A preliminary study on the effects of Xiang shao granules on reproductive endocrinology in drugged ovariectomised rats

A preliminary study on the effects of Xiang shao granules on reproductive endocrinology in drugged ovariectomised rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A preliminary study on the effects of Xiang shao granules on reproductive endocrinology in drugged ovariectomised rats Huimin Tang, Qiucheng Jia, Wanying Chen, Yihan Wu, Weiwei Wei, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4161365/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Oct, 2024 Read the published version in Journal of Ovarian Research → Version 1 posted 9 You are reading this latest preprint version Abstract Objective: To establish a rat model of pharmacological ovariectomy by GnRH-a injection, and to preliminarily investigate the reproductive endocrine effects of Xiangshao granules on pharmacological ovariectomised rats. Methods: A rat model of pharmacological ovariectomy was established by injecting female rats with GnRH-a. The rats were randomly divided into four groups: GnRH-a injected saline group (GnRH-a + NS); GnRH-a injected oestradiol group (GnRH-a + E2); GnRH-a injected Xiang shao granule group (GnRH-a + Xiang shao), and the control group of saline injected rats (NS + NS). according to the observation of the vaginal smear of the rats to determine the success of the modelling, after the success of the modelling of the corresponding drug gavage intervention for 28 days, every other day to weigh the body weight of the rats and measure the anal temperature, according to the changes in body weight of the rats to adjust the amount of drug intervention. Plasma sex hormone levels (E2, FSH, LH), uterine weight, uterine index and endometrial histomorphological changes, and ovarian weight, ovarian index and ovarian histomorphological changes were measured in each group after gavage. Results: (1) Vaginal cell smears of rats in the control group (NS+NS) showed changes in the estrous cycle, whereas vaginal cell smears of rats in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao groups showed no changes in the estrous cycle; (2) The body mass gain of rats in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao groups was significantly higher than that of the NS+NS group, whereas intervention with estradiol (E2) and peony granules significantly slowed down the GnRH-a induced body mass gain. NS group, while the intervention of estradiol (E2) and Xiang shao granules could significantly delay the trend of GnRH-a-induced body mass gain in rats; (3) The anal temperature of rats after GnRH-a injection showed an overall increasing trend, and compared with GnRH-a+NS, the body temperature of rats in GnRH-a+E2 and GnRH-a+Xiang shao groups showed a gradual decreasing trend, and the decreasing of the temperature in Xiang shao granules compared with that of rats in E2 group was (4) Plasma sex hormone levels (E2, FSH, LH) were significantly lower in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao groups than in the NS+NS group (P<0.001), and the levels of E2 in the GnRH-a+E2 and GnRH-a+Xiang shao groups were significantly higher than those in the GnRH-a+NS group (P<0.001, P<0.05), and the levels of E2 in the GnRH-a+NS group were significantly lower than those in the GnRH-a+Xiang shao group (P<0.001, P<0.05). 0.05), and the E2 level in GnRH-a+E2 group was higher than that in GnRH-a+Xiangshao Granules group (P<0.05); the FSH level in GnRH-a+E2 group was significantly lower than that in GnRH-a+ Xiangshao granules group (P 0.05); LH levels in the GnRH-a+E2 group were significantly lower than those in the GnRH-a+NS and GnRH-a+Xiang shao groups (P0.05); (5) compared with the NS+NS group, GnRH-a injected rats in each model, uterine weight and uterine index, ovarian weight and ovarian index were significantly decreased (P<0.001); comparing between the groups, the uterine weight and uterine index, ovarian weight and ovarian index of GnRH-a+ E2 and GnRH-a+Xiang shao groups were significantly higher than those of GnRH-a+NS group (P<0.001, P<0.05); uterine weight and uterine index, ovarian weight and ovarian index of GnRH-a+E2 group were significantly higher than those of GnRH-a+NS group (P<0.001, P<0.05); and uterine weight and uterine index, ovarian weight and ovarian index were elevated compared with the GnRH-a+Xiang shao group (P<0.05); (6) compared with the NS+NS group, the number of primordial follicles was significantly higher and the number of growing follicles and mature follicles was significantly lower in the GnRH-a+NS, GnRH-a+E2 and GnRH-a+Xiang shao groups; (7) the number of rats' uterine wall was significantly higher and the number of rats' uterine wall was significantly lower in the NS+NS group than in the GnRH-a NS+NS group and GnRH-a group, the uterine wall of rats in each group was significantly thinner, the endothelial layer was atrophied, the thickness of the uterine wall increased in the GnRH-a+E2 and GnRH-a+Xiang shao groups, and the number of vaginal folds and blood vessels also increased. Among them, the improvement of uterus and vagina was more obvious in GnRH-a+E2 than in GnRH-a+NS and GnRH-a+Xiangshao groups. Conclusion: GnRH-a injection could reduce the levels of sex hormones E2, FSH and LH in rats, causing perimenopausal symptoms such as hot flashes, while E2 and Xiang shao granules significantly improved such symptoms, and Xiang shao granules had a slight oestrogenic effect, but to a lesser extent than E2. xiangshao granules GnRH-a pharmacological oophorectomy perimenopausal syndrome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Endometriosis usually refers to the appearance of endometrial tissue (glands and mesenchyme) in tissues outside the uterine cavity, and infiltrate and grow in them, shedding and bleeding with the menstrual cycle, and it is a gynecological chronic inflammatory disease that affects 5%-10% of women of childbearing age globally [1,2] , and its clinical symptoms mainly include dysmenorrhoea, non-menstrual pelvic pain, and infertility, etc., and it is estimated that about 50%-80% of dysmenorrhoeic According to statistics, about 50%-80% of women with dysmenorrhoea and 50% of women with infertility suffer from endometriosis [3,4] , which seriously affects the quality of life of women. Although endometriosis is a benign disease, it has the malignant biological behaviours of infiltration, metastasis and recurrence, which seriously troubles the clinicians. As endometriosis is an estrogen-dependent disease, there is often a lack of effective clinical treatments, and at present, the more unified view is that the purpose of the treatment of endometriosis is to eliminate the foci, alleviate the pain, solve the problem of fertility, and reduce the recurrence of endometriosis, etc. [5,6], and the treatment mainly includes drug therapy and surgery. Treatment mainly includes drug therapy and surgery, drug therapy aims to inhibit the growth of ectopic endometrium [7], but it is often ineffective for patients with moderate-to-severe endometriosis, and surgery is the most common means of treatment for endometriosis, but it has a high rate of postoperative recurrence, and it has been reported that the recurrence rate of endometriosis 5 years after surgery can reach 18.9-40% [8-10], and the rate of patients who are readmitted to hospital for surgery 4 years after surgery is about 27% [7] , and the recurrence rate of patients who are readmitted to hospital for surgery is about 20%. The recurrence rate of endometriosis is reported to be 18.9%-40% at 5 years after surgery [8-10] and 27% at 4 years after surgery [11,12] , therefore, it is extremely important to prevent its recurrence by postoperative drug treatment. Gonadotropin-releasing hormone agonist (GnRH-a) is currently recognised as the most effective drug for the treatment of endometriosis [13-15] , GnRH-a is an analogue of the hypothalamic hormone GnRH, which is a synthetic decapeptide analogue, and in physiological state GnRH regulates the secretion of steroid hormones by the pituitary through the stimulation of release of follicle-stimulating hormone (FSH) and luteinising hormone (LH). GnRH-a has a high affinity for and binds to pituitary GnRH receptors, leading to a decrease in pituitary secretion of hormones, thus reducing ovarian oestrogen production, and this down-regulation of pituitary function is constant, thus leading to severe hypo-estrogenism, and thus temporary amenorrhoea, which is medically suppressed, and is referred to as "pharmacological oophorectomy" [16,17] . Due to the lack of oestrogen in the endometriotic cells leading to atrophy of the ectopic endometrial tissue, it is because of this down-regulation of the pituitary gland and the inhibition of gonadotropin release to maintain the hypo-estrogenic state that patients tend to suffer from perimenopausal symptoms, such as hot flashes and sweats, irritability, poor sleep, and osteoporosis [18] . Hot flashes and sweats have been reported in about 90% of women, and bone loss occurs in 2.5% to 5% of women after 4 - 6 months of treatment [19] . Atrophic vaginitis, insomnia, peripheral oedema and nervousness have also been reported, but the prevalence is not known at present [20] , in view of which the safety and efficacy of GnRH-a treatment beyond 6 months is controversial, which undoubtedly limits the clinical efficacy of GnRH-a [21] . Therefore, it is necessary to seek complementary or alternative therapies to reduce the side effects of GnRH-a in endometriosis treatment. The proposed reverse addition theory can remedy this deficiency by adding low-dose estrogen and progesterone, which, on the one hand, will not cause ectopic endometrial growth and, on the other hand, can improve perimenopausal symptoms and quality of life due to GnRH-a, while progesterone prevents estrogen-induced endometrial overgrowth. Although the theory of reverse addition can effectively alleviate perimenopausal symptoms, long-term hormone use can also trigger some adverse effects, such as affecting coagulation levels and increasing the risk of venous thrombosis; increasing the chances of endometrial cancer, breast cancer and so on [22] . Influenced by traditional thinking, hormone replacement therapy is less accepted in China, and patient compliance is poor. Therefore, an urgent problem is to find other safe and effective reverse additive drugs. Xiangshao granule is a compound preparation composed of 10 traditional Chinese medicines, such as Fragrant herb and white peony [23] , which is a national class III new drug (traditional Chinese medicine). Shaoxing granules are effective in relieving perimenopausal symptoms in women. However, the current studies are based on the treatment of Xiang shao granules in healthy naturally menopausal and perimenopausal women. There are few studies on the treatment of pharmacological menopause, so we hypothesised that the Fragrance Granules could also rapidly relieve perimenopausal symptoms such as hot flashes and sweating caused by pharmacological menopause. In this study, it was proposed to establish a rat model of pharmacological oophorectomy induced by GnRH-a treatment by means of GnRH-a injection, with physiological saline (NS) injection as a control. The intervention was carried out by using paeoniflora granules and estradiol drug to further understand the effect of paeoniflora granules on female reproductive endocrine secretion. Materials and methods、 Materials and methods 1.Materials 1.1 Experimental animals Twenty-four female Sprague-Dawley (SD) clean-grade rats, with an average weekly age of 6-8 weeks, were selected and purchased from Shanghai Bicaikewing Biotechnology Co., Ltd. and approved by the Ethics Committee for Laboratory Animal Welfare, and the experiments were formally begun after the rats had been acclimated to the rearing environment for 1 week. All experimental operators of the experimental animals in this study possessed the Laboratory Animal Induction Certificate, and all experimental operations complied with the Regulations of the People's Republic of China on the Management of Laboratory Animals issued by the National Science and Technology Commission of China and the Regulations on the Management of Laboratory Animals issued by the Jiangsu Provincial Government. 1.2 Experimental reagents (1) Gonadotropin-releasing hormone agonist (GnRH-a): generic name treprostinil acetate, trade name Dafylline, its main ingredient is treprostinil acetate, excipients: polypropylene cross ester ethyl cross ester copolymer, mannitol, carboxymethylcellulose, polysorbate 80, specification 3.75mg/capsule, Manufacturer: Beaufor Ipsen Pharmaceuticals Limited, France. (2) Xiangshao granules: main ingredients: white peony, fragrant herbs, neem seeds (fried), chaihu, chuanxiong rhizome, citrus aurantium, half-sia (ginger-made), cardamom, woodruff, liquorice. Excipients: sucrose, betacyclodextrin, dextrin. Specification: 4g/bag. Manufacturer: Yangzijiang Pharmaceutical Group Sichuan Hairong Pharmaceutical Co., Ltd, Batch No.: State Standard Z20050425. (3) Estradiol Valerate: generic name: Estradiol Valerate Tablets, trade name: Supplement Jiale, main ingredient: Estradiol Valerate (E2), specification: 1mg/capsule, manufacturer: Bayer Healthcare Co., Ltd. Guangzhou Branch. (4) Others 1.3 Enzyme-linked immunosorbent assay (ELISA) reagents 1.4 HE staining reagents 2. Methods 2.1 Animal modelling (1) GnRH-a injectable drug ovarian denudation animal modelling: 18 SD adult female rats, GnRH-a (tretinoin acetate for injection, dafilgrastim) was administered intramuscularly at 0.06 mg /kg for 7 consecutive days, and thereafter maintained for 21 days in accordance with the maintenance injections at 1/5 of the starting dose. (2) Saline injection control group: 6 adult female SD rats were injected with saline 0.06 mg/kg intramuscularly for 7 consecutive days, after which the injection was maintained at 1/5 of the starting dose for 21 days. The saline control group was designed to reduce the error associated with drug injection. 2.2 Modelling results determination: In order to determine the success of GnRH-a injection drug modelling, vaginal smears of rats were observed at a fixed time selected every day 7 days after GnRH-a injection, and the vaginal secretion cells were observed under the microscope to determine the motility cycle. 2.3 Experimental drug gavage intervention: Pharmacological interventions were started after successful modelling (2 weeks of continuous GnRH-a injection) in each group. Estradiol valerate (E2) and Xiang shao granules were processed and dissolved in sterile saline to form a turbid solution for rats to be gavaged, and rats were weighed every other day, and the dosage of the drug was adjusted according to the changes in body mass. 2.4 Experimental programme and grouping: After the modelling of the experimental rats was completed, 24 rats were randomly divided into 4 groups of 6 rats each, depending on the different drug intervention protocols: GnRH-a injected saline group (GnRH-a+NS): 2 weeks after GnRH-a injection, 0.06 mg/kg saline was used for 28 d of continuous gavage. GnRH-a injected oestradiol group (GnRH-a+E2): 2 weeks after GnRH-a injection, 0.8 mg/kg oestradiol valerate E2 was used by continuous gavage for 28d. GnRH-a injection of Xiang shao granules group (GnRH-a + Xiang shao): after 2 weeks of GnRH-a injection, 60 mg/kg of Xiang shao granules were used for 28 d of continuous gavage. Physiological saline injection control group physiological saline group (NS+NS): rats saline 0.06 mg/kg intramuscular injection for 2 weeks, then use 0.06 mg/kg physiological saline, continuous gavage for 28d. 2.5 Body temperature and body weight measurements of rats in each group: After successful modelling, the anal temperature of each group was measured and the body weight of rats was weighed every other day. The average body temperature of each group was calculated and plotted as a "temperature-time curve"; at the same time, the body weights of the rats were weighed and recorded every other day, and a "body weight-time curve" was plotted according to the average body weights of the rats. 2.6 Experimental rat execution, sample collection and testing: At the end of the drug intervention in each group, rats in each group were injected intraperitoneally with 1% pentobarbital sodium and then decapitated to take rat specimens. Blood (serum and plasma, respectively) was taken from the abdominal vein of the rats, and the uterus and ovaries were removed by saline irrigation. Uterine weight and uterine index: After the execution of rats in each group, the intact uterus was dissected and removed, the wet weight of the uterus was weighed and the uterine index was calculated according to the uterine index formula (uterine index (UMI) = wet weight of the uterus / body weight (%)), the uterine index of each rat was calculated, and the data were statistically analysed. Ovary weight and ovarian coefficient: After the rats in each group were executed, the intact bilateral ovaries of the rats were dissected and removed. The ovarian coefficient was calculated according to the formula of ovarian coefficient (ovarian coefficient = wet weight of ovary/body weight (%)), and the ovarian coefficient of each rat was calculated and the data were statistically analysed. ELISA assay to detect changes in the expression levels of E2, FSH and LH in plasma. HE staining to detect morphological changes in the endometrium. HE staining to detect morphological changes in the ovary and observe follicular development. 2. All data in this study were statistically analysed using SPSS27.0 statistical software. All measurements were expressed as means plus or minus standard deviation (士s), and one-way ANOVA was used to compare the means of multiple samples within a group; if significant differences existed, the Turkey test was used for further between-group comparisons. Statistical significance was taken as P<0.05. Graph Pad Prism 10 software was used to draw the relevant graphs for this study. Results 1. Analysis of vaginal smear results The results showed that the vaginal cell smears of rats in the control group (NS+NS group) showed changes in the estrous cycle (see Figure 1-1), and the cells in the smears were mostly polygonal superficial cells with less cytoplasm and a few epithelial cells; on the contrary, there were no changes in the estrous cycle in the vaginal cell smears of the rats in the GnRH-a model group, which only stayed in the inter-estrous phase, and the cell smears were more homogeneous, mainly with leukocytes and rounded epithelial cells (see Figure 1-2). On the other hand, in the GnRH-a model group, the vaginal cell smears showed no changes in the estrous cycle and only remained in the inter-estrous phase. 2.Trends of body weight changes in rats During the 2-week feeding period of rat modelling, the body weight of GnRH-a-injected rats (GnRH-a+NS, GnRH-a+E2, GnRH-a+ Xiang shao) increased compared with that of the control group (NS+NS), and the body weight of rats in the three groups of rats with pharmacological interventions increased more significantly in the GnRH-a+NS than in the GnRH-a+E2, GnRH-a+Xiang shao groups. As shown in Figure 2 (the "weight-time trend graph" of rats was plotted based on the recording results), within 14 days of rat modelling, the weight of rats in the GnRH-a injection group increased significantly compared with that of the control (NS+NS) group, whereas during the period of drug intervention (18d-42d), rats in the GnRH-a+E2, GnRH-a+Xiang shao group increased significantly. a + Xiang shao granules group showed a slower increase in body weight than the GnRH-a+NS group. This phenomenon suggests that E2 and paeoniflora granules can effectively slow down the trend of GnRH-a-induced weight gain in rats. 3.Trends in rat body temperature During the 2-week feeding period of rat modelling, the body temperatures of GnRH-a-injected rats (GnRH-a+NS, GnRH-a+E2, GnRH-a+ Xiang shao) increased significantly compared with those of the control group (NS+NS), and among the three groups of rats intervened with the drug, the body temperatures of rats in the GnRH-a+NS group rose significantly compared with those of rats in the GnRH-a+E2 and GnRH-a+ Xiang shao groups, as shown in Fig. 3 (the "time trend" graph of rats was plotted based on the recorded results). See Fig. 3 for details (plotting the "body temperature-time trend graph" of rats based on the recording results). As shown in Fig. 3, within 14 days of rat modelling, the body temperature of rats in the GnRH-a injection group increased significantly compared with that of the control (NS+NS) group, and during the period of pharmacological interventions (20d-42d), the body temperature of rats in the GnRH-a+E2, GnRH-a+Xiang shaos group increased significantly compared with that in the GnRH-a+E2, GnRH-a+Xiang shaos group. GnRH-a+Xiangshao group showed a significant decreasing trend in body temperature, and the decreasing trend of body temperature was similar in GnRH-a+E2, GnRH-a+Xiangshao groups. This phenomenon suggests that GnRH-a injection can cause the rats to have a response of increased body surface temperature similar to perimenopausal hot flashes, and the intervention of E2 and Xiang shao granules can rapidly improve this situation, and Xiang shao granules can improve the perimenopausal hot flashes symptoms induced by GnRH-a to the same extent as E2. 4. Serum sex hormone levels after different drug interventions in each group of rats At the end of the drug gavage intervention, the serum E2 levels of all three groups of GnRH-a-injected rats (GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiangshao) were all significantly all lower than those of the NS+NS group (P<0.001), and when compared between the groups, the E2 levels of the GnRH-a+E2, and GnRH-a+Xiang shao groups were significantly higher than those of the GnRH-a+NS group ( P<0.001, P<0.05), and E2 levels were significantly lower in the GnRH-a+ Xiangshao group than in the GnRH-a+E2 group (P<0.05); at the end of the pharmacological intervention, serum FSH levels in the GnRH-a+NS, GnRH-a+E2 and GnRH-a+ Xiangshao groups were significantly lower than those of the NS+NS group (P0.05), FSH levels in the GnRH-a+E2 group were significantly lower compared with those in the GnRH-a+ Xiangshao granules group (P0.05). (P>0.05); at the end of the drug intervention, the serum LH levels of rats in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+ Xiangshao groups were all significantly lower than those in the NS+NS group (P<0.01, P<0.001, P<0.001), and the LH levels in the GnRH-a+E2 group were significantly lower than those in the GnRH-a+NS versus GnRH-a+ Fragrant peony granule group (P<0.001, P0.05). The test results suggested that GnRH-a injection could achieve the expected effect of establishing a perimenopausal rat model, and this animal model successfully achieved a significant decrease in the levels of E2, FSH and LH in rats, meanwhile, the fragrant peony granules could increase the level of E2 in vivo, which indicated that the fragrant peony granules might have a certain stimulating effect on the ovary or have a certain estrogen-like effect, which could produce a small amount of estrogen, but its effect was It can produce a small amount of estrogen, but its effect is significantly lower than that of estrogen, indicating that the estrogen-like effect of Xiang shao granules is relatively weak, and Xiang shao granules will not increase the levels of FSH and LH. Table 4.1 Comparison of plasma sex hormone (E2, FSH, LH) levels after drug intervention in each group of rats Group N E2 (pg/ml) FSH (IU/L) LH (IU/L) NS+NS 6 34.20±8.60 2.72±0.32 2.03±0.25 GnRH-a+NS 6 1.45±0.38***### 1.45±0.38*** 1.55±0.24***### GnRH-a+E2 6 1.16±0.34** 1.16±0.34*** 0.91±0.13*** GnRH-a+ Xiangshao 6 1.70±0.36***# 1.70±0.36***# 1.48±0.46***## *P<0.05, **P<0.01 ***P＜0.001vs NS+NS，#P＜0.05，##P＜0.01，###P＜0.001 vs GnRH-a+E2 5.Morphological changes in uterine index and endometrial tissue of rats in all groups after drug intervention 5.1 Uterine wet weight and uterine index in rats after drug intervention Group N Uterus wet weight Uterus index NS+NS 6 0.5355±0.1314 0.192±0.051278 GnRH-a+NS 6 0.17425±0.01846***### 0.055±0.005353***### GnRH-a+E2 6 0.354625±0.048718 0.122±0.020585*** GnRH-a+ Xiangshao 6 0.28025±0.029305***# 0.091±0.011101***# ***P＜0.001vs NS+NS；#P＜0.05，###P＜0.001 vs GnRH-a+E2 After the drug intervention, the uterine index of all three groups of rats injected with GnRH-a (GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao) was significantly smaller than that of the NS+NS group (P<0.001), and the uterine index of the rats in the GnRH-a+E2 and GnRH-a+Xiang shao groups was significantly increased compared with that of the GnRH-a+NS (P<0.001 and P<0.05), and the uterine index of rats in the GnRH-a+E2 group was significantly higher than that in the GnRH-a+ Xiangshao granule group (P<0.05). 5.2 Morphological changes of endometrial tissue after drug intervention At the end of drug intervention, endometrial tissues of rats in each group were subjected to pathological section while stained with HE staining, and the results showed that: endometrium of rats in NS+NS group consisted of a single layer of columnar epithelial cells, tubular glands were distributed in the lamina propria, glandular epithelium was a single layer of columnar shape, cell morphology had no abnormality, there was no oedema, no atrophy or hyperplasia. The uterine wall of rats in the GnRH-a injection group was significantly thinner, and the endometrial layer was atrophied. The epithelial cells were flattened and partially atrophied. The uterine wall of rats in the GnRH-a+E2, GnRH-a+ Xiangshao granules group showed all the increases in the thickness of the uterine wall compared with that of rats in the GnRH-a+NS group, the endometrial epithelial cells were in the form of columns or cubes, and the cells were not edematous, but there were different degrees of atrophic thinning in the GnRH-a+NS group compared to the GnRH-a+NS group. 6. Morphological changes in ovarian index and sub-ovarian tissues of rats in various groups after drug intervention 6.1 Ovarian wet weight, ovarian index in rats after drug intervention Group N Ovarian wet weight Ovarian index NS+NS 6 0.204625±0.027682 0.0732±0.010204 GnRH-a+NS 6 0.093625±0.017353***### 0.0294±0.004776***### GnRH-a+E2 6 0.13675±0.01836*** 0.0468±0.005377*** GnRH-a+ Xiangshao 6 01185±0.015793***# 0.0385±0.006576***# ***P＜0.001vs NS+NS；#P＜0.05，###P＜0.001 vs GnRH-a+E2 After the drug intervention, the ovarian indices of all three groups of GnRH-a-injected rats (GnRH-a+NS, GnRH-a+E2, GnRH-a+ Xiangshao) were significantly smaller than those of the NS+NS group (P<0.001), and the ovarian indices of the rats in the GnRH-a+E2, and GnRH-a+ Xiangshao groups were significantly higher when compared with those in the GnRH-a+NS (P<0.001 and P<0.05), and the uterine index of rats in the GnRH-a+E2 group was significantly higher than that in the GnRH-a+ Xiangshao granule group (P<0.05). 6.2 Morphological changes of ovarian tissue after drug intervention At the end of drug intervention, complete ovaries of rats in each group were taken out, and pathological sections were stained with HE staining at the same time, and morphological changes of ovarian groups were observed under the microscope, which showed that: follicle structure was normal in the NS+NS group, with visible follicles at all levels; follicle structure was normal in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao granules groups, and the number of follicular growth and mature follicles was obviously reduced. Discussion The drug GnRH-a, which inhibits oestrogen synthesis, can be used to control recurrence after endometriosis surgery. However, long-term use of GnRH-a can lead to secondary perimenopausal reactions, which can cause discomfort such as hot flashes and sweats, emotional irritability, and poor sleep, and is the main reason for patients to give up the treatment. Reverse-addition therapy, although a good solution to perimenopausal symptoms, but long-term use of hormones may lead to liver function damage, venous embolism, vascular disease, and increase the risk of endometrial cancer, ovarian cancer, breast cancer and other diseases. Therefore, the search for effective and safe counter-additive drugs is an urgent problem to be solved. Xiangshao granule is a new class III new drug (traditional Chinese medicine) that has been newly listed in China in recent years, which is extracted from 10 purely natural plant medicines, such as Fragrance, White peony, Neem, Chaihu, Chuanxiong, Citrus aurantium, Cardamom and so on [ 24 ] , and its main effects have the effects of dredging the liver and regulating the qi, calming the liver and relieving the pain. Clinical phase II and III studies have proved [ 25 ] that the fragrant peony granules have good efficacy in perimenopausal syndrome and premenstrual syndrome, and can rapidly relieve the symptoms of perimenopausal syndrome such as hot flashes and sweating, depression, and emotional irritability, etc. However, little research has been done on perimenopausal symptoms caused by pharmacological menopause, and in view of the secondary, we can deduce that it also antagonises the symptoms of postoperative GnRH-a treatment for patients with endometriosis. This study aims to investigate the clinical efficacy of Xiang shao granules in antagonising perimenopausal symptoms caused by postoperative GnRH-a treatment in patients with endometriosis using oestradiol as a reference, in the hope that it can become a new, safer and more reliable alternative to reverse-addition therapy after GnRH-a treatment. In this study, GnRH-a injection was used to establish a pharmacological ovariectomy rat model, and vaginal smears were given to observe the changes in the vaginal cells of rats in order to determine the success of the modelling. In this study, the vaginal smears of rats showed that the changes in the estrous cycle of rats in the GnRH-a group had disappeared, and the vaginal cells showed a persistent inter-estrous phase, with the cells mainly being the rounded outer primordial layer with a large rounded nucleus, which means that the modelling was successful, and it was further confirmed that GnRH-a could induce the hypoestrogenic state in the body of the rats. In addition, the results of this study showed that the body temperature of rats injected with GnRH-a was on the rise, and the body temperature of rats with E2 and Xiang shao granules was on the decline, and the degree of decline of both was similar, which further indicated that GnRH-a could cause perimenopausal hot flashes, sweating and other discomforts, and Xiang shao granules and oestradiol could improve this symptom, and the degree of improvement of hot flashes was similar to the degree of improvement of estradiol in the perimenopausal period. The degree of improvement of hot flushes by Xiang shao granules is similar to that of estradiol, which can rapidly relieve the symptoms of perimenopausal hot flushes. The results of this study showed that GnRH-a injection could increase the body weight of rats significantly, and at the same time, it could reduce the levels of serum sex hormones E2, FSH and LH, and lower the uterine index and ovarian index of rats, and it was found that the endometrium of the rats injected with GnRH-a was obviously atrophied, the number of follicular growth follicles and mature follicles was reduced significantly, and the primordial follicles were increased, and the body weight of rats was significantly reduced after the interventions of E2 and Paeoniae chinensis granules, and the body weight of rats was increased. After the intervention of E2 and Xiang shao granules, the body weight of rats decreased significantly, which indicated that Xiang shao granules had the same effect as estradiol in alleviating the perimenopausal weight gain induced by GnRH-a. At the same time, after the intervention of Xiang shao granules, the level of E2 in the rats increased, but to a lesser extent than that of the E2 group, which further indicated that Xiang shao granules might have the effect of stimulating or improving the function of ovary, or have a slight estrogenic effect, which was consistent with the study by Li et al. This is the same as the results of Li et al. who studied the effects of Xiang shao granules on perimenopausal ovarian function [ 23 ] . However, the results of HE staining of rat endometrium showed that there was no significant endometrial hyperplasia in the Xiang shao granules group, which indicated that the estrogen-like effect of Xiang shao granules may be weak and have a small effect on the endometrium, and there was no difference in the serum levels of FSH and LH between rats and the control group, which indicated that Xiang shao granules may not exert anti-perimenopausal effects through estrogen-like effects, so what pathway is Xiang shao granules acting on perimenopausal symptoms? The pathway through which the Xiang shao granule exerts its menopause-relieving effect is still unclear, and needs to be further investigated, which is also the focus of our next study. In summary, GnRH-a injection can cause low FSH, LH and E2 levels in perimenopausal rats, which can lead to perimenopausal-like symptoms such as hot flashes and weight gain, which are more in line with the perimenopausal clinical discomfort caused by GnRH-a. It was found that Xiang shao granules can improve the symptoms such as hot flashes quickly and effectively, and although Xiang shao granules can cause estrogen elevation in rats, according to endometrial HE staining, it does not cause endometrial hyperplasia. Although this study found that Xiang shao granules can cause the increase of estrogen in rats, according to the HE staining of endometrium, Xiang shao granules do not cause endometrial hyperplasia. Therefore, although Xiang shao granules have the estrogen-like effect, its estrogen-like effect is weak, and it will not increase the risk of endometrial hyperplasia, breast cancer and other related risks, but it can achieve the same as estrogen to alleviate the symptoms of peri-menopausal period. Meanwhile, as mentioned above, both Xiang shao granules and estradiol can alleviate perimenopausal symptoms such as hot flashes, which have certain commonalities, but the difference is that estradiol improves the symptoms through direct exogenous estrogen, while Xiang shao granules have a small amount of estrogenic effect, but its effect may be through other pathways, which shows that Xiang shao granules can effectively alleviate the perimenopausal symptoms due to GnRH-a, and will not increase the risk of endometrial cancer and breast cancer. It is a safer alternative to GnRH-a as it can effectively relieve perimenopausal symptoms without increasing the risk of endometrial cancer, breast cancer, renal vein thrombosis, and so on. Conclusion GnRH-a injection can reduce the levels of E2, FSH and LH in rats, causing perimenopausal symptoms such as hot flushes, which can be significantly ameliorated by E2 and Xiang shao granules, and Xiang shao granules do not exert their anti-perimenopausal symptomatic effects through oestrogen-like effects, but rather, they act through other pathways. Declarations Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding details Changzhou Municipal Health Commission Science and Technology Project (ZD202314); Changzhou "14th Five-Year Plan" High-level Talent Cultivation Project (2022CZBJ074); Jiangsu Province Maternal and Child Health Key Talent Project (RC202101); Jiangsu Province Maternal and Child Health Scientific Research Project (F202138). Author Contribution "Huimin Tang and Qiucheng Jia wrote the main manuscript text，Wanying Chen and Yihan Wu Analysied data and mapping，Weiwei Wei and Hong Zheng literatured review，Jiming Chen Designed Experiments and Instruction，All authors reviewed the manuscript." References Wang PH ,Yang ST,Chang WH et al. Endometriosis: Part I. Basic concept .Taiwan J Obstet Gynecol, 2022, 61: 927-934. Bulun SE, Yilmaz BD, Sison C, et al. Endometriosis. Endocr Rev. 2019;40(4):1048-1079. doi:10.1210/er.2018-00242 Taylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet. 2021 Feb 27;397(10276):839-852. Zondervan KT, Becker CM, Missmer SA. Endometriosis. N Engl J Med. 2020;382(13):1244-1256. doi:10.1056/NEJMra1810764 Allaire C, Bedaiwy MA, Yong PJ. Diagnosis and management of endometriosis. CMAJ. 2023 Mar 14;195(10):E363-E371. Chapron C, Marcellin L, Borghese B, Santulli P. Rethinking mechanisms, diagnosis and management of endometriosis. Nat Rev Endocrinol. 2019;15(11):666-682. doi:10.1038/s41574-019-0245-z Perrone U, Evangelisti G, Laganà AS, et al. A review of phase II and III drugs for the treatment and management of endometriosis. Expert Opin Emerg Drugs. 2023;28(4):333-351. doi:10.1080/14728214.2023.2296080 Fedele L, Bianchi S, Zanconato G, Berlanda N, Raffaelli R, Fontana E (2006) Laparoscopic excision of recurrent endometriomas: long-term outcome and comparison with primary surgery. Fertil Steril 85(3):694–699. Wheeler JM, Malinak LR,Recurrent endometriosis: incidence, management, and prognosis. Am J Obstet Gynecol 146(3):247–253. Fedele L, Bianchi S, Di Nola G, Candiani M, Busacca M, Vignali M (1994) The recurrence of endometriosis. Ann N Y Acad Sci 734:358–364. Cheong Y,Tay P,Luk F et al. Laparoscopic surgery for endometriosis: How often do we need to re-operate? J Obstet Gynaecol, 2008, 28: 82-5. Saunders PTK, Horne AW. Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell. 2021;184(11):2807-2824. doi:10.1016/j.cell.2021.04.041 Donnez J, Dolmans MM. GnRH Antagonists with or without Add-Back Therapy: A New Alternative in the Management of Endometriosis? Int J Mol Sci. 2021 Oct 20;22(21):11342. Chen LH, Lo WC, Huang HY, Wu HM. A Lifelong Impact on Endometriosis: Pathophysiology and Pharmacological Treatment. Int J Mol Sci. 2023;24(8):7503. Published 2023 Apr 19. doi:10.3390/ijms24087503 Donnez J, Dolmans MM. Endometriosis and Medical Therapy: From Progestogens to Progesterone Resistance to GnRH Antagonists: A Review. J Clin Med. 2021;10(5):1085. Published 2021 Mar 5. doi:10.3390/jcm10051085 Suga S, Akaishi T, Sakuma Y. GnRH inhibits neuronal activity in the ventral tegmental area of the estrogen-primed ovariectomized rat. Neurosci Lett. 1997 May 30;228(1):13-6. Horne AW, Missmer SA. Pathophysiology, diagnosis, and management of endometriosis. BMJ. 2022;379:e070750. Published 2022 Nov 14. doi:10.1136/bmj-2022-070750 Vannuccini S, Clemenza S, Rossi M, Petraglia F. Hormonal treatments for endometriosis: The endocrine background. Rev Endocr Metab Disord. 2022;23(3):333-355. doi:10.1007/s11154-021-09666-w Perry CM, Brogden RN. Goserelin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in benign gynaecological disorders. Drugs. 1996 Feb;51(2):319-46. Friedman AJ, Hoffman DI, Comite F, Browneller RW, Miller JD. Treatment of leiomyomata uteri with leuprolide acetate depot: a double-blind, placebo-controlled, multicenter study. The Leuprolide Study Group. Obstet Gynecol. 1991 May;77(5):720-5. Donnez J, Dolmans MM. GnRH Antagonists with or without Add-Back Therapy: A New Alternative in the Management of Endometriosis?. Int J Mol Sci. 2021;22(21):11342. Published 2021 Oct 20. doi:10.3390/ijms222111342 Capezzuoli T, Rossi M, La Torre F, Vannuccini S, Petraglia F. Hormonal drugs for the treatment of endometriosis. Curr Opin Pharmacol. 2022;67:102311. doi:10.1016/j.coph.2022.102311 唐瑞怡,王亚平,张绍芬等.香芍颗粒治疗围绝经期女性情绪障碍的临床研究.实用妇产科杂志,2023,39(03):210-216. 陈蓉,郁琦.香芍颗粒临床应用指导建议.中国实用妇科与产科杂志,2015,31(05):419-420. 乔明琦,张惠云,姜坤等.经前平颗粒多中心、随机双盲双模拟对照治疗经前期综合征肝气逆证403例.中国新药杂志,2002,(05):389-392. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Oct, 2024 Read the published version in Journal of Ovarian Research → Version 1 posted Editorial decision: Revision requested 06 May, 2024 Reviews received at journal 26 Apr, 2024 Reviews received at journal 16 Apr, 2024 Reviewers agreed at journal 07 Apr, 2024 Reviewers agreed at journal 05 Apr, 2024 Reviewers invited by journal 05 Apr, 2024 Editor assigned by journal 26 Mar, 2024 Submission checks completed at journal 25 Mar, 2024 First submitted to journal 25 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4161365","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284729896,"identity":"fd5e897c-99e2-4349-980b-d07795600c93","order_by":0,"name":"Huimin Tang","email":"","orcid":"","institution":"The Affiliated Changzhou Second People's Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Tang","suffix":""},{"id":284729897,"identity":"a51cc6cd-8e6c-4c59-a461-ceada85a629e","order_by":1,"name":"Qiucheng Jia","email":"","orcid":"","institution":"The Affiliated Changzhou Second People's Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiucheng","middleName":"","lastName":"Jia","suffix":""},{"id":284729898,"identity":"15f529ec-e0aa-429f-9dba-00713529562e","order_by":2,"name":"Wanying Chen","email":"","orcid":"","institution":"The Affiliated Changzhou Second People's Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wanying","middleName":"","lastName":"Chen","suffix":""},{"id":284729899,"identity":"47c6fbd4-022c-4828-831a-5e3ff5e0f6ed","order_by":3,"name":"Yihan Wu","email":"","orcid":"","institution":"The Affiliated Changzhou Second People's Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yihan","middleName":"","lastName":"Wu","suffix":""},{"id":284729900,"identity":"8d1652ed-820c-41cb-a74b-9a82793603f6","order_by":4,"name":"Weiwei Wei","email":"","orcid":"","institution":"The Affiliated Changzhou Second People's Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Wei","suffix":""},{"id":284729901,"identity":"6b94481e-2ae8-4aa6-b420-16fccf933617","order_by":5,"name":"Hong Zheng","email":"","orcid":"","institution":"The Affiliated Changzhou Second People's Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zheng","suffix":""},{"id":284729902,"identity":"886ccef5-235e-497e-bf91-a690db732348","order_by":6,"name":"Jiming Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYFCCA0DIYMPDz95AmpY0GcmeA6RZddjG4IYDkWr5G08nHi74dZ6H4QYD44ePOURokThwdsPhmX23eRhnNzBLztxGhBYDBqAW3p7bPMwyB9iYeUnQco6HTSKBFC08Pw7w8BCtBewX3oZkHgmeg83E+YV/xtnNn3n+2NnbH28++OEjMVqA1jAwMLaBWIwNxKgHWQNS+IdIxaNgFIyCUTAyAQChZTvL1QqUuQAAAABJRU5ErkJggg==","orcid":"","institution":"The Affiliated Changzhou Second People's Hospital of Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jiming","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-03-25 07:20:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4161365/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4161365/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13048-024-01531-z","type":"published","date":"2024-10-18T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53632216,"identity":"da9ec972-3a41-4bef-b5e8-3f65cdaf6519","added_by":"auto","created_at":"2024-03-28 10:07:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":165874,"visible":true,"origin":"","legend":"\u003cp\u003e1-1 Vaginal cell smears of rats in NS+NS group\u003c/p\u003e\n\u003cp\u003e1-2 Vaginal cell smears of rats in the GnRH-a injection modelling group\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4161365/v1/15e4e26528d2637291089ca1.png"},{"id":53632208,"identity":"f917ea97-b633-4453-a70e-9cc83897bcd0","added_by":"auto","created_at":"2024-03-28 10:07:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108581,"visible":true,"origin":"","legend":"\u003cp\u003e\"Body weight - 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Although endometriosis is a benign disease, it has the malignant biological behaviours of infiltration, metastasis and recurrence, which seriously troubles the clinicians. As endometriosis is an estrogen-dependent disease, there is often a lack of effective clinical treatments, and at present, the more unified view is that the purpose of the treatment of endometriosis is to eliminate the foci, alleviate the pain, solve the problem of fertility, and reduce the recurrence of endometriosis, etc. [5,6], and the treatment mainly includes drug therapy and surgery. Treatment mainly includes drug therapy and surgery, drug therapy aims to inhibit the growth of ectopic endometrium [7], but it is often ineffective for patients with moderate-to-severe endometriosis, and surgery is the most common means of treatment for endometriosis, but it has a high rate of postoperative recurrence, and it has been reported that the recurrence rate of endometriosis 5 years after surgery can reach 18.9-40% [8-10], and the rate of patients who are readmitted to hospital for surgery 4 years after surgery is about 27%\u003csup\u003e\u0026nbsp;[7]\u003c/sup\u003e, and the recurrence rate of patients who are readmitted to hospital for surgery is about 20%. The recurrence rate of endometriosis is reported to be 18.9%-40% at 5 years after surgery\u003csup\u003e\u0026nbsp;[8-10]\u003c/sup\u003e and 27% at 4 years after surgery \u003csup\u003e[11,12]\u003c/sup\u003e, therefore, it is extremely important to prevent its recurrence by postoperative drug treatment.\u003c/p\u003e\n\u003cp\u003eGonadotropin-releasing hormone agonist (GnRH-a) is currently recognised as the most effective drug for the treatment of endometriosis \u003csup\u003e[13-15]\u003c/sup\u003e, GnRH-a is an analogue of the hypothalamic hormone GnRH, which is a synthetic decapeptide analogue, and in physiological state GnRH regulates the secretion of steroid hormones by the pituitary through the stimulation of release of follicle-stimulating hormone (FSH) and luteinising hormone (LH). GnRH-a has a high affinity for and binds to pituitary GnRH receptors, leading to a decrease in pituitary secretion of hormones, thus reducing ovarian oestrogen production, and this down-regulation of pituitary function is constant, thus leading to severe hypo-estrogenism, and thus temporary amenorrhoea, which is medically suppressed, and is referred to as \"pharmacological oophorectomy\" \u003csup\u003e[16,17]\u003c/sup\u003e. Due to the lack of oestrogen in the endometriotic cells leading to atrophy of the ectopic endometrial tissue, it is because of this down-regulation of the pituitary gland and the inhibition of gonadotropin release to maintain the hypo-estrogenic state that patients tend to suffer from perimenopausal symptoms, such as hot flashes and sweats, irritability, poor sleep, and osteoporosis\u003csup\u003e\u0026nbsp;[18]\u003c/sup\u003e. Hot flashes and sweats have been reported in about 90% of women, and bone loss occurs in 2.5% to 5% of women after 4 - 6 months of treatment\u003csup\u003e\u0026nbsp;[19]\u003c/sup\u003e. Atrophic vaginitis, insomnia, peripheral oedema and nervousness have also been reported, but the prevalence is not known at present\u003csup\u003e\u0026nbsp;[20]\u003c/sup\u003e, in view of which the safety and efficacy of GnRH-a treatment beyond 6 months is controversial, which undoubtedly limits the clinical efficacy of GnRH-a\u003csup\u003e\u0026nbsp;[21]\u003c/sup\u003e. Therefore, it is necessary to seek complementary or alternative therapies to reduce the side effects of GnRH-a in endometriosis treatment. The proposed reverse addition theory can remedy this deficiency by adding low-dose estrogen and progesterone, which, on the one hand, will not cause ectopic endometrial growth and, on the other hand, can improve perimenopausal symptoms and quality of life due to GnRH-a, while progesterone prevents estrogen-induced endometrial overgrowth. Although the theory of reverse addition can effectively alleviate perimenopausal symptoms, long-term hormone use can also trigger some adverse effects, such as affecting coagulation levels and increasing the risk of venous thrombosis; increasing the chances of endometrial cancer, breast cancer and so on\u003csup\u003e\u0026nbsp;[22]\u003c/sup\u003e. Influenced by traditional thinking, hormone replacement therapy is less accepted in China, and patient compliance is poor. Therefore, an urgent problem is to find other safe and effective reverse additive drugs.\u003c/p\u003e\n\u003cp\u003eXiangshao granule is a compound preparation composed of 10 traditional Chinese medicines, such as Fragrant herb and white peony\u003csup\u003e\u0026nbsp;[23]\u003c/sup\u003e, which is a national class III new drug (traditional Chinese medicine). Shaoxing granules are effective in relieving perimenopausal symptoms in women. However, the current studies are based on the treatment of Xiang shao granules in healthy naturally menopausal and perimenopausal women. There are few studies on the treatment of pharmacological menopause, so we hypothesised that the Fragrance Granules could also rapidly relieve perimenopausal symptoms such as hot flashes and sweating caused by pharmacological menopause. In this study, it was proposed to establish a rat model of pharmacological oophorectomy induced by GnRH-a treatment by means of GnRH-a injection, with physiological saline (NS) injection as a control. The intervention was carried out by using paeoniflora granules and estradiol drug to further understand the effect of paeoniflora granules on female reproductive endocrine secretion.\u003c/p\u003e\n\u003cp\u003eMaterials and methods、\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e1.Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.1 Experimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four female Sprague-Dawley (SD) clean-grade rats, with an average weekly age of 6-8 weeks, were selected and purchased from Shanghai Bicaikewing Biotechnology Co., Ltd. and approved by the Ethics Committee for Laboratory Animal Welfare, and the experiments were formally begun after the rats had been acclimated to the rearing environment for 1 week. All experimental operators of the experimental animals in this study possessed the Laboratory Animal Induction Certificate, and all experimental operations complied with the Regulations of the People's Republic of China on the Management of Laboratory Animals issued by the National Science and Technology Commission of China and the Regulations on the Management of Laboratory Animals issued by the Jiangsu Provincial Government.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2 Experimental reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(1) Gonadotropin-releasing hormone agonist (GnRH-a): generic name treprostinil acetate, trade name Dafylline, its main ingredient is treprostinil acetate, excipients: polypropylene cross ester ethyl cross ester copolymer, mannitol, carboxymethylcellulose, polysorbate 80, specification 3.75mg/capsule, Manufacturer: Beaufor Ipsen Pharmaceuticals Limited, France.\u003c/p\u003e\n\u003cp\u003e(2) Xiangshao granules: main ingredients: white peony, fragrant herbs, neem seeds (fried), chaihu, chuanxiong rhizome, citrus aurantium, half-sia (ginger-made), cardamom, woodruff, liquorice. Excipients: sucrose, betacyclodextrin, dextrin. Specification: 4g/bag. Manufacturer: Yangzijiang Pharmaceutical Group Sichuan Hairong Pharmaceutical Co., Ltd, Batch No.: State Standard Z20050425.\u003c/p\u003e\n\u003cp\u003e(3) Estradiol Valerate: generic name: Estradiol Valerate Tablets, trade name: Supplement Jiale, main ingredient: Estradiol Valerate (E2), specification: 1mg/capsule, manufacturer: Bayer Healthcare Co., Ltd. Guangzhou Branch.\u003c/p\u003e\n\u003cp\u003e(4) Others\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.3 Enzyme-linked immunosorbent assay (ELISA) reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.4 HE staining reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1 Animal modelling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(1) GnRH-a injectable drug ovarian denudation animal modelling: 18 SD adult female rats, GnRH-a (tretinoin acetate for injection, dafilgrastim) was administered intramuscularly at 0.06 mg /kg for 7 consecutive days, and thereafter maintained for 21 days in accordance with the maintenance injections at 1/5 of the starting dose.\u003c/p\u003e\n\u003cp\u003e(2) Saline injection control group: 6 adult female SD rats were injected with saline 0.06 mg/kg intramuscularly for 7 consecutive days, after which the injection was maintained at 1/5 of the starting dose for 21 days. The saline control group was designed to reduce the error associated with drug injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Modelling results determination:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to determine the success of GnRH-a injection drug modelling, vaginal smears of rats were observed at a fixed time selected every day 7 days after GnRH-a injection, and the vaginal secretion cells were observed under the microscope to determine the motility cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Experimental drug gavage intervention:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePharmacological interventions were started after successful modelling (2 weeks of continuous GnRH-a injection) in each group. Estradiol valerate (E2) and Xiang shao granules were processed and dissolved in sterile saline to form a turbid solution for rats to be gavaged, and rats were weighed every other day, and the dosage of the drug was adjusted according to the changes in body mass.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Experimental programme and grouping:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the modelling of the experimental rats was completed, 24 rats were randomly divided into 4 groups of 6 rats each, depending on the different drug intervention protocols:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eGnRH-a injected saline group (GnRH-a+NS): 2 weeks after GnRH-a injection, 0.06 mg/kg saline was used for 28 d of continuous gavage.\u003c/li\u003e\n \u003cli\u003eGnRH-a injected oestradiol group (GnRH-a+E2): 2 weeks after GnRH-a injection, 0.8 mg/kg oestradiol valerate E2 was used by continuous gavage for 28d.\u003c/li\u003e\n \u003cli\u003eGnRH-a injection of Xiang shao granules group (GnRH-a + Xiang shao): after 2 weeks of GnRH-a injection, 60 mg/kg of Xiang shao granules were used for 28 d of continuous gavage.\u003c/li\u003e\n \u003cli\u003ePhysiological saline injection control group physiological saline group (NS+NS): rats saline 0.06 mg/kg intramuscular injection for 2 weeks, then use 0.06 mg/kg physiological saline, continuous gavage for 28d.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Body temperature and body weight measurements of rats in each group:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter successful modelling, the anal temperature of each group was measured and the body weight of rats was weighed every other day. The average body temperature of each group was calculated and plotted as a \"temperature-time curve\"; at the same time, the body weights of the rats were weighed and recorded every other day, and a \"body weight-time curve\" was plotted according to the average body weights of the rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Experimental rat execution, sample collection and testing:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the drug intervention in each group, rats in each group were injected intraperitoneally with 1% pentobarbital sodium and then decapitated to take rat specimens. Blood (serum and plasma, respectively) was taken from the abdominal vein of the rats, and the uterus and ovaries were removed by saline irrigation.\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eUterine weight and uterine index: After the execution of rats in each group, the intact uterus was dissected and removed, the wet weight of the uterus was weighed and the uterine index was calculated according to the uterine index formula (uterine index (UMI) = wet weight of the uterus / body weight (%)), the uterine index of each rat was calculated, and the data were statistically analysed.\u003c/li\u003e\n \u003cli\u003eOvary weight and ovarian coefficient: After the rats in each group were executed, the intact bilateral ovaries of the rats were dissected and removed. The ovarian coefficient was calculated according to the formula of ovarian coefficient (ovarian coefficient = wet weight of ovary/body weight (%)), and the ovarian coefficient of each rat was calculated and the data were statistically analysed.\u003c/li\u003e\n \u003cli\u003eELISA assay to detect changes in the expression levels of E2, FSH and LH in plasma.\u003c/li\u003e\n \u003cli\u003eHE staining to detect morphological changes in the endometrium.\u003c/li\u003e\n \u003cli\u003eHE staining to detect morphological changes in the ovary and observe follicular development.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e2. All data in this study were statistically analysed using SPSS27.0 statistical software. All measurements were expressed as means plus or minus standard deviation (士s), and one-way ANOVA was used to compare the means of multiple samples within a group; if significant differences existed, the Turkey test was used for further between-group comparisons. Statistical significance was taken as P\u0026lt;0.05. Graph Pad Prism 10 software was used to draw the relevant graphs for this study.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1.\u0026nbsp; \u0026nbsp;Analysis of vaginal smear results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results showed that the vaginal cell smears of rats in the control group (NS+NS group) showed changes in the estrous cycle (see Figure 1-1), and the cells in the smears were mostly polygonal superficial cells with less cytoplasm and a few epithelial cells; on the contrary, there were no changes in the estrous cycle in the vaginal cell smears of the rats in the GnRH-a model group, which only stayed in the inter-estrous phase, and the cell smears were more homogeneous, mainly with leukocytes and rounded epithelial cells (see Figure 1-2). On the other hand, in the GnRH-a model group, the vaginal cell smears showed no changes in the estrous cycle and only remained in the inter-estrous phase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.Trends of body weight changes in rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the 2-week feeding period of rat modelling, the body weight of GnRH-a-injected rats (GnRH-a+NS, GnRH-a+E2, GnRH-a+ Xiang shao) increased compared with that of the control group (NS+NS), and the body weight of rats in the three groups of rats with pharmacological interventions increased more significantly in the GnRH-a+NS than in the GnRH-a+E2, GnRH-a+Xiang shao groups. As shown in Figure 2 (the \u0026quot;weight-time trend graph\u0026quot; of rats was plotted based on the recording results), within 14 days of rat modelling, the weight of rats in the GnRH-a injection group increased significantly compared with that of the control (NS+NS) group, whereas during the period of drug intervention (18d-42d), rats in the GnRH-a+E2, GnRH-a+Xiang shao group increased significantly. a + Xiang shao granules group showed a slower increase in body weight than the GnRH-a+NS group. This phenomenon suggests that E2 and paeoniflora granules can effectively slow down the trend of GnRH-a-induced weight gain in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.Trends in rat body temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the 2-week feeding period of rat modelling, the body temperatures of GnRH-a-injected rats (GnRH-a+NS, GnRH-a+E2, GnRH-a+ Xiang shao) increased significantly compared with those of the control group (NS+NS), and among the three groups of rats intervened with the drug, the body temperatures of rats in the GnRH-a+NS group rose significantly compared with those of rats in the GnRH-a+E2 and GnRH-a+ Xiang shao groups, as shown in Fig. 3 (the \u0026quot;time trend\u0026quot; graph of rats was plotted based on the recorded results). See Fig. 3 for details (plotting the \u0026quot;body temperature-time trend graph\u0026quot; of rats based on the recording results). As shown in Fig. 3, within 14 days of rat modelling, the body temperature of rats in the GnRH-a injection group increased significantly compared with that of the control (NS+NS) group, and during the period of pharmacological interventions (20d-42d), the body temperature of rats in the GnRH-a+E2, GnRH-a+Xiang shaos group increased significantly compared with that in the GnRH-a+E2, GnRH-a+Xiang shaos group. GnRH-a+Xiangshao group showed a significant decreasing trend in body temperature, and the decreasing trend of body temperature was similar in GnRH-a+E2, GnRH-a+Xiangshao groups. This phenomenon suggests that GnRH-a injection can cause the rats to have a response of increased body surface temperature similar to perimenopausal hot flashes, and the intervention of E2 and Xiang shao granules can rapidly improve this situation, and Xiang shao granules can improve the perimenopausal hot flashes symptoms induced by GnRH-a to the same extent as E2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Serum sex hormone levels after different drug interventions in each group of rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the drug gavage intervention, the serum E2 levels of all three groups of GnRH-a-injected rats (GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiangshao) were all significantly all lower than those of the NS+NS group (P\u0026lt;0.001), and when compared between the groups, the E2 levels of the GnRH-a+E2, and GnRH-a+Xiang shao groups were significantly higher than those of the GnRH-a+NS group ( P\u0026lt;0.001, P\u0026lt;0.05), and E2 levels were significantly lower in the GnRH-a+ Xiangshao group than in the GnRH-a+E2 group (P\u0026lt;0.05); at the end of the pharmacological intervention, serum FSH levels in the GnRH-a+NS, GnRH-a+E2 and GnRH-a+ Xiangshao groups were significantly lower than those of the NS+NS group (P\u0026lt;0.001). FSH levels in the GnRH-a+E2 group showed a slight downward trend compared with those in the GnRH-a+NS group, but the difference was not statistically significant (P\u0026gt;0.05), FSH levels in the GnRH-a+E2 group were significantly lower compared with those in the GnRH-a+ Xiangshao granules group (P\u0026lt;0.05), and there was no statistically significant difference between FSH levels in the GnRH-a+NS and GnRH-a+ Xiangshao groups (P\u0026gt;0.05). (P\u0026gt;0.05); at the end of the drug intervention, the serum LH levels of rats in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+ Xiangshao groups were all significantly lower than those in the NS+NS group (P\u0026lt;0.01, P\u0026lt;0.001, P\u0026lt;0.001), and the LH levels in the GnRH-a+E2 group were significantly lower than those in the GnRH-a+NS versus GnRH-a+ Fragrant peony granule group (P\u0026lt;0.001, P\u0026lt;0.05), and the serum LH level in the GnRH-a+ Fragrant peony granule group was slightly decreased compared with that in the GnRH-a+ NS group, but statistically there was no statistical difference (P\u0026gt;0.05). The test results suggested that GnRH-a injection could achieve the expected effect of establishing a perimenopausal rat model, and this animal model successfully achieved a significant decrease in the levels of E2, FSH and LH in rats, meanwhile, the fragrant peony granules could increase the level of E2 in vivo, which indicated that the fragrant peony granules might have a certain stimulating effect on the ovary or have a certain estrogen-like effect, which could produce a small amount of estrogen, but its effect was It can produce a small amount of estrogen, but its effect is significantly lower than that of estrogen, indicating that the estrogen-like effect of Xiang shao granules is relatively weak, and Xiang shao granules will not increase the levels of FSH and LH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.1 Comparison of plasma sex hormone (E2, FSH, LH) levels after drug intervention in each group of rats\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"549\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.27007299270073%\" valign=\"top\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.75912408759124%\" valign=\"top\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003eE2\u003c/p\u003e\n \u003cp\u003e(pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.985401459854014%\" valign=\"top\"\u003e\n \u003cp\u003eFSH\u003c/p\u003e\n \u003cp\u003e(IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003eLH\u003c/p\u003e\n \u003cp\u003e(IU/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.27007299270073%\" valign=\"top\"\u003e\n \u003cp\u003eNS+NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.75912408759124%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003e34.20\u0026plusmn;8.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.985401459854014%\" valign=\"top\"\u003e\n \u003cp\u003e2.72\u0026plusmn;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003e2.03\u0026plusmn;0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.27007299270073%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.75912408759124%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003e1.45\u0026plusmn;0.38***###\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.985401459854014%\" valign=\"top\"\u003e\n \u003cp\u003e1.45\u0026plusmn;0.38***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003e1.55\u0026plusmn;0.24***###\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.27007299270073%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+E2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.75912408759124%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003e1.16\u0026plusmn;0.34**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.985401459854014%\" valign=\"top\"\u003e\n \u003cp\u003e1.16\u0026plusmn;0.34***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003e0.91\u0026plusmn;0.13***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.27007299270073%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+ Xiangshao\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.75912408759124%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003e1.70\u0026plusmn;0.36***#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.985401459854014%\" valign=\"top\"\u003e\n \u003cp\u003e1.70\u0026plusmn;0.36***#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.99270072992701%\" valign=\"top\"\u003e\n \u003cp\u003e1.48\u0026plusmn;0.46***##\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e*P\u0026lt;0.05, **P\u0026lt;0.01 ***P＜0.001vs NS+NS，#P＜0.05，##P＜0.01，###P＜0.001 vs GnRH-a+E2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.Morphological changes in uterine index and endometrial tissue of rats in all groups after drug intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.1 Uterine wet weight and uterine index in rats after drug intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"557\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.014336917562723%\" valign=\"top\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.21505376344086%\" valign=\"top\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.72043010752688%\" valign=\"top\"\u003e\n \u003cp\u003eUterus wet weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.05017921146953%\" valign=\"top\"\u003e\n \u003cp\u003eUterus index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.014336917562723%\" valign=\"top\"\u003e\n \u003cp\u003eNS+NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.21505376344086%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.72043010752688%\" valign=\"top\"\u003e\n \u003cp\u003e0.5355\u0026plusmn;0.1314\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.05017921146953%\" valign=\"top\"\u003e\n \u003cp\u003e0.192\u0026plusmn;0.051278\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.014336917562723%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.21505376344086%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.72043010752688%\" valign=\"top\"\u003e\n \u003cp\u003e0.17425\u0026plusmn;0.01846***###\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.05017921146953%\" valign=\"top\"\u003e\n \u003cp\u003e0.055\u0026plusmn;0.005353***###\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.014336917562723%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+E2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.21505376344086%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.72043010752688%\" valign=\"top\"\u003e\n \u003cp\u003e0.354625\u0026plusmn;0.048718\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.05017921146953%\" valign=\"top\"\u003e\n \u003cp\u003e0.122\u0026plusmn;0.020585***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.014336917562723%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+ Xiangshao\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.21505376344086%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.72043010752688%\" valign=\"top\"\u003e\n \u003cp\u003e0.28025\u0026plusmn;0.029305***#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.05017921146953%\" valign=\"top\"\u003e\n \u003cp\u003e0.091\u0026plusmn;0.011101***#\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e***P＜0.001vs NS+NS；#P＜0.05，###P＜0.001 vs GnRH-a+E2\u003c/p\u003e\n\u003cp\u003eAfter the drug intervention, the uterine index of all three groups of rats injected with GnRH-a (GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao) was significantly smaller than that of the NS+NS group (P\u0026lt;0.001), and the uterine index of the rats in the GnRH-a+E2 and GnRH-a+Xiang shao groups was significantly increased compared with that of the GnRH-a+NS (P\u0026lt;0.001 and P\u0026lt;0.05), and the uterine index of rats in the GnRH-a+E2 group was significantly higher than that in the GnRH-a+ Xiangshao granule group (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2 Morphological changes of endometrial tissue after drug intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of drug intervention, endometrial tissues of rats in each group were subjected to pathological section while stained with HE staining, and the results showed that: endometrium of rats in NS+NS group consisted of a single layer of columnar epithelial cells, tubular glands were distributed in the lamina propria, glandular epithelium was a single layer of columnar shape, cell morphology had no abnormality, there was no oedema, no atrophy or hyperplasia. The uterine wall of rats in the GnRH-a injection group was significantly thinner, and the endometrial layer was atrophied. The epithelial cells were flattened and partially atrophied. The uterine wall of rats in the GnRH-a+E2, GnRH-a+ Xiangshao granules group showed all the increases in the thickness of the uterine wall compared with that of rats in the GnRH-a+NS group, the endometrial epithelial cells were in the form of columns or cubes, and the cells were not edematous, but there were different degrees of atrophic thinning in the GnRH-a+NS group compared to the GnRH-a+NS group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Morphological changes in ovarian index and sub-ovarian tissues of rats in various groups after drug intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.1 Ovarian wet weight, ovarian index in rats after drug intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"546\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.80952380952381%\" valign=\"top\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\" valign=\"top\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.61538461538461%\" valign=\"top\"\u003e\n \u003cp\u003eOvarian wet weight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.967032967032964%\" valign=\"top\"\u003e\n \u003cp\u003eOvarian index\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.80952380952381%\" valign=\"top\"\u003e\n \u003cp\u003eNS+NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.61538461538461%\" valign=\"top\"\u003e\n \u003cp\u003e0.204625\u0026plusmn;0.027682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.967032967032964%\" valign=\"top\"\u003e\n \u003cp\u003e0.0732\u0026plusmn;0.010204\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.80952380952381%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+NS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.61538461538461%\" valign=\"top\"\u003e\n \u003cp\u003e0.093625\u0026plusmn;0.017353***###\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.967032967032964%\" valign=\"top\"\u003e\n \u003cp\u003e0.0294\u0026plusmn;0.004776***###\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.80952380952381%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+E2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.61538461538461%\" valign=\"top\"\u003e\n \u003cp\u003e0.13675\u0026plusmn;0.01836***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.967032967032964%\" valign=\"top\"\u003e\n \u003cp\u003e0.0468\u0026plusmn;0.005377***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.80952380952381%\" valign=\"top\"\u003e\n \u003cp\u003eGnRH-a+ Xiangshao\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.608058608058608%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.61538461538461%\" valign=\"top\"\u003e\n \u003cp\u003e01185\u0026plusmn;0.015793***#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.967032967032964%\" valign=\"top\"\u003e\n \u003cp\u003e0.0385\u0026plusmn;0.006576***#\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e***P＜0.001vs NS+NS；#P＜0.05，###P＜0.001 vs GnRH-a+E2\u003c/p\u003e\n\u003cp\u003eAfter the drug intervention, the ovarian indices of all three groups of GnRH-a-injected rats (GnRH-a+NS, GnRH-a+E2, GnRH-a+ Xiangshao) were significantly smaller than those of the NS+NS group (P\u0026lt;0.001), and the ovarian indices of the rats in the GnRH-a+E2, and GnRH-a+ Xiangshao groups were significantly higher when compared with those in the GnRH-a+NS (P\u0026lt;0.001 and P\u0026lt;0.05), and the uterine index of rats in the GnRH-a+E2 group was significantly higher than that in the GnRH-a+ Xiangshao granule group (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.2 Morphological changes of ovarian tissue after drug intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of drug intervention, complete ovaries of rats in each group were taken out, and pathological sections were stained with HE staining at the same time, and morphological changes of ovarian groups were observed under the microscope, which showed that: follicle structure was normal in the NS+NS group, with visible follicles at all levels; follicle structure was normal in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao granules groups, and the number of follicular growth and mature follicles was obviously reduced.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe drug GnRH-a, which inhibits oestrogen synthesis, can be used to control recurrence after endometriosis surgery. However, long-term use of GnRH-a can lead to secondary perimenopausal reactions, which can cause discomfort such as hot flashes and sweats, emotional irritability, and poor sleep, and is the main reason for patients to give up the treatment. Reverse-addition therapy, although a good solution to perimenopausal symptoms, but long-term use of hormones may lead to liver function damage, venous embolism, vascular disease, and increase the risk of endometrial cancer, ovarian cancer, breast cancer and other diseases. Therefore, the search for effective and safe counter-additive drugs is an urgent problem to be solved.\u003c/p\u003e \u003cp\u003eXiangshao granule is a new class III new drug (traditional Chinese medicine) that has been newly listed in China in recent years, which is extracted from 10 purely natural plant medicines, such as Fragrance, White peony, Neem, Chaihu, Chuanxiong, Citrus aurantium, Cardamom and so on \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and its main effects have the effects of dredging the liver and regulating the qi, calming the liver and relieving the pain. Clinical phase II and III studies have proved \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e that the fragrant peony granules have good efficacy in perimenopausal syndrome and premenstrual syndrome, and can rapidly relieve the symptoms of perimenopausal syndrome such as hot flashes and sweating, depression, and emotional irritability, etc. However, little research has been done on perimenopausal symptoms caused by pharmacological menopause, and in view of the secondary, we can deduce that it also antagonises the symptoms of postoperative GnRH-a treatment for patients with endometriosis. This study aims to investigate the clinical efficacy of Xiang shao granules in antagonising perimenopausal symptoms caused by postoperative GnRH-a treatment in patients with endometriosis using oestradiol as a reference, in the hope that it can become a new, safer and more reliable alternative to reverse-addition therapy after GnRH-a treatment.\u003c/p\u003e \u003cp\u003eIn this study, GnRH-a injection was used to establish a pharmacological ovariectomy rat model, and vaginal smears were given to observe the changes in the vaginal cells of rats in order to determine the success of the modelling. In this study, the vaginal smears of rats showed that the changes in the estrous cycle of rats in the GnRH-a group had disappeared, and the vaginal cells showed a persistent inter-estrous phase, with the cells mainly being the rounded outer primordial layer with a large rounded nucleus, which means that the modelling was successful, and it was further confirmed that GnRH-a could induce the hypoestrogenic state in the body of the rats. In addition, the results of this study showed that the body temperature of rats injected with GnRH-a was on the rise, and the body temperature of rats with E2 and Xiang shao granules was on the decline, and the degree of decline of both was similar, which further indicated that GnRH-a could cause perimenopausal hot flashes, sweating and other discomforts, and Xiang shao granules and oestradiol could improve this symptom, and the degree of improvement of hot flashes was similar to the degree of improvement of estradiol in the perimenopausal period. The degree of improvement of hot flushes by Xiang shao granules is similar to that of estradiol, which can rapidly relieve the symptoms of perimenopausal hot flushes. The results of this study showed that GnRH-a injection could increase the body weight of rats significantly, and at the same time, it could reduce the levels of serum sex hormones E2, FSH and LH, and lower the uterine index and ovarian index of rats, and it was found that the endometrium of the rats injected with GnRH-a was obviously atrophied, the number of follicular growth follicles and mature follicles was reduced significantly, and the primordial follicles were increased, and the body weight of rats was significantly reduced after the interventions of E2 and Paeoniae chinensis granules, and the body weight of rats was increased. After the intervention of E2 and Xiang shao granules, the body weight of rats decreased significantly, which indicated that Xiang shao granules had the same effect as estradiol in alleviating the perimenopausal weight gain induced by GnRH-a. At the same time, after the intervention of Xiang shao granules, the level of E2 in the rats increased, but to a lesser extent than that of the E2 group, which further indicated that Xiang shao granules might have the effect of stimulating or improving the function of ovary, or have a slight estrogenic effect, which was consistent with the study by Li et al. This is the same as the results of Li et al. who studied the effects of Xiang shao granules on perimenopausal ovarian function \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. However, the results of HE staining of rat endometrium showed that there was no significant endometrial hyperplasia in the Xiang shao granules group, which indicated that the estrogen-like effect of Xiang shao granules may be weak and have a small effect on the endometrium, and there was no difference in the serum levels of FSH and LH between rats and the control group, which indicated that Xiang shao granules may not exert anti-perimenopausal effects through estrogen-like effects, so what pathway is Xiang shao granules acting on perimenopausal symptoms? The pathway through which the Xiang shao granule exerts its menopause-relieving effect is still unclear, and needs to be further investigated, which is also the focus of our next study.\u003c/p\u003e \u003cp\u003eIn summary, GnRH-a injection can cause low FSH, LH and E2 levels in perimenopausal rats, which can lead to perimenopausal-like symptoms such as hot flashes and weight gain, which are more in line with the perimenopausal clinical discomfort caused by GnRH-a. It was found that Xiang shao granules can improve the symptoms such as hot flashes quickly and effectively, and although Xiang shao granules can cause estrogen elevation in rats, according to endometrial HE staining, it does not cause endometrial hyperplasia. Although this study found that Xiang shao granules can cause the increase of estrogen in rats, according to the HE staining of endometrium, Xiang shao granules do not cause endometrial hyperplasia. Therefore, although Xiang shao granules have the estrogen-like effect, its estrogen-like effect is weak, and it will not increase the risk of endometrial hyperplasia, breast cancer and other related risks, but it can achieve the same as estrogen to alleviate the symptoms of peri-menopausal period. Meanwhile, as mentioned above, both Xiang shao granules and estradiol can alleviate perimenopausal symptoms such as hot flashes, which have certain commonalities, but the difference is that estradiol improves the symptoms through direct exogenous estrogen, while Xiang shao granules have a small amount of estrogenic effect, but its effect may be through other pathways, which shows that Xiang shao granules can effectively alleviate the perimenopausal symptoms due to GnRH-a, and will not increase the risk of endometrial cancer and breast cancer. It is a safer alternative to GnRH-a as it can effectively relieve perimenopausal symptoms without increasing the risk of endometrial cancer, breast cancer, renal vein thrombosis, and so on.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eGnRH-a injection can reduce the levels of E2, FSH and LH in rats, causing perimenopausal symptoms such as hot flushes, which can be significantly ameliorated by E2 and Xiang shao granules, and Xiang shao granules do not exert their anti-perimenopausal symptomatic effects through oestrogen-like effects, but rather, they act through other pathways.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eFunding details \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChangzhou Municipal Health Commission Science and Technology Project (ZD202314); Changzhou \u0026quot;14th Five-Year Plan\u0026quot; High-level Talent Cultivation Project (2022CZBJ074); Jiangsu Province Maternal and Child Health Key Talent Project (RC202101); Jiangsu Province Maternal and Child Health Scientific Research Project (F202138).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026quot;Huimin Tang and Qiucheng Jia wrote the main manuscript text，Wanying Chen and Yihan Wu Analysied data and mapping，Weiwei Wei and Hong Zheng literatured review，Jiming Chen Designed Experiments and Instruction，All authors reviewed the manuscript.\u0026quot;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWang PH ,Yang ST,Chang WH et al. Endometriosis: Part I. Basic concept .Taiwan J Obstet Gynecol, 2022, 61: 927-934.\u003c/li\u003e\n \u003cli\u003eBulun SE, Yilmaz BD, Sison C, et al. Endometriosis. Endocr Rev. 2019;40(4):1048-1079. doi:10.1210/er.2018-00242\u003c/li\u003e\n \u003cli\u003eTaylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet. 2021 Feb 27;397(10276):839-852.\u003c/li\u003e\n \u003cli\u003eZondervan KT, Becker CM, Missmer SA. Endometriosis. N Engl J Med. 2020;382(13):1244-1256. doi:10.1056/NEJMra1810764\u003c/li\u003e\n \u003cli\u003eAllaire C, Bedaiwy MA, Yong PJ. Diagnosis and management of endometriosis. CMAJ. 2023 Mar 14;195(10):E363-E371.\u003c/li\u003e\n \u003cli\u003eChapron C, Marcellin L, Borghese B, Santulli P. Rethinking mechanisms, diagnosis and management of endometriosis. 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Published 2021 Mar 5. doi:10.3390/jcm10051085\u003c/li\u003e\n \u003cli\u003eSuga S, Akaishi T, Sakuma Y. GnRH inhibits neuronal activity in the ventral tegmental area of the estrogen-primed ovariectomized rat. Neurosci Lett. 1997 May 30;228(1):13-6.\u003c/li\u003e\n \u003cli\u003eHorne AW, Missmer SA. Pathophysiology, diagnosis, and management of endometriosis. BMJ. 2022;379:e070750. Published 2022 Nov 14. doi:10.1136/bmj-2022-070750\u003c/li\u003e\n \u003cli\u003eVannuccini S, Clemenza S, Rossi M, Petraglia F. Hormonal treatments for endometriosis: The endocrine background. Rev Endocr Metab Disord. 2022;23(3):333-355. doi:10.1007/s11154-021-09666-w\u003c/li\u003e\n \u003cli\u003ePerry CM, Brogden RN. Goserelin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in benign gynaecological disorders. Drugs. 1996 Feb;51(2):319-46.\u003c/li\u003e\n \u003cli\u003eFriedman AJ, Hoffman DI, Comite F, Browneller RW, Miller JD. Treatment of leiomyomata uteri with leuprolide acetate depot: a double-blind, placebo-controlled, multicenter study. The Leuprolide Study Group. Obstet Gynecol. 1991 May;77(5):720-5.\u003c/li\u003e\n \u003cli\u003eDonnez J, Dolmans MM. GnRH Antagonists with or without Add-Back Therapy: A New Alternative in the Management of Endometriosis?. Int J Mol Sci. 2021;22(21):11342. Published 2021 Oct 20. doi:10.3390/ijms222111342\u003c/li\u003e\n \u003cli\u003eCapezzuoli T, Rossi M, La Torre F, Vannuccini S, Petraglia F. Hormonal drugs for the treatment of endometriosis. Curr Opin Pharmacol. 2022;67:102311. doi:10.1016/j.coph.2022.102311\u003c/li\u003e\n \u003cli\u003e唐瑞怡,王亚平,张绍芬等.香芍颗粒治疗围绝经期女性情绪障碍的临床研究.实用妇产科杂志,2023,39(03):210-216.\u003c/li\u003e\n \u003cli\u003e陈蓉,郁琦.香芍颗粒临床应用指导建议.中国实用妇科与产科杂志,2015,31(05):419-420.\u003c/li\u003e\n \u003cli\u003e乔明琦,张惠云,姜坤等.经前平颗粒多中心、随机双盲双模拟对照治疗经前期综合征肝气逆证403例.中国新药杂志,2002,(05):389-392.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"xiangshao granules, GnRH-a, pharmacological oophorectomy, perimenopausal syndrome","lastPublishedDoi":"10.21203/rs.3.rs-4161365/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4161365/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To establish a rat model of pharmacological ovariectomy by GnRH-a injection, and to preliminarily investigate the reproductive endocrine effects of Xiangshao granules on pharmacological ovariectomised rats.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A rat model of pharmacological ovariectomy was established by injecting female rats with GnRH-a. The rats were randomly divided into four groups: GnRH-a injected saline group (GnRH-a + NS); GnRH-a injected oestradiol group (GnRH-a + E2); GnRH-a injected Xiang shao granule group (GnRH-a + Xiang shao), and the control group of saline injected rats (NS + NS). according to the observation of the vaginal smear of the rats to determine the success of the modelling, after the success of the modelling of the corresponding drug gavage intervention for 28 days, every other day to weigh the body weight of the rats and measure the anal temperature, according to the changes in body weight of the rats to adjust the amount of drug intervention. Plasma sex hormone levels (E2, FSH, LH), uterine weight, uterine index and endometrial histomorphological changes, and ovarian weight, ovarian index and ovarian histomorphological changes were measured in each group after gavage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e(1) Vaginal cell smears of rats in the control group (NS+NS) showed changes in the estrous cycle, whereas vaginal cell smears of rats in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao groups showed no changes in the estrous cycle; (2) The body mass gain of rats in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao groups was significantly higher than that of the NS+NS group, whereas intervention with estradiol (E2) and peony granules significantly slowed down the GnRH-a induced body mass gain. NS group, while the intervention of estradiol (E2) and Xiang shao granules could significantly delay the trend of GnRH-a-induced body mass gain in rats; (3) The anal temperature of rats after GnRH-a injection showed an overall increasing trend, and compared with GnRH-a+NS, the body temperature of rats in GnRH-a+E2 and GnRH-a+Xiang shao groups showed a gradual decreasing trend, and the decreasing of the temperature in Xiang shao granules compared with that of rats in E2 group was (4) Plasma sex hormone levels (E2, FSH, LH) were significantly lower in the GnRH-a+NS, GnRH-a+E2, and GnRH-a+Xiang shao groups than in the NS+NS group (P\u0026lt;0.001), and the levels of E2 in the GnRH-a+E2 and GnRH-a+Xiang shao groups were significantly higher than those in the GnRH-a+NS group (P\u0026lt;0.001, P\u0026lt;0.05), and the levels of E2 in the GnRH-a+NS group were significantly lower than those in the GnRH-a+Xiang shao group (P\u0026lt;0.001, P\u0026lt;0.05). 0.05), and the E2 level in GnRH-a+E2 group was higher than that in GnRH-a+Xiangshao Granules group (P\u0026lt;0.05); the FSH level in GnRH-a+E2 group was significantly lower than that in GnRH-a+ Xiangshao granules group (P\u0026lt;0.05), and there was a slight downward trend in the FSH level of GnRH-a+E2 group compared to that of GnRH-a+NS, but the difference was not statistically significant (P\u0026gt; 0.05); LH levels in the GnRH-a+E2 group were significantly lower than those in the GnRH-a+NS and GnRH-a+Xiang shao groups (P\u0026lt;0.001, P=0.001), whereas there was no significant difference in the LH and FSH levels between the two groups, GnRH-a+NS and GnRH-a+ Xiang shao groups (P\u0026gt;0.05); (5) compared with the NS+NS group, GnRH-a injected rats in each model, uterine weight and uterine index, ovarian weight and ovarian index were significantly decreased (P\u0026lt;0.001); comparing between the groups, the uterine weight and uterine index, ovarian weight and ovarian index of GnRH-a+ E2 and GnRH-a+Xiang shao groups were significantly higher than those of GnRH-a+NS group (P\u0026lt;0.001, P\u0026lt;0.05); uterine weight and uterine index, ovarian weight and ovarian index of GnRH-a+E2 group were significantly higher than those of GnRH-a+NS group (P\u0026lt;0.001, P\u0026lt;0.05); and uterine weight and uterine index, ovarian weight and ovarian index were elevated compared with the GnRH-a+Xiang shao group (P\u0026lt;0.05); (6) compared with the NS+NS group, the number of primordial follicles was significantly higher and the number of growing follicles and mature follicles was significantly lower in the GnRH-a+NS, GnRH-a+E2 and GnRH-a+Xiang shao groups; (7) the number of rats' uterine wall was significantly higher and the number of rats' uterine wall was significantly lower in the NS+NS group than in the GnRH-a NS+NS group and GnRH-a group, the uterine wall of rats in each group was significantly thinner, the endothelial layer was atrophied, the thickness of the uterine wall increased in the GnRH-a+E2 and GnRH-a+Xiang shao groups, and the number of vaginal folds and blood vessels also increased. Among them, the improvement of uterus and vagina was more obvious in GnRH-a+E2 than in GnRH-a+NS and GnRH-a+Xiangshao groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eGnRH-a injection could reduce the levels of sex hormones E2, FSH and LH in rats, causing perimenopausal symptoms such as hot flashes, while E2 and Xiang shao granules significantly improved such symptoms, and Xiang shao granules had a slight oestrogenic effect, but to a lesser extent than E2.\u003c/p\u003e","manuscriptTitle":"A preliminary study on the effects of Xiang shao granules on reproductive endocrinology in drugged ovariectomised rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-28 10:07:03","doi":"10.21203/rs.3.rs-4161365/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-06T14:29:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-26T15:45:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-16T20:12:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4ae3dee5-7da4-42c7-a266-484079a41319","date":"2024-04-07T11:03:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4aa6d0da-7642-418f-9e91-faf4885e32e3","date":"2024-04-05T14:29:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-05T14:28:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-26T16:40:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-25T07:29:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2024-03-25T07:17:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9fd0232a-cdb9-4ee2-9c04-6ceac982ed25","owner":[],"postedDate":"March 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T16:11:48+00:00","versionOfRecord":{"articleIdentity":"rs-4161365","link":"https://doi.org/10.1186/s13048-024-01531-z","journal":{"identity":"journal-of-ovarian-research","isVorOnly":false,"title":"Journal of Ovarian Research"},"publishedOn":"2024-10-18 15:57:02","publishedOnDateReadable":"October 18th, 2024"},"versionCreatedAt":"2024-03-28 10:07:03","video":"","vorDoi":"10.1186/s13048-024-01531-z","vorDoiUrl":"https://doi.org/10.1186/s13048-024-01531-z","workflowStages":[]},"version":"v1","identity":"rs-4161365","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4161365","identity":"rs-4161365","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}