hippocampus were decreased(P<0.01 or P<0.05);IL-1? and IL-6 levels in plasma were obvious increased(P<0.01 or P<0.05).2 Results of transcriptome of peripheral blood leukocytes in model rats with liver-qi stagnation syndrome.(1)Leukocyte transcriptomics in the model group:compared with the normal group,there were 800 differentially expressed genes in the model group,including 557 up-regulated genes and 243 down-regulated genes.The function of differential genes mainly involved metabolism,apoptosis,autophagy,oxidative stress,endoplasmic reticulum stress,and coagulation function,cell adhesion,immune function,norepinephrine secretion,cytokine production,central nervous development,5-HT transport,and the functions of circulation,reproduction and digestive system,and were closely related to the occurrence of cancer,systemic lupus erythematosus,diabetes,autoimmune thyroid disease and cardiovascular disease.(2)Leukocyte transcriptomics in the intervention group of drugs:1)Compared with the model group,there were 31 differential genes in the Chaihu-Shugan-San group,including 12 up-regulated genes and 19 down-regulated genes.Differential genes functions mainly included improving stress response,immune inflammatory response and regulation of leukocytes,and were associated with rejection,viral infection,type I diabetes,autoimmune thyroid disease,viral myocarditis,cancer and HTLU-I infection.2)Compared with the model group,there were 59 differential genes in the fluoxetine group,including 19 up-regulated genes and 40 down-regulated genes.Differential genes functions mainly involved improving stress response,immune function,coagulation function,neurogenesis,tissue and organ development,cell damage,cell differentiation and signal transduction pathway,and were associated with amebiasis and atherosclerosis.(3)Bioinformatics analysis between intervention groups:there were 49 differentially expressed genes of Chaihu-Shugan-San intervention of liver-qi stagnation syndrome,including 6 genes with significant therapeutic effects(RT1-CE3,Lta4h,Als2crl2,LOC100364500,AABR07051689.1,AABR07065782.1);The function of differential genes involved DNA expression regulation,immune regulation,neuronal apoptosis,glucose metabolism,stress response,biosynthesis,and were associated with systemic lupus erythematosus and viral carcinogenesis.There were 81 effective differentially expressed genes of fluoxetine intervention,among which 5 genes were significantly therapeutic(Tppp3,Fst,Pmp22,Kdr,AABR07051689.1).The differential genes functions involved DNA and transcriptional regulation,glycolipid metabolism,cell adhesion,endoplasmic reticulum stress,nerve cell migration,development of tissue and cell.There were 10 common effective differential genes of the two drugs in the treatment of liver-qi stagnation syndrome,among which 1 gene was significantly therapeutic(AABR07051689.1),The differential genes functions involved cell metabolism,neuronal apoptosis,apoptotic signaling pathway,endoplasmic reticulum stimulation,stress response,and viral carcinogenesis.(4)Molecular pathway and target analysis of correlation between liver-qi stagnation syndrome and Chaihu-Shugan-San:The correlation between protein interaction,GO analysis,KEGG Pathway analysis and connotation of liver-qi stagnation syndrome were analyzed.The key genes of the model and drug function were verified by PCR.The results showed that the potential biomarkers of peripheral blood leukocytes in rat model of liver-qi stagnation syndrome were Src,Kdr,Tal1,Fn1,Id2,Gpr183,LTA4H,Rab4a and Cyp2s1;The effective target genes of Chaihu-Shugan-San were Lta4h,Cyp2s1,Wfs1;the effective target gene of fluoxetine was Pmp22;the target of the two drugs was Src.Conclusion:(1)There was a disorder of the NEI network in the rat model with liver-qi stagnation syndrome.(2)The characteristics of gene transcription at peripheral blood leukocytes in the rat model of liver-qi stagnation syndrome involved NEI disorder,accompanied by abnormal changes in blood circulation and cell metabolism,and related with digestion,reproduction,circulatory system,as well as relating to the occurrence of cancer,systemic lupus erythematosus,diabetes,cardiovascular diseasecancer,systemic Lupus erythematosus,diabetes,and cardiovascular disease;Src,Kdr,Tall,Fnl,Id2,Gpr183,LTA4H,Rab4a,and Cyp2sl which were involving regulation of nerve,immunity,hematopoiesis and metabolic function were potential biomarkers in peripheral blood leukocytes of liver-qi stagnation syndrome.(3)Chaihu-Shugan-San and fluoxetine had effects on the appearance of TCM syndromes,related laboratory indicators and NEI in the rat model with liver-qi stagnation syndrome,and were more extensive on the leukocytes transcriptome of model rats.The common effects of which were related to the nervous system,metabolism and intracellular damage as well as stress response,the role of Chaihu-Shugan-San seemd to be more extensive and accurate,especially the regulation of the immune link.(4)The leukocyte genes Lta4h,Cyp2sl and Wfsl involved in the regulation of neuro-immune function may be the molecular basis of the association between Chaihu-Shugan-San and liver-qi stagnation syndrome.This study explored the physiological connotation of liver-qi stagnation syndrome from the perspective of NEI network.The transcriptome technique was used to analyze the transcriptomics of leukocyte genes in rats with liver-qi stagnation syndrome,the effects of Chaihu-Shugan-san and fluoxetine on this model rats,as well as the potential molecular markers,the role of drug intervention and its molecular targets of leukocyte in the model rats.This study provided a understanding of the modern biological connotation of liver-qi stagnation syndrome,and the molecular basis of correlation between the liver-qi stagnation syndrome and Chaihu-Shugan-San.