| Objective1.To develop Jiuweichushi formula as a stable,safe and effective granule to benefit more children with precocious puberty.2.To explore the mechanism and target of Jiuweichushi formula in the treatment of CPP.In this study,female CPP model rats established by NMA were selected as the research object,and the effects of Jiuweichushi formula on vaginal opening,gonadal development,adverse psychological behavior,serum LH,FSH,E2 expression levels,and GnRH,SIRT1,Kiss-1,GPR54 gene as well as protein expression in hypothalamus and ovarian tissue of CPP rats were investigated.To investigate whether the intervention of Jiuweichushi formula in CPP rats is related to SIRT1/Kiss-1 signaling pathway,and to further clarify the mechanism and target of Jiuweichushi formula.Methods1.The preparation process and quality standard of Jiuweichushi formula were studied to determine the extraction process and quality standard of Jiuweichushi formula,and to provide the relevant pharmacological quality control basis for Jiuweichushi formula widely used as a hospital preparation in children with clinical precocious puberty.2.26-day-old SPF female SD rats were randomly divided into normal control group(hereinafter referred to as normal group),CPP model group(hereinafter referred to as model group),Jiuweichushi formula high-dose,medium-dose and low-dose groups(hereinafter referred to as high-dose,medium-dose and low-dose groups),and Gonadorelin group(hereinafter referred to as western medicine group),with 8 rats in each group.Except the normal group,the other groups were subcutaneously injected with NMA(40mg/kg,dissolved in 0.2ml normal saline)at 14:00 and 16:00,respectively,to establish the CPP rat model.The normal group was given 0.9%sodium chloride injection subcutaneously at the same time(0.2ml/time).At the same time,the western medicine group was given subcutaneous injection of Gonadorelin,and the Chinese medicine group was given high,medium and low doses of Jiuweichushi formula by intragastric administration.During the intervention period,the experimental conditions of each group were as consistent as possible,except for the specific programs of special intervention treatment.The rats in the normal group,model group and high-dose,medium-dose and low-dose groups were given subcutaneous injection of normal saline at the same time and place as those in the western medicine group.The rats in the normal group,model group and western medicine group were given gavage of normal saline at the same time as those in high-dose,medium-dose and low-dose groups.After the intervention,the general condition,body weight,vaginal opening and estrous cycle of the rats were observed,and forced swimming experiment,sugar water preference experiment and open field experiment were carried out.The opening condition of the vaginal opening of the model group rats was observed one by one every day,and the vaginal shedding cells of the opened rats were examined by smear to determine the sexual cycle,drug withdrawal and execution time.According to the test requirements,hypothalamus,uterus,ovarian tissue and peripheral blood were collected.HE staining was used to observe and measure the endometrial thickness,luteal number and follicle number of the ovary in each group.The expression levels of serum LH,FSH and E2 in each group were detected by ELISA.The expression of GnRH gene and protein in hypothalamus and ovary were detected by RT-qPCR and WB.3.26-day-old SPF female SD rats were randomly divided into normal control group(hereinafter referred to as normal group),CPP model group(hereinafter referred to as model group)and Jiuweichushi formula group(hereinafter referred to as traditional Chinese medicine group).Gonadorelin group(hereinafter referred to as Western medicine group),SIRT1 activator group(hereinafter referred to as activator group),Jiuweichushi formula+SIRT1 inhibitor group(hereinafter referred to as inhibitor group),8 animals in each group.The molding method is the same as before.At the same time,the western medicine group was given subcutaneous injection of Gonadorelin,the Chinese medicine group was given gavage of Jiuweichushi formula(2.87ml/Kg),the activator group was given gavage of resveratrol solution(100mg/kg),and the inhibitor group was given gavage of Jiuweichushi formula(2.87ml/Kg)combined with EX-527(5mg/kg).The time of drug withdrawal and execution was the same as before.The hypothalamus and ovarian tissues of rats were collected according to the test requirements.The expressions of SIRT1,Kiss-1 and GPR54 genes and proteins in hypothalamus and ovary were detected by RT-qPCR and WB.Results1.Study on the preparation technology and quality standard of Jiuweichushi formula1.1The raw material of Jiuweichushi formula was extracted by decocting method.The content of paeoniflorin and the extraction rate were taken as evaluation indexes.In the verification test,the extraction rate of 3 batches of prescription medicinal materials was stable,which was consistent with the expectation of the optimal extraction process,indicating that the optimal extraction process was stable and feasible.1.2The amount of dextrin and concentration of wetting agent were investigated by wet granulation method,and the forming process of Jiuweichushi formula was optimized as follows:Jiuweichushi formula extract was added with dextrin in a ratio of 1:1,mixed,granulated,and then,after drying,the whole granules were screened with 12 mesh and packed according to 10g per bag to get the finished product.1.3A TLC identification method was established for the two main medicinal materials in Jiuweichushi formula.The results showed that the spots were clear,negative and without interference,and the presence of Phellodendri phellodendri and Bupleuri vinegar in Jiuweichushi formula could be quickly identified.Quantitative analysis:The representative effective component paeoniflorin was selected as the evaluation index to establish HPLC content determination method.The linear relationship between paeoniflorin content and peak area was good in the range of 19.1-286.5 μg/mL,and the regression equation was Y=12745X+5610(r=0.9999).The method of HPLC determination of paeoniflorin was proved to be accurate,simple and feasible.The content of paeoniflorin in three batches of Jiuweichushi formula ranged from 1.4327 to 1.4979mg/g,with an average of 1.4620mg/g and RSDS of 1.47%.According to the content determination results of tested products,combined with the content of paeoniflorin in this batch of raw materials and the content limit requirements of Chinese Pharmacopoeia(2020 edition)on Chinese herbal decoction pieces of Paeoniflorin,the content limit of this product is tentatively determined to be no less than 4.0mg per bag(10g)containing paeoniflorin(C23H28O11).The properties,particle size,water content,solubility,difference of dosage and microbial limit of the preparation were examined,and the results were in accordance with the provisions of Chinese Pharmacopoeia(2020 edition).2.Effects of Jiuweichushi formula on the general behavior,psychological behavior and histomorphology of female CPP rats2.1 General Status of rats in each groupIn the process of modeling,a small amount of blood oosed from the injection site was occasionally observed in each group of rats,which could recover by itself after cleaning with 75%medical disinfection alcohol;During the experiment,the feeding,drinking and activity of the rats in each group were normal,the growth condition was good,the hair was smooth and glossy,the mental state was good,the body weight kept increasing,and there was no abnormal death.2.2 Body Mass of rats in each groupBefore intervention,there was no significant difference in body mass of rats among all groups.After intervention,the body weight of rats in model group and low-dose group was significantly higher than that in normal group.Compared with model group,body weight of western medicine group,high dose group and medium dose group was significantly lower.The above differences were statistically significant(P<0.05).2.3 The rat’s vaginal opening time in each groupCompared with the normal group,the opening time of the vaginal opening of rats in the model group and the low-dose group was significantly earlier;Compared with model group,the opening time of vulva in western medicine group,high-dose group and medium-dose group was significantly delayed.The above differences were statistically significant(P<0.05).2.4 Smear of vaginal exfoliated cells of rats in model groupModel group of rats establish a stable and regular estrous cycle.2.5 Endometrial thickness,luteal number and follicle number in rats in each groupCompared with normal group,endometrial thickness,luteal number and follicle number of rats in model group were significantly increased;Compared with model group,endometrial thickness in western medicine group,high dose group and medium dose group was significantly reduced.The above differences were statistically significant(P<0.05).Compared with normal group,the corpus luteum number in model group and low dose group was significantly increased.Compared with model group,corpus luteum number in western medicine group,high-dose group and medium-dose group was significantly reduced.Compared with the medium dose group,the corpus luteum number in the low dose group was significantly increased.The above differences were statistically significant(P<0.05).Compared with the normal group,the number of ovarian follicles in the model group was significantly increased.Compared with model group,the number of ovarian follicles in western medicine group,high dose group and medium dose group was significantly reduced.The above differences were statistically significant(P<0.05).2.6 Bad psychological behavior2.6.1 Forced swimming experimentCompared with the normal group,the immobile time of rats in the model group and the low-dose group was significantly increased;Compared with model group,immobile time in western medicine group,high-dose group,medium-dose group and low-dose group was significantly shortened.The above differences were statistically significant(P<0.05).2.6.2 Sugar preference experimentCompared with the normal group,the sucrose preference rate of the model group was significantly decreased(P<0.05).Compared with the model group,the sucrose preference rate of the western medicine group,the high dose group,the middle dose group and the low dose group increased slightly,but the difference was not statistically significant(P>0.05).2.6.3 Open-field experimentCompared with normal group,the total distance of rats was significantly reduced in model group,western medicine group,high-dose group,medium-dose group and low-dose group;Compared with model group,the total distance of exercise in high-dose and medium-dose groups was significantly increased.Compared with western medicine group,the total distance of exercise in high-dose and medium-dose groups was significantly increased.Compared with the high-dose group,the total distance of exercise in the low-dose group was significantly reduced.Compared with the medium dose group,the total exercise distance of rats in the low dose group was significantly reduced,The above differences were statistically significant(P<0.05),indicating that Jiuweichushi formula has dose-effect relationship in regulating the total exercise distance of CPP rats.Compared with normal group,the residence time in the central region of rats in model group,western medicine group,high dose group,medium dose group and low dose group was significantly reduced;Compared with model group,the residence time in the central region of rats in high-dose and medium-dose groups was significantly increased.Compared with western medicine group,the retention time in the central region of rats in high-dose and medium-dose groups was significantly increased.Compared with the high-dose group,the residence time in the central region of rats in the low-dose group was significantly reduced.Compared with the medium-dose group,the residence time in the central region of the low-dose group was significantly reduced,The above differences were statistically significant(P<0.05),which indicated that Jiuweichushi formula could regulate the residence time in the central region of CPP rats.3.Effect of Jiuweichushi formula on the expression levels of serum LH,FSH and E2 in female CPP rats3.1 Serum LH levels in rats in each groupCompared with normal group,the expression level of serum LH in model group,western medicine group,medium dose group and low dose group was significantly increased;The expression level of LH in western medicine group,high-dose group and medium-dose group was significantly decreased compared with that in model group.Compared with the high-dose group,the LH expression level in the low-dose group was significantly increased.The above differences were statistically significant(P<0.05).3.2 Serum FSH levels in rats in each groupCompared with normal group,FSH expression level in model group,medium dose group and low dose group was significantly increased;The expression level of FSH in western medicine group,high-dose group,medium-dose group and low-dose group was significantly decreased compared with model group.Compared with western medicine group,the expression level of FSH in medium dose group and low dose group was significantly increased.Compared with the high-dose group,the expression level of FSH in the low-dose group was significantly increased.Compared with the medium dose group,the expression level of FSH in the low dose group was significantly increased.The above differences were statistically significant(P<0.05).3.3 Serum E2 levels in rats in each groupCompared with the normal group,the expression level of E2 in model group,high-dose group,medium-dose group and low-dose group was significantly increased;The expression level of E2 in western medicine group,high-dose group and medium-dose group was significantly decreased compared with that in model group.Compared with western medicine group,the expression level of E2 in high-dose,medium-dose and low-dose groups was significantly increased;Compared with the high-dose group,the expression level of E2 in the low-dose group was significantly increased.The above differences were statistically significant(P<0.05).4.Effect of Jiuweichushi Formula on GnRH gene and protein expression in hypothalamus and ovary of female CPP rats4.1 Comparison of GnRH gene expression levels in hypothalamus of rats in each groupCompared with normal group,the expression level of GnRH gene in hypothalamus of rats in model group,western medicine group,high-dose group,medium-dose group and low-dose group was significantly increased;The expression level of GnRH gene in hypothalamus of western medicine group,high-dose group and medium-dose group was significantly decreased compared with model group.Compared with western medicine group,GnRH gene expression level in hypothalamus of rats in low-dose group was significantly increased;Compared with high-dose group,GnRH gene expression in hypothalamus of rats in low-dose group was significantly increased.Compared with the medium-dose group,the GnRH gene expression level in the hypothalamus of rats in the low-dose group was significantly increased,indicating that Jiuweichushi formula can regulate the GnRH gene expression level in the hypothalamus of CPP rats.The above differences were statistically significant(P<0.05).4.2 Comparison of GnRH gene expression level in ovary of rats in eachgroupCompared with the normal group,GnRH gene expression level in ovary of rats in model group,western medicine group,high-dose group,medium-dose group and low-dose group was significantly increased;The expression level of GnRH gene in ovary of western medicine group,high-dose group,medium-dose group and low-dose group was significantly decreased compared with model group.Compared with western medicine group,GnRH gene expression in ovary of rats in high-dose,medium-dose and low-dose groups was significantly increased;Compared with the high-dose group,the expression level of GnRH gene in ovary of rats in low-dose group was significantly increased.Compared with the medium-dose group,the expression level of GnRH gene in the ovary of rats in the low-dose group was significantly increased,The above differences were statistically significant(P<0.05),indicating that Jiuweichushi formula has dose-effect relationship in regulating the expression level of GnRH gene in the ovary of CPP rats..4.3 Comparison of GnRH protein expression level in hypothalamus of rats in each groupCompared with normal group,GnRH protein expression level in hypothalamus of rats in model group,western medicine group,high-dose group,medium-dose group and low-dose group was significantly increased;The expression level of GnRH protein in hypothalamus of western medicine group,high-dose group,medium-dose group and low-dose group was significantly decreased compared with model group.Compared with western medicine group,GnRH protein expression level in hypothalamus of rats in medium dose group and low dose group was significantly increased.The above differences were statistically significant(P<0.05).4.4 Comparison of GnRH protein expression level in ovary of rats in each groupCompared with the normal group,GnRH protein expression level in ovary of rats in model group,western medicine group,high-dose group,medium-dose group and low-dose group was significantly increased;Compared with model group,the expression level of GnRH protein in ovary in western medicine group,high-dose group and medium-dose group was significantly decreased.Compared with western medicine group,the expression level of GnRH protein in ovary of rats in high-dose,medium-dose and low-dose groups was significantly increased.The above differences were statistically significant(P<0.05).5.Effects of Jiuweichushi formula on SIRT1/Kiss-1 signaling pathway of female CPP rats5.1 Expression levels of SIRT1 gene in hypothalamus and ovary of rats in each groupCompared with normal group,the expression level of SIRT1 gene in hypothalamus of rats in model group,western medicine group,Chinese medicine group,inhibitor group and activator group was significantly lower;The expression level of SIRT1 gene in hypothalamus of rats in western medicine group,Chinese medicine group,inhibitor group and activator group was significantly higher than that in model group.Compared with the western medicine group,the expression level of SIRT1 gene in hypothalamus of rats in the activator group was significantly higher,while that in the inhibitor group was significantly lower.The expression level of SIRT1 gene in hypothalamus of rats in inhibitor group was significantly lower than that in TCM group.Compared with the inhibitor group,the expression level of SIRT1 gene in hypothalamus of rats in the activator group was significantly higher.The above differences were statistically significant(P<0.05).Compared with normal group,the expression level of SIRT1 gene in ovary in model group,western medicine group,Chinese medicine group,inhibitor group and activator group was significantly lower.The expression level of SIRT1 gene in ovary of western medicine group,Chinese medicine group,inhibitor group and activator group was significantly higher than that of model group.Compared with western medicine group,the expression level of SIRT1 gene in ovary of rats in activator group was significantly higher,while that in inhibitor group was significantly lower.Compared with the traditional Chinese medicine group,the expression level of SIRT1 gene in ovary of rats in the activator group was significantly higher,while that in the inhibitor group was significantly lower.eompared with the inhibitor group,the expression level of SIRT1 protein in ovary of rats in the activator group was significantly higher.The above differences were statistically significant(P<0.05).5.2 Expression level of Kiss-1 gene in hypothalamus and ovary of rats in each groupCompared with normal group,Kiss-1 gene expression level in hypothalamus and ovary of rats was significantly higher in model group,western medicine group,Chinese medicine group,inhibitor group and activator group;The expression level of Kiss-1 gene in hypothalamus of western medicine group,Chinese medicine group,inhibitor group and activator group was significantly lower than that of model group.Compared with western medicine group,Kiss-1 gene expression level in hypothalamus of rats in inhibitor group was significantly higher;Compared with the Chinese medicine group,Kiss-1 gene expression level in the hypothalamus of rats in the inhibitor group was significantly higher.Compared with the inhibitor group,the expression level of Kiss-1 gene in the hypothalamus of rats in the activator group was significantly lower.The above differences were statistically significant(P<0.05).Compared with normal group,Kiss-1 gene expression level in ovary of rats in model group,western medicine group,Chinese medicine group,inhibitor group and activator group was significantly higher;The expression level of Kiss-1 gene in ovary of western medicine group,Chinese medicine group,inhibitor group and activator group was significantly lower than that of model group.Compared with western medicine group,the expression level of Kiss-1 gene in ovary of rats in Chinese medicine group,inhibitor group and activator group was higher;Compared with the Chinese medicine group,the expression level of Kiss-1 gene in ovary of rats in the inhibitor group was significantly higher.Compared with the inhibitor group,the expression level of Kiss-1 gene in the ovary of rats in the activator group was significantly lower.The above differences were statistically significant(P<0.05).5.3 Expression of GPR54 gene in hypothalamus and ovary of rats in each groupCompared with normal group,GPR54 gene expression level in hypothalamus and ovary of rats was significantly higher in model group,Chinese medicine group,inhibitor group and activator group;The expression level of GPR54 gene in hypothalamus of western medicine group,Chinese medicine group and activator group was significantly lower than that of model group.Compared with western medicine group,GPR54 gene expression level in hypothalamus of rats in inhibitor group and activator group was significantly higher;Compared with the Chinese medicine group,the expression level of GPR54 gene in the hypothalamus of rats in the inhibitor group was significantly higher.Compared with the inhibitor group,the expression level of GPR54 gene in hypothalamus of rats in the activator group was significantly lower.The above differences were statistically significant(P<0.05).Compared with normal group,the expression level of GPR54 gene in ovary in model group,western medicine group,Chinese medicine group,inhibitor group and activator group was significantly higher;The expression level of GPR54 gene in ovary of western medicine group,Chinese medicine group and activator group was significantly lower than that of model group.Compared with western medicine group,the expression level of GPR54 gene in ovary of rats in Chinese medicine group,inhibitor group and activator group was significantly higher;Compared with the Chinese medicine group,the expression level of GPR54 gene in ovary of rats in the inhibitor group was significantly higher.Compared with the inhibitor group,the expression level of GPR54 gene in ovary of rats in the activator group was significantly lower.The above differences were statistically significant(P<0.05).5.4 SIRT1 protein expression levels in hypothalamus and ovary of rats in each groupCompared with normal group,SIRT1 protein expression level in hypothalamus of rats in model group,western medicine group,Chinese medicine group,inhibitor group and activator group was significantly lower;The expression level of SIRT1 protein in hypothalamus of western medicine group,Chinese medicine group,inhibitor group and activator group was significantly higher than that of model group.Compared with the western medicine group,the expression level of SIRT1 protein in hypothalamus of rats in the activator group was significantly higher than that in the inhibitor group;Compared with the Chinese medicine group,the expression level of SIRT1 protein in hypothalamus of rats in the activator group was significantly higher.Compared with inhibitor group,SIRT1 protein expression level in hypothalamus of rats in activator group was significantly higher.The above differences were statistically significant(P<0.05).Compared with normal group,the expression level of SIRT1 protein in ovary in model group,western medicine group,Chinese medicine group and inhibitor group was significantly lower.Compared with model group,western medicine group,Chinese medicine group,inhibitor group and activator group had significantly higher expression levels of SIRT1 protein in ovary.Compared with western medicine group,the expression level of SIRT1 protein in ovary of rats in TCM group and activator group was significantly higher,while that in inhibitor group was significantly lower.Compared with the Chinese medicine group,the expression level of SIRT1 protein in ovary of rats in the activator group was significantly higher,and that in the inhibitor group was significantly lower.Compared with the inhibitor group,the expression level of SIRT1 protein in ovary of rats in the activator group was significantly higher.The above differences were statistically significant(P<0.05).5.5 Expression level of Kiss-1 protein in hypothalamus and ovary of rats in each groupCompared with normal group,Kiss-1 protein expression level in hypothalamus and ovary of rats was significantly higher in model group,western medicine group,Chinese medicine group,inhibitor group and activator group;The expression level of Kiss-1 protein in hypothalamus of western medicine group,Chinese medicine group and activator group was significantly lower than that of model group.Compared with western medicine group,the expression level of Kiss-1 protein in hypothalamus of rats in Chinese medicine group,inhibitor group and activator group was significantly higher;Compared with the traditional Chinese medicine group,Kiss-1 protein expression level in the hypothalamus of rats in the inhibitor group was significantly higher.The above differences were statistically significant(P<0.05).Compared with normal group,the expression level of Kiss-1 protein in ovary of model group,western medicine group,Chinese medicine group,inhibitor group and activator group was significantly higher;The expression level of Kiss-1 protein in ovary of western medicine group,Chinese medicine group and activator group was significantly lower than that of model group.Compared with western medicine group,the expression level of Kiss-1 protein in ovary of rats in Chinese medicine group,inhibitor group and activator group was significantly higher;Compared with the Chinese medicine group,the expression level of Kiss-1 protein in ovary of rats in the inhibitor group was significantly higher.Compared with the inhibitor group,the expression level of Kiss-1 protein in the ovary of rats in the activator group was significantly lower.The above differences were statistically significant(P<0.05).5.6 Expression level of GPR54 protein in hypothalamus and ovary of rats in each groupCompared with normal group,GPR54 protein expression levels in hypothalamus and ovary of rats were significantly higher in model group,Chinese medicine group,inhibitor group and activator group;Compared with model group,western medicine group,Chinese medicine group,inhibitor group and activator group,the expression level of GPR54 protein in hypothalamus was significantly lower;Compared with western medicine group,the expression level of GPR54 protein in hypothalamus of rats in inhibitor group was significantly higher;Compared with the Chinese medicine group,the expression level of GPR54 protein in the hypothalamus of rats in the inhibitor group was significantly higher.Compared with the inhibitor group,the expression level of GPR54 protein in the hypothalamus of rats in the activator group was significantly lower.The above differences were statistically significant(P<0.05).Compared with normal group,ovary GPR54 protein expression level of model group,inhibitor group and activator group was significantly higher;The expression level of GPR54 protein in ovary of western medicine group,Chinese medicine group and activator group was significantly lower than that of model group.Compared with western medicine group,the expression level of GPR54 protein in ovary of inhibitor group was significantly higher.Compared with the Chinese medicine group,the expression level of GPR54 protein in ovary of rats in the inhibitor group was significantly higher.Compared with the inhibitor group,the expression level of GPR54 protein in the ovary of rats in the activator group was significantly lower.The above differences were statistically significant(P<0.05).Conclusion1.Through the study of preparation technology and quality standard,Jiuweichushi formula with reasonable drug compatibility,scientific and feasible preparation technology and stable and controllable quality were developed.2.In CPP rats,vaginal opening is advanced,regular estrous cycle is established,endometrial thickness,luteal number and follicle number are increased,and adverse psychological behaviors occur.Jiuweichushi formula can delay vaginal opening,inhibit gonadal development and improve adverse psychological behavior in CPP rats.3.The secretion of LH and FSH in the pituitary gland of CPP rats is increased,and Jiuweichushi formula can significantly reduce the expression levels of LH,FSH and E2 in serum of CPP rats,regulate the function of Pituitary-gonadal axis,and inhibit gonad development.4.The expression levels of GnRH gene and protein in hypothalamus and ovary of CPP rats were significantly increased.Jiuweichushi formula played a therapeutic role in CPP by inhibiting the activity of GnRH neurons in hypothalamus,reducing the secretion of GnRH and regulating the function of HPGA.5.In hypothalamus and ovarian tissues of CPP rats,Kiss-1/GPR54 system expression was significantly increased,while SIRT1 gene and protein expression levels were significantly decreased.SIRT1 could regulate Kiss-1 and GPR54 gene and protein expression levels,thus affecting GnRH secretion,regulating HPGA function,and participating in the development of CPP.Jiuweichushi formula can activate SIRT1 activity,regulate Kiss-1/GPR54 system expression,inhibit GnRH neuron activity,reduce GnRH secretion,and exert epigenetic regulation on CPP rats,which may be the potential mechanism of CPP treatment with traditional Chinese medicine. |
PDF Full Text Request