The Hyper-prolactin And The Mechanism Of Neuro-Endocrine-immune Of The Model Rats Of Liver-qi Stagnation And The Effection Of Relevant Formulas

This study base on the "the correlation between formula and syndrome’" and premised on the basis of relevance among Liver-gi stagnation(GY),prolactin(PRL)and neuroendocrine immune network(NEIN)and curative effects of chaihu shugan powder(CHSGS)to the model of Liver-gi stagnation rats.Base on the etiology of Chinese traditional medicine,this study use related pathogeny,the same batch rats to form the GY model rats.The first part of this study from the perspective that GY syndrome may exist immunologic dysfunction and PRL abnormality secretion and PRL might be involve in immune function regulation,to explore the state of immune function,the level of PRL and the changes of relevant regulators at different times and probes,then to discuss the modern connotation of GY syndrome in TCM from the perspective of the correlation between PRL and NEIN.The second part of the research based on the relevance among GY syndrome,PRL,cellular immunity and molecular signaling pathways to explore the PRL,immunocyte functions,PRLR and associated proteins and target gene in the signaling pathways from the perspective of systems biology and molecular biology,then to observe and compare the effects of CHSGS on the changes in the above exploration of GY models in Rat.In addition to this aspect to discuss the immune abnormality mechanism in GY models rats with hyperpro-lactinemia and the effects of CHSGS.The study falls into literature review and experimental research.Literature research mainly involved in the GY syndrome and its connotation of modern Nerve-endocrine-immunology and the experimental and clinical research of prolactin immune adjustment.The experimental research including research part one and research part two.The first part of the research focused on the level of prolactin secretion and the state of Nerve-endocrine-immunology at different times and probes.The second part of the research focused on the immune abnormality mechanism in GY models rats with hyperpro-lactinemia and the effects of CHSGS.Section A:The function of immune system and the change of prolactin in rats of liver qi stagnationMethods:Rats were randomly divided into normal control group,GY group,36 rats in each group.Adopted by chronic restraint stress to form the model.Observe and record all of the emotional behavior in rats,hair color,diet,appearance of urine,and so on.Measured the body weight every week.On the 15th,22th,29th day,the 10%of chloral hydrate anesthesia animals,abdominal aortic blood,separate hypothalamus at low temperature respectively.Elisa method to test Plasma NE,E,IL-2,IL-6,y-IFN,TNF-a and the content of 5-HT、DA in hypothalamus.Radioimmunoassay method to test plasma CORT,PRL and the content of TRH in hypothalamus.Flow cytometry method to test CD3+T、CD4+T、CD4+T/CD8+T in blood.Statistical methods:the data were described by(x±SD)and the number of samples compared between groups used independent-samples T test:non-normal data using non-parametric statistical analysis.All data used SPSS17.0 statistics.Experimental results:Stimulation after two weeks,GY groups starts to irritability,gradually turning indifference and struggle to reduce depression,expression,and companion see diet reduce,defecate is dry.Compared with normal group,the model rats shown the abnormal changes of behavior and the decrease of their weight at the end of 2wth,3wth and 4wth(p<0.05).The score of activity and emotional in GY rats increased markedly at the end of 2wth,3wth and 4wth(p<0.05 or p<0.01),the score of hair increased markedly at the end of 4wth(p<0.05).the sucrose consumption rate increased markedly at the end of 2Wth and 4Wth(p<0.01).The content of NE,E,CORT,PRL,TNF-α,γ-IFN,CD3,CD4 in blood increased markedly,the content of IL-6 in plasma and the content of 5-HT and DA in hypothalamus declined markedly in model rats at the end of 2Wth(p<0.05 or p<0.05).The content of NE,E,CORT,PRL in plasma and the content of 5-HT and TRH in hypothalamus increased markedly,the content of IL-2,IL-6,TNF-α,γ-IFN,CD3,CD4,CD4/CD8 in blood declined markedly in model rats at the end of 3Wth and 4Wth(p<0.05 or p<0.01).Section B:The hyper-prolactin and the mechanism of neuro-endocrine-immune of the model rats of liver-qi stagnation and the effection of relevant formulasMethods:Rats were randomly divided into normal control group,GY group,high dosage CHSGS group,low dosage CHSGS group,bromocriptine group and fluoxetine group,12 rats in each group.Adopted by chronic restraint stress to form the model.Observe and record all of the emotional behavior in rats,hair color,diet,appearance of urine,and so on.Measured the body weight every week.On the 15th day,high dosage CHSGS group rats were given CHSGS 1.24 g·1000g-1·d-1,low dosage CHSGS group rats were given CHSGS 0.31 g·1000g-1·d-1,bromocriptine group rats were given bromocriptine 0.22mg· 1000g-1·d-1 and fluoxetine group rats were given fluoxetine 1.8 mg·1000g-1·d-1 accordingly.On the 29th day,the 10%of chloral hydrate anesthesia animals,abdominal aortic blood,separate hypothalamus at low temperature respectively.Elisa method to test Plasma NE,E,IL-2，IL-6,y-IFN,TNF-a and the content of 5-HT、DA in hypothalamus.Radioimmunoassay method to test Plasma CORT,PRL and the content of TRH in hypothalamus.Flow cytometry method to test CD3+T、CD4+T、CD4+T/CD8+T in blood.Protein immunoblot method(Western blotting)to test the expression of STAT5 protein and PRLR protein.Real-time fluorescent quantitative PCR method(RQ-PCR)to test the expression of IRF-1 gene in T lymphocytes.The data were described by(x±SD)and the number of samples compared between groups used one-way ANOVA(one-way ANOVA)test;non-normal data using non-parametric statistical analysis.All data used SPSS17.0 statistics software.Experimental results:Stimulation after two weeks,GY groups starts to irritability,gradually turning indifference and struggle to reduce depression,expression,and companion see diet reduce,defecate is dry.Compared with normal group,the model rats shown the abnormal changes of behavior and the decrease of their weight at the end of lwth,2wth,3wth and 4wth(p<0.01).The score of activity and emotional in GY rats increased markedly at the end of 2wth,3wth and 4wth(p<0.05 or p<0.05),the score of hair increased markedly at the end of 4wth(p<0.05).the sucrose consumption rate increased markedly at the end of 2wth and 4wth(p<0.01).The the crossed squares,number of rearing,the total distance of movement and the stay period in the central zone show increased(p<0.01),the decorating time show decreased(p<0.01)at the end of 2Wth.The crossed squares,number of rearing,the total distance of movement and the decorating time show decreased(p<0.05 or p<0.05),the stay period in the central zone show increased(p<0.01).The content of NE,E,CORT,PRL in plasma,the content of 5-HT and TRH in hypothalamus and expression of PRL in hypophysis increased markedly,the content of IL-2,IL-6,TNF-α,γ-IFN,CD3,CD4,CD4/CD8 in blood and the expression of STAT5 protein,PRLR protein and IRF-1 gene in T lymphocytes declined markedly in model rats at the end of 4wth(p<0.05 or p<0.01).Compared with model groups,the mood improved obviously,activity increased slightly,diet has increased in high dosage CHSGS group and fluoxetine group,there is no obvious changes in low dosage CHSGS group and bromocriptine group.The weight,sucrose consumption rate,crossed squares,the total distance of movement and the content of IL-2,IL-6,TNF-α,γ-IFN,CD3,CD4,CD4/CD8 in blood and the expression of STAT5 protein,PRLR protein and IRF-1 gene in T lymphocytes all improved obviously and the score of activity,emotional and hair,the stay period in the central zone and the content of NE,E,CORT,PRL in plasma,the content of 5-HT and TRH in hypothalamus and expression of PRL in hypophysis decreased markedly in high dosage CHSGS group rats(p<0.05 or p<0.01).The weight,sucrose consumption rate,crossed squares,the total distance of movement,the decorating time and the content of IL-6,TNF-α,γ-IFN,CD4/CD8 in blood all improved obviously,the score of activity,emotional and hair,the stay period in the central zone and the content of NE,E,CORT,PRL in plasma and expression of PRL in hypophysis,the expression of IRF-1 gene in T lymphocytes all decreased markedly in fluoxetine group rats(p<0.05 or p<0.01).The content of PRL in plasma and TRH in hypothalamus and expression of PRL in hypophysis decreased markedly,the content of IL-6,CD3,CD4,CD4/CD8 in blood and the expression of STAT5 protein,PRLR protein and IRF-1 gene in T lymphocytes all improved obviously in bromocriptine group(p<0.05 or p<0.01).Compared with high dosage CHSGS group,the score of emotional,crossed squares,the total distance of movement and the number of CD3,CD4,CD4/CD8 in blood and the expression of STAT5 protein,PRLR protein and IRF-1 gene in T lymphocytes all decreased obviously,the stay period in the central zone and sucrose consumption rate increased obviously in fluoxetine group(p<0.05 or p<0.01).The score of activity,emotional,the stay period in the central zone,the content of PRL in plasma and TRH in hypothalamus all increased obviously,the sucrose consumption rate,crossed squares,the total distance of movement,the content of IL-6,CD3,CD4,CD4/CD8 in blood and the expression of PRLR protein and IRF-1 gene in T lymphocytes all decreased obviously(p<0.05 or p<0.01)in low dosage CHSGS group.The score of activity,emotional and the content of 5-HT in hypothalamus all increased obviously,the sucrose consumption rate and the expression of PRLR protein all decreased obviously in bromocriptine group(p<0.05 or p<0.01).Conclusion:The research based on the exploration of the level of the state of immune function,the level of PRL and the changes of relevant regulators at different times and probes in GY model rats due to chronic restraint stress,then observed that the GY model rats exist persistent elevated prolactin,over-active HPA axis and the abnormal state of Nerve-endocrine-immunology(central single amine neurotransmitter appear to reduce gradually recovering,reduced immune function after rising first).Based on this research,the section B based on the relevance among GY syndrome,PRL,cellular immunity and molecular signaling pathways to explore the PRL,immunocyte functions,PRLR and associated proteins and target gene in the signaling pathways from the perspective of systems biology and molecular biology,then to purpose the abnormal immune mechanism of GY model rats.The results suggest that the change of the body’s immune function involved in T cell and its abnormal conduction of JAK-STAT signal pathway in GY model,the high levels of PRL caused PRL on the T cell membrane receptor expression reduced may play important roles.Moreover,the over-active HPA axis may also influence the immune function of GY models,but the exact regulation mechanism is still to be further exploration.The CHSGS had comprehensive effect on the function of the syndrome of GY and disease physiological indexes(Nerve-endocrine-immunology)on this model.The fluoxetine had comprehensive effect on the function of the syndrome of GY,had effect on the function of part of the disease physiological indexes,had no obvious effect on immune system.The bromocriptine had no obvious effect on the function of the syndrome of GY,had effect on the hyperprolactinemia and immune system.The CHSGS and fluoxetine all had comprehensive effect on the function of the syndrome of GY and part indexes of Nerve-endocrine,prompt that there are some doublication in the connotation between GY model and depression in Western medicine.The CHSGS and bromocriptine all had comprehensive effect on immune system and the effect involved in PRL central adjustment and the adjustment of the PRL-JAK-STAT signaling pathway of T lymphocytes,the results show that there is abnormal adjustment in PRL-immune system-T lymphocytes-PRL-JAK-STAT in GY model rats,also this founding provide strong evidence for the hypothesis that low immune function in GY syndrome has relation to the abnormal secretion of PRL.