Effect Of Xihuang Pill On H-P-O Axis Isolated Cells And Proliferating Mamary Epithelial Cells

Objective:By studying the effect of rat serum containing Xihuang Pills on the function of hormones and receptors secreted by hypothalamic neurons cells,adenohypophysis cells,and ovarian granulosa cells on the hypothalamic-pituitary-ovarian axis.The effect of rat serum containing Xihuang Pills on mammary epithelial cells intervened by E₂and P.To explore the regulatory effect of Xihuangwan on hypothalamic-pituitary-ovarian axis,and to elucidate the mechanism of anti-mammary hyperplasia.Methods:SD female non-pregnant rats were divided into blank group,1.8 mg?kg^-1?d^-1dose group of tamoxifen group,Xihuang Pill 2.16 g?kg^-1?d^-1dose group,Xihuang Pill 1.08 g?kg^-1?d^-1dose group,Xihuang Pill 0.54 g?kg^-1?d^-1dose group,and rats were given gavage 7 days,blood is taken to prepare drug-containing serum.Hypothalamus neuron cells,adenohypophysis cells and ovarian granulosa cells cultured in vitro were divided into blank group,tamoxifen group,Xihuang Pill high,medium and low dose groups.After the administration of serum containing drugs,the following tests were performed on hypothalamic neurons:CCK-8 method to detect cell survival rate;ELISA method to detect Gn RH content;Western blot detection of kisspeptins-1,GPR54 protein expression.After administration,the following tests were performed on the adenohypophysis cells:CCK-8 method to detect survival rate,ELISA method to detect FSH and LH content,and Western blot to detect Gn RHR protein expression.Ovarian granulosa cells were subjected to the following tests:CCK-8 method to detect cell survival rate;ELISA method to detect E₂and P content;Western blot to detect FSHR and LHR protein expression.The isolated and cultured mammary epithelial cells were given E₂and P interventions,and the optimal proliferation concentration was screened using the CCK-8 method.Cells were divided into blank group,proliferation group,tamoxifen group,Xihuang Pill high,medium and low dose groups,except for the blank group,the optimal concentration of E₂and P intervention was given,and the following tests were performed after administration:CCK-8 methods to detect cell inhibition,Giemsa staining was used to observe changes in cell morphology,flow cytometry was used to detect the apoptosis rate,immunohistochemistry was used to detect the expression of Bax and Bcl^-2,and Western Blot was used to detect the expression of ERα,ERβand PR protein.Results:1.The rat serum containing Xihuang Pill had no effect on the survival rate of hypothalamic neuron cells,adenohypophysis cells and ovarian granulosa cells.Xihuangwan high,middle,low and tamoxifen groups can reduce the level of Gn RH hormone in hypothalamic neurons（P<0.01）,inhibit the expression of kisspeptins^-1and GPR54（P<0.01）;it can reduce adenohypophysis cells FSH hormone level（P<0.01）,increase LH hormone level（P<0.01）,inhibit Gn RHR protein expression（P<0.01）;can reduce E₂hormone content in ovarian granulosa cells（P<0.01）,increase P hormone Content（P<0.01）,inhibit FSHR protein expression（P<0.01）,promote LHR protein expression（P<0.01）.2.The rat serum containing Xihuang Pill can inhibit the growth of rat mammary epithelial cells intervened by E₂and P（P<0.05）,induce its apoptosis,and affect the expression of apoptosis-related proteins Bcl-2 and Bax（P<0.05）.And inhibit the expression of estrogen receptor ERα,ERβ.Conclusion:1.Xihuang Pill can affect the secretory function of hypothalamic neuron cells,adenohypophysis cells and ovarian granulosa cells.2.Xihuang Pill inhibits the proliferation of mammary epithelial cells and promotes the apoptosis of mammary epithelial cells.3.Xihuang Pill regulates the hypothalamic-pituitary-ovarian axis to play a role in preventing breast hyperplasia.