Experience Summary Of Treating Precocious Puberty In Girls Based On "Homogeny Of Liver And Kidney" And Mechanism Study Of Shugan Xiehuo Formula In Treating CPP

Objective:1.Summarize the tutor's clinical experience and medication rules in the treatment of girls'precocious puberty based on "homogeny of liver and kidney";2.To explore the mechanism of Shugan Xiehuo Formula in the treatment of CPP.Based on the pituitary GnRH receptor signal transduction system,around the post GnRH receptor PKC-MAPK signal pathway and estrogen feedback mechanism,using pathological.and RT-qPCR?Western Blot?ELISA?IHC,from in vivo to in vitro cell experiments,to explore the regulatory effect of Shugan Xiehuo Formula on pituitary,reveal its effect on the vertical GTH secretion and estrogen feedback regulation,and further clarify the regulatory mechanism of Shugan Xiehuo Formula on HPG axis.Method:1.Experience summary:by means of outpatient consultation with teachers and teacher guidance,and using data analysis technology,the clinical experience and medication rules of tutors in the treatment of girls' precocious puberty were summarized.2.Animal experiment:rats were divided into control group,model group,leuprorelin group and high,medium and low dose groups of Shugan Xiehuo Formula(SXF).CPP model was established by subcutaneous injection of NMA acid,and leuprorelin was used as positive control.High,medium and low doses of Shugan Xiehuo Formula were given by gavage respectively.The rats in CPP group were killed at the first proestrus,and the other groups were killed at the same time according to the ratio of 1:1.The hypothalamus,pituitary,ovary,uterus and peripheral blood were collected.The body weight and sex organ weight of rats were measured.The follicle maturity and uterine wall thickness were observed.The expression levels of FSH,LH,PRL and E₂ in peripheral blood were detected by ELISA.The mRNA and protein expression levels of GnRH,GnRHR,ERa and GPR30 were detected by RT-qPCR and Western blot.The expression of GnRH in hypothalamus and GnRHR in ovary were detected by immunohistochemistry method.3.Cell experiment:in cell experiment,cell viability was calculated by CCK-8 method.Firstly,the effect of leptin(LEP)on pituitary cells was analyzed,and the optimal concentration of leuprorelin(LP)for treatment of pituitary cells in vitro was determined.To investigate the effect of SXF on pituitary cells induced by LEP,rats were divided into three groups:LEP group,LEP+LP group(model+leuproline positive drug intervention group)and LEP+SXF group(model+serum containing SXF drug intervention group).In order to study the effect of SXF on pituitary cells treated with LEP through PKC-MAPK pathway,they were divided into 2 groups:LEP group(CPP model group),LEP+SXF group(model+serum containing SXF intervention group),LEP+SXF+HY-n2180 group(model+serum containing SXF intervention+PKC activator group).The levels of FSH and LH were detected by ELISA.The mRNA and protein levels of GnRHR,ER? and GPR30 were detected by RT-qPCR and Western blot.The expression levels of PKC,p38 MAPK,EPK and JNK were detected by Western blot.Result:1.Based on the theory of "homogeny of liver and kidney",Professor Xiong lie put forward the treatment concept of "treating girls' precocious puberty from liver,regulating treatment as a whole and differentiating treatment by stages",and Shugan Xiehuo Formula is an effective prescription for treating girls' precocious puberty.2.Animal experiment:compared with the normal development group,the bilateral ovaries and uterus of the model group were significantly larger,the mature follicles became more and the uterine wall became thicker;while the SXF group and the positive drug group could inhibit the development of sexual organs.In the model group,the expression levels of FSH,LH,PRL,E₂,GnRH protein in hypothalamus,GnRH,GnRHR,ER? and GPR30 in pituitary were detected Compared with the model group,the above indexes were alleviated in leuproline group and SXF high,medium and low dose groups,in which CPP-HD-SXF group and CPP-LP group had the most significant remission.At the same time,it is suggested that the above effects of Shugan Xiehuo Formula are dose-dependent.3.Cell experiment:for group 1,the secretion levels of FSH and LH,the mRNA and protein expression levels of GnRHR,er ? and GPR30,the transcription and protein expression levels of PKC,p38MAPK,EPK and JNK in LEP group were significantly higher than those in LEP+LP group and LEP+SXF group,and the above indexes in LEP+LP group and LEP+SXF group were significantly lower than those in LEP group.For group 2,the protein expression levels of PKC,p38 MAPK,EPK and JNK in LEP+SXF group and the expression levels of FSH and LH in medium were significantly lower than those in LEP group,while the above indexes in LEP+SXF+HY-n2180 group were significantly higher than those in LEP+SXF group.At the same time,the results showed that hy-n2180 could partially reverse the inhibitory effect of SXF on PKC-MAPK pathway in pituitary cells.SXF alleviated the secretion of FSH and LH by inhibiting PKC-MAPK pathway.Conclusion1.Shugan Xiehuo Formula is an effective prescription for treating precocious puberty of girls based on the theory of "homogeny of liver and kidney".The core drugs are chinese thorowax root,white paeony root,common selfheal fruit-spik,chinese angelica,glossy privet fruit,yerbadetajo herb,peppermint,which reflect the characteristics of treating from the liver and regulating as a whole.The tutor is good at using Yunnan local drugs for addition and subtraction,which reflects the characteristics of adjusting measures to local conditions.2.Shugan Xiehuo Formula may inhibit the development of sexual organs and the activation of HPG axis by inhibiting the expression of GnRH in hypothalamus and pituitary,regulating the expression of ERa and GPR30,thus inhibiting the secretion of estrogen,FSH and LH.3.Shugan Xiehuo Formula may alleviate CPP by inhibiting PKC-MAPK signaling pathway and reducing the expression of estrogen receptor and gonadotropin..