Therapeutic effects of Huayu Jiedu formula on endometriosis via downregulating GATA 6 expression.

Background: Endometriosis (EMs) is a common chronic inflammatory disease which is characterized by multiple clinical symptoms and high recurrence rate due to the absence of effective therapies. Huayu Jiedu Formula (HYJDF), is a traditional Chinese medicine prescription with five major herbs. It has been used as traditional medicine to treat EMs for more than twenty years and exerted a good therapeutic effect. However, the underlying mechanism is unclear. Here we aim to observe the effects of HYJDF on EMs and investigate the therapeutic mechanism. Methods: The extract components of HYJDF were identified and quantified by an UHPLC-QE-MS method. Network pharmacology was used to obtain the core targets of HYJDF for the treatment of EMs and the specific biologic processes involved. A total of 68 EMs cases were randomly divided into control (gestrinone) and observation (HYJDF) groups. The overall effectiveness, pain scores, cyst-size changes, serum CA125 levels, quality-of-life scores, safety, and adverse events were evaluated before and after treatment. For the mechanism research, DNA methylation-chip analysis was performed to determine the differential genes. EMs mice models and human ectopic stromal cells (ESCs) were treated with HYJDF and its pharmaceutical serum, respectively. The ectopic foci was measured via H&E staining while the expressions of the target genes were verified by real-time PCR and Western blot analysis. The inflammatory cytokine levels in the peritoneal fluid of mice were detected by ELISA. The proliferative potential of cells was analyzed by MTS whereas the apoptosis and cell cycle were determined through flow analysis. Results: The total number of components detected in positive and negative ion modes was 839 and 597, respectively. Network pharmacology suggested that HYJDF treated EMs through DNA methylation. We found that HYJDF and gestrinone exerted good therapeutic effect with no obvious difference, but the HYJDF treatment group had fewer side effects. GATA 6, which was hypomethylated and abundant in endometriotic cells, potently induced inflammatory response. This finding indicated the important role of GATA 6 in EMs development. Moreover, HYJDF ameliorated inflammatory response (i.e., reduced the levels of IL-1ß and PGE2 in peritoneal fluid), suppressed ESCs proliferation, and increased cell apoptosis by down-regulating GATA 6 expression. Conclusion: We demonstrated that HYJDF has anti-inflammation activity and increased cell apoptosis through the reduction of GATA 6 expression in ectopic tissues, which showed good therapeutic effect without any obvious side effects. These findings suggest that HYJDF may be a new and efficient traditional Chinese medicine for the treatment of EMs.

A Network Pharmacology Study on the Similarities and Differences in the Mechanisms of Zuo Gui Wan/You Gui Wan for the Treatment of Premature Ovarian Failure.

BACKGROUND: Premature ovarian failure is a heterogeneous disease that severely affects the quality of life of women in their reproductive years. The ancient classical Chinese medicine compounds Zuo Gui Wan and You Gui Wan have great potential to treat premature ovarian failure, but the similarities and differences in their pharmacological mechanisms for treating POF are not yet clear. METHODS: In this study, the public database was used to screen the active ingredients and potential targets of Zuo Gui Wan and You Gui Wan. The similarities and differences in the potential targets of both pills for the treatment of POF were analysed using the POF-related genes obtained from OMIM and GeneCards. The protein-protein interaction network was established and collated to form a drug-active ingredient-target gene network using STRING. Finally, the drug-target-pathway network was constructed by enrichment analysis to find the differences in target enrichment on the same pathway. RESULTS: Pharmacological analysis of the network showed that Zuo Gui Wan contains 72 active ingredients, while You Gui Wan has 112. A total of 62 common compositions, such as quercetin and kaempferol, were identified. Amongst them were 10 unique compounds, such as hydroxyproline and cholesterol, in Zuo Gui Wan and 50 exclusive compounds, such as Karanjin and betacarotene, in You Gui Wan. In addition, 14 overlapping targets, including MAPK1, CXCL8, TNF, IL6, and EGFR, were determined amongst the first 20 targets in the treatment of POF by both pills, demonstrating that the core mechanism of POF treatment is similar between the two. Pathway enrichment analysis showed 87 identical and significant pathways between Zuo Gui Wan and You Gui Wan, including IL-17, TNF, PI3K-Akt, oestrogen, VEGF, and other pathways. Zuo Gui Wan has 12 special pathways, such as natural killer cell-mediated cytotoxicity and intestinal immune network for IgA production. You Gui Wan has nine unique pathways, such as insulin secretion and glucagon signalling pathway. CONCLUSION: Zuo Gui Wan and You Gui Wan could treat POF by inhibiting oxidative stress and inflammation, regulating hormone levels, improving ovarian function, and promoting follicular development. Zuo Gui Wan is inclined to immune regulation, while You Gui Wan prefers insulin regulation. Therefore, similarities and differences clearly exist in the specific mechanisms of Zuo Gui Wan and You Gui Wan in the treatment of POF.

Huayu Jiedu Fang Protects Ovarian Function in Mouse with Endometriosis Iron Overload by Inhibiting Ferroptosis.

Endometriosis (EM) is a common chronic inflammatory disease in women. Sampson's retrograde menstruation theory is the most widely accepted theory of EM pathogenesis. The periodic bleeding of ectopic lesions is an important pathological feature of this disease, and the occurrence and progression of EM are closely associated with the iron overload caused by ectopic lesions. However, animal models that simulate menstrual-blood reflux and hemorrhage from EM lesions are lacking. In this study, we performed intraperitoneal injection of endometrial fragments and periodic intraperitoneal blood injection to simulate the real cause and disease state of EM and successfully constructed a mouse model of EM iron overload. Our research found that the number, size, and degree of adhesion of EM lesions in the iron-overload model mouse were significantly higher than those in the model mouse. Moreover, the iron concentration in the abdominal fluid and ovary significantly increased, and the level of malondialdehyde (MDA) in the ovary increased. Conversely, GPX4, GSH, and other anti-ferroptosis-related proteins were downregulated, proving the occurrence of ferroptosis. Huayu Jiedu Fang (HYJDF) is an empirical prescription for EM treatment. This study combined animal experiments, UHPLC-QE-MS analysis, and network pharmacology to analyze whether HYJDF can inhibit ferroptosis to slow down the progression of EM and protect ovarian function. Based on the constructed iron-overload model, HYJDF can reduce the volume of EM lesions and the degree of adhesion, downregulate the total iron concentration in the peritoneal fluid and ovary, upregulate GPX4 expression and GSSG in the ovary, downregulate the level of MDA in the ovary, and promote the development of follicles. We further confirmed that HYJDF can inhibit the progression of EM disease and improve the ovarian function of the model mouse by inhibiting ferroptosis. Finally, through UHPLC-QE-MS and network pharmacology analysis, the natural compounds in HYJDF were identified and verified and the regulatory effect of HYJDF on the EM ferroptosis pathway through the IL-6/hepcidin pathway was preliminarily elucidated.

Alpha-linolenic acid regulates the gut microbiota and the inflammatory environment in a mouse model of endometriosis.

PROBLEM: This study aims to investigate the effects of alpha-linolenic acid (ALA) on the gut microbiota (GM) and the abdominal environment in mice with endometriosis (EMS). METHODS: The effects of faecal microbiota transplantation (FMT) from EMS mice on mice treated with antibiotic cocktail were conducted. The 16S rRNA sequencing and PICRUSt software were used to detect the structure and function of GM respectively. The protein levels of Claudin 4 and ZO-2 in the intestinal wall were detected using the western blotting. The level of LPS in the abdominal cavity was detected using enzyme-linked immunosorbent assay (ELISA). The content of macrophages in the abdominal cavity was detected using flow cytometry. RESULTS: The exogenous supplementation of ALA could restore the abundance of Firmicutes and Bacteroidota in EMS mice. After the ALA treatment, the abundance of 125 functional pathways and 50 abnormal enzymes related to GM in EMS mice was significantly improved (p < .05). The expression of the ZO-2 protein in the intestinal wall was decreased, and the level of LPS in the abdominal cavity was significantly increased after FMT from EMS mice (p < .05). ALA could increase the expression of the ZO-2 protein in the intestinal wall of EMS mice, reduce the level of LPS in the abdominal cavity (p < .05) and reduce the aggregation of peritoneal macrophages (p < .05). CONCLUSION: Alpha-linolenic acid can improve the GM, intestinal wall barrier and abdominal inflammatory environment and reduce the level of LPS in mice with EMS.

Impact of Buzhong Yiqi Prescription on the Gut Microbiota of Patients with Obesity Manifesting Polycystic Ovarian Syndrome.

Gut microbiota disorders are closely related to polycystic ovarian syndrome (PCOS). Buzhong Yiqi prescription (BZYQ) has a significant clinical effect on the treatment of patients with obesity exhibiting PCOS and phlegm-dampness syndrome caused by spleen deficiency (SPSD). Hence, this study aimed to explore gut microbiota and fecal metabolite alterations in such patients treated with BZYQ. Fifty eligible patients with obesity manifesting PCOS and SPSD participated and agreed to undergo 3 months of BZYQ treatment. Results showed that BZYQ significantly alleviated the serum dehydroepiandrosterone sulfate (p < 0.001) and testosterone levels (p < 0.001) and markedly changed the gut microbiota structure in these patients. Furthermore, 106 differential fecal metabolites and 14 KEGG enrichment pathways were quantified. The phylum Spirochaetae and the genera [Eubacterium]_rectale_group, Escherichia-Shigella, and Fusicatenibacter were significantly more abundant, but Megamonas was significantly less abundant after treatment than before treatment. Disorders in the gut microbiota and fecal metabolites of these patients were closely related to hyperandrogenemia and insulin resistance. In conclusion, BZYQ could ameliorate the serum androgen level and had an impact on the gut microbiota and metabolites in patients with obesity manifesting PCOS and SPSD.

Xiaotan Jieyu Prescription Alleviates Breast Precancerous Lesions through PI3K/Akt Signaling Pathway.

METHODS: The successfully established breast precancerous lesion rat model and normal healthy rats were randomly assigned into the blank (BLA), model (MOD), XTJY-low (LD), XTJY-medium (MD), XTJY-high (HD), and tamoxifen (TAM) groups. Different concentrations of XTJY and saline were supplied by intragastric administration for 4 consecutive weeks to assess the protective effect of XTJY on the progress of the breast precancerous lesion in rats involving the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. RESULTS: In this study, it determined that 10 mg/each rat DMBA-combined estrogen and progesterone induction for 10 weeks was the optimal condition for the establishment of the breast precancerous lesion rat model. In vivo administration of XTJY or TAM was found to inhibit the development of the breast precancerous lesion, and the occurrence rate of breast invasive carcinomas was decreased by about 50%. Furthermore, XTJY or TAM markedly reduced protein expressions of PI3K and p-Akt and increased protein expressions of PTEN. CONCLUSION: These data indicated that XTJY can significantly alleviate the development of breast precancerous lesions by inhibiting the activation of the PI3K/Akt signaling pathway. XTJY may be a promising drug for the treatment of precancerous lesions in breast cancer.

Transferrin Insufficiency and Iron Overload in Follicular Fluid Contribute to Oocyte Dysmaturity in Infertile Women With Advanced Endometriosis.

Purpose: To screen out specific protein with different concentration in follicular fluid from advanced endometriosis and determine its direct effect on mouse oocytes matured in vitro. Methods: FF samples were obtained from 25 patients (EMS group, n = 15; control group, n = 10) to screen the differential proteins by using iTRAQ Labeling and 2D LC-MS. Transferrin (TRF) in was found significantly decreased in EMS group, which was verified using ELISA in enlarged FF samples (EMS group, n = 31; control group, n = 27). The contents of ferric ion in FFs were detected by ELISA and TRF saturations were calculated in two groups. Germinal vesicle (GV) oocytes of mouse were maturated in vitro interfered with the FFs in five groups, whose concentrations of TRF were modulated, and maturation in vitro rates were compared among groups. Results: The reduced concentration of TRF with three analogs and increased concentration of ferric ion were found in the FF of the EMS group (p < 0.05). The numerical values of TSAT was 54.8% in EMS group, indicating iron overload in the FF. The EMS-FF showed significantly decreased maturation in vitro rate (p < 0.05) of mouse oocytes, which was improved with the supplementation of TRF, compared with the control-FF. The effect was blocked by the TRF antibody (p < 0.05). Conclusions: Being aware of the relatively small sample size, our results possibly suggest that TRF insufficiency and iron overload in FF from advanced EMS contribute to oocytes dysmaturity, which may be a cause of EMS-related infertility.

Efficacy of "Pinggan Formula" in Controlling Acute Type B Aortic Dissection Perioperative Blood Pressure: A Randomized Controlled Clinical Trial.

OBJECTIVE: To explore a new treatment that can proceed from the whole, control blood pressure smoothly and coordinate the treatment of multiple factors causing blood pressure fluctuations. METHOD: We conducted a single-center, double-blinded, and randomized controlled clinical trial. 48 patients with acute Type B aortic dissection were randomly assigned into two groups: the experimental group, who received pinggan formula treatment, and the control group, who received placebo treatment. The drug was taken orally after meals three times a day. Only when the patients' blood pressure fluctuated, conventional antihypertensive drugs were given to maintain the blood pressure within the target range and the dosage was recorded to convert the DDD value. Meanwhile, the international standardized score was used to evaluate the defecation, sleep, pain, anxiety, and depression of patients in the two groups during the hospitalization. RESULT: Univariate analysis was conducted on variables that might affect the assessment results, and it was found that grouping factors had a significant impact on the outcome variables, that is, after the intervention, the mean value of DDDs used in the perioperative period in the control group was 2.19 (0.38, 4.00). (P=0.0219), defecation score (2.13 (1.59, 2.67); P < 0.0001), sleep score (0.95 (0.40, 1.50); P=0.0014), pain score (1.77 (0.61, 2.93); P=0.0045), depression score (4.04 (2.95, 5.12); and P < 0.0001) were significantly higher than that of the experimental group, and the difference was statistically significant. CONCLUSION: Pinggan formula has a clear therapeutic regulation effect on the overall hemodynamics of acute Stanford type B aortic dissection during the perioperative period and can be recommended as an auxiliary drug for conventional antihypertensive drugs at the current stage.

Baicalin inhibits recruitment of GATA1 to the HSD3B2 promoter and reverses hyperandrogenism of PCOS.

High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) can be effectively reversed if the herb Scutellaria baicalensis is included in traditional Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum concentration and intervention time for baicalin treatment of NCI-H295R cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and enzyme-linked immunosorbent assays. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique, and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, Baicalin decreased the testosterone concentrations in a dose-and time-dependent manner in NCI-H295R cells. GEP revealed that 3ß-hydroxysteroid dehydrogenase type II (HSD3B2) was the key enzyme of androgen biosynthesis, and baicalin inhibited the expression of HSD3B2 by regulating the binding of transcription factor GATA-binding factor 1 (GATA1) to the HSD3B2 promoter. Hyperandrogenic PCOS model rats treated with baicalin significantly reversed the high androgen levels of serum and the abnormal ovarian status, restored the estrous cyclicity, and decreased the expression of HSD3B2 in ovarian. In summary , our data revealed that GATA1 is an important transcription factor activating the HSD3B2 promoter in steroidogenesis, and baicalin potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue.

Consensus on the integrated traditional Chinese and Western medicine criteria of diagnostic classification in polycystic ovary syndrome (draft).

Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder of women, with complex pathogenesis and heterogeneous manifestations. Professor Jin Yu recently wrote an article entitled "Proposal of Diagnosis and Diagnostic Classification of PCOS in Integrated Traditional Chinese and Western Medicine."From this, the Obstetrics and Gynecology branches of the Chinese Association of Integrative Medicine and the China Association of Chinese Medicine collaborated with the Gynecology branch of the Chinese Association for Research and Advancement of Chinese Medicine to draft a report on the consensus of criteria for the diagnosis and classification of PCOS in integrated traditional Chinese and Western medicine. The diagnosis for PCOS includes all three features: (1) oligo-ovulation or anovulation; (2) clinical and/or laboratory evidence of hyperandrogenism;(3) PCOS is classified into four types: types Ia,Ib, IIa, and IIb. Syndrome differentiation types for PCOS in traditional Chinese medicine are as follows: Kidney deficiency with phlegm blockage syndrome, Kidney Yin deficiency with phlegm blockage and blood stasis syndrome, and Kidney deficiency with Liver Qi stagnation syndrome.

Quercetin Decreases Insulin Resistance in a Polycystic Ovary Syndrome Rat Model by Improving Inflammatory Microenvironment.

Insulin resistance (IR) is a clinical feature of polycystic ovary syndrome (PCOS). Quercetin, derived from Chinese medicinal herbs such as hawthorn, has been proven practical in the management of IR in diabetes. However, whether quercetin could decrease IR in PCOS is unknown. This study aims to observe the therapeutic effect of quercetin on IR in a PCOS rat model and explore the underlying mechanism. An IR PCOS rat model was established by subcutaneous injection with dehydroepiandrosterone. The body weight, estrous cycle, and ovary morphology of the quercetin-treated rats were observed. Serum inflammatory cytokines were analyzed using enzyme-linked immunosorbent assay. In ovarian tissues, the expression of key genes involved in the inflammatory signaling pathway was detected through Western blot, real-time polymerase chain reaction, or immunohistochemistry. The nuclear translocation of nuclear factor κB (NF-κB) was also observed by immunofluorescence. The estrous cycle recovery rate of the insulin-resistant PCOS model after quercetin treatment was 58.33%. Quercetin significantly reduced the levels of blood insulin, interleukin 1ß, IL-6, and tumor necrosis factor α. Quercetin also significantly decreased the granulosa cell nuclear translocation of NF-κB in the insulin-resistant PCOS rat model. The treatment inhibited the expression of inflammation-related genes, including the nicotinamide adenine dinucleotide phosphate oxidase subunit p22phox, oxidized low-density lipoprotein, and Toll-like receptor 4, in ovarian tissue. Quercetin improved IR and demonstrated a favorable therapeutic effect on the PCOS rats. The underlying mechanism of quercetin potentially involves the inhibition of the Toll-like receptor/NF-κB signaling pathway and the improvement in the inflammatory microenvironment of the ovarian tissue of the PCOS rat model.

Meta-Analysis of Chinese Traditional Medicine Bushen Huoxue Prescription for Endometriosis Treatment.

OBJECTIVES: To evaluate the efficacy and safety of Bushen Huoxue prescription (BSHXP) for endometriosis. METHODS: A meta-analysis was performed, and studies were searched from the seven databases from the date of database establishment to April 30, 2017. Randomized controlled trials (RCTs) that explored the efficacy and safety of BSHXP for patients with endometriosis were included. Two assessors independently reviewed each trial. The Cochrane Risk of Bias assessment tool was used for quality assessment. RESULTS: In the 13 included studies, the total effectiveness rates of BSHXP were higher than those of Western medicine (RR, 1.55; 95% CI, 1.03-2.32; P = 0.04), but the dysmenorrhea alleviation rates of the two treatments did not significantly differ (RR, 1.28; 95% CI, 0.70-2.34; P = 0.42). The pregnancy rates of BSHXP were also higher than those of hormone therapy (RR, 1.99; 95% CI, 1.17-3.39; P = 0.01). However, whether BSHXP is more effective than Western medicine in diminishing endometriotic cyst remains unknown. CONCLUSIONS: Our study provides evidence that BSHXP is effective and safe for endometriosis, but this evidence is inconclusive because of the low methodological quality of the included RCTs. Our findings suggest that BSHXP is an alternative drug for endometriosis, but it should be further examined in future clinical research.

The effects and possible mechanisms of puerarin to treat endometriosis model rats.

Objective. To explore the effects of puerarin to treat endometriosis (EMT) model rats and the possible regulatory mechanisms. Methods. EMT model rats were surgically induced by autotransplantion of endometrial tissues. The appropriate dosage of puerarin to treat EMT model rats was determined by observing the pathologic morphology of ectopic endometrial tissues and by detecting the levels of estradiol (E2) and prostaglandin E2 (PGE2) of both serum and ectopic endometrial tissues. The related genes and proteins of ectopic endometrial tissues were analyzed by Real-time PCR and immunohistochemistry (IHC) to explore the possible mechanisms. Results. Puerarin could reduce the levels of E2 and PGE2 and prevent the growth of ectopic endometrium tissues by inhibiting the expression of aromatase cytochrome P450 (p450arom) and cyclooxygenase-2 (cox-2); puerarin could adjust the anabolism of E2 by upregulating the expression of 17ß-hydroxysteroid-2 (17ß-hsd-2) and downregulating the expression of 17ß-hydroxysteroid-1 (17ß-hsd-1) of the ectopic endometrium tissues; puerarin could increase the expression of ERß and improve the inflammatory microenvironment of EMT model rats. Conclusions. Our data suggest that puerarin has a therapeutic effect on EMT model rats and could be a potential therapeutic agent for the treatment of EMT in clinic.

Wrist-ankle acupuncture and ginger moxibustion for preventing gastrointestinal reactions to chemotherapy: A randomized controlled trial.

OBJECTIVE: To evaluate the effects of wrist-ankle acupuncture combined with ginger moxibustion against gastrointestinal tract reactions (nausea, vomiting, and constipation) to chemotherapy in cancer patients. METHODS: A total of 60 patients with gynecological tumors treated by chemotherapy were randomly divided into two groups. The treatment group (30 cases) underwent wrist-ankle acupuncture and ginger moxibustion, whereas tropisetron hydrochloride and dexamethasone were intravenously administered to the control group (30 cases) during chemotherapy. RESULTS: The frequency of nausea in the treatment group was significantly less than that of the control group from the 2nd to the 5th day of chemotherapy (P<0.01). The anti-emetic effect in the treatment group was significantly better than that in the control group on the 3rd day of therapy (P<0.05). The incidence rate of constipation was significantly lower in the treatment group than that in the control group (P<0.01). Furthermore, the cost of therapy for the treatment group was significantly lower than that of the control group (P<0.01). Only 1 patient manifested a post-acupuncture side effect in the form of subcutaneous blood stasis. CONCLUSION: Wrist-ankle acupuncture combined with ginger moxibustion could prevent gastrointestinal tract reactions to chemotherapy in cancer patients. In addition, the proposed method had fewer side effects, lower cost, and less risk.

Role of JNK Activation and Mitochondrial Bax Translocation in Allicin-Induced Apoptosis in Human Ovarian Cancer SKOV3 Cells.

Background. Allicin, the major component of freshly crushed garlic, is one of the most biologically active compounds of garlic; it has been reported to induce apoptosis in cancer cells; however, the mechanism by which allicin exerts its apoptotic effects is not fully understood. The aim of the present study was to further elucidate the apoptotic pathways induced by allicin in the human ovarian cancer cell line SKOV3. Methods. Cell proliferation and apoptosis were measured by cell-counting assay and flow cytometry analysis. Activation of the signaling pathway was screened by human phospho-kinase array analysis, and the activated pathway and its related proteins were further confirmed by western blot analysis. Results. Allicin induced SKOV3 cell apoptosis and JNK phosphorylation in a time- and dose-dependent manner, but these were significantly blocked by SP600125 (an inhibitor of JNK). The findings suggest that JNK phosphorylation is related to the action of allicin on SKOV3 cells. Furthermore, JNK activation induced Bcl-2 family activation, triggered mitochondria-mediated signaling pathways, and led to the translocation of a considerable amount of Bax and cytochrome c release. Conclusions. JNK activation and mitochondrial Bax translocation are involved in allicin-induced apoptosis in SKOV3 cells. Our data input new insights to the literature of allicin-induced apoptosis.

Cryptotanshinone Reverses Reproductive and Metabolic Disturbances in PCOS Model Rats via Regulating the Expression of CYP17 and AR.

Objective. To explore the effect of Cryptotanshinone on reversing the reproductive and metabolic disturbances in polycystic ovary syndrome (PCOS) model rats and the possible regulatory mechanisms. Methods. PCOS model rats were induced by subcutaneous injection of dehydroepiandrosterone (DHEA) and verified by histological screening of vaginal exfoliated cells. After Cryptotanshinone intervention, the rats' body weight and ovary morphological were observed; the serum biochemical assessments were analyzed by radioimmunoassay (RIA) and key genes and proteins related with anabolism of androgen and insulin were detected by Real-Time PCR and Immunohistochemical (IHC). Results. The estrous cyclicity of PCOS model rats was significantly recovered by Cryptotanshinone. The body weight, ovarian coefficient, and ovarian morphology had been improved and the serum biochemical indicators including testosterone (T), androstenedione (A2), luteinizing hormone (LH), LH/follicle stimulating hormone (FSH), sexual binding globulin (SHBG), low density cholesterol (LDL-C), fasting insulin (FINS) were reversed after Cryptotanshinone intervention. Specifically, the levels of Cytochrome P450, 17-a hydroxylase/17,20 lyase (CYP17), and androgen receptor (AR) were downregulated significantly. Conclusions. Our data suggest that Cryptotanshinone could rebalance reproductive and metabolic disturbances in PCOS model rats and could be a potential therapeutic agent for the treatment of PCOS.

Puerarin suppresses proliferation of endometriotic stromal cells in part via differential recruitment of nuclear receptor coregulators to estrogen receptor-α.

BACKGROUND AND OBJECTIVES: Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17ß-estradiol and producing an anti-estrogenic effect. In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae. This study was to investigate whether puerarin could suppress the proliferation of endometriotic stromal cells (ESCs) and to further elucidate the potential mechanism. METHODS AND RESULTS: The ESCs were successfully established. The effects of puerarin on the proliferation of ESCs, cell cycle and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry. The mRNA and protein levels of cyclin D1 and cdc25A were detected by real-time PCR and Western blot analysis. Coimmunoprecipitation was applied to examine the recruitment of nuclear receptor coregulators to the estrogen receptor-α. We found that puerarin can suppress estrogen-stimulated proliferation partly through down-regulating the transcription of cyclin D1 and cdc25A by promoting the recruitment of corepressors to estrogen receptor-α as well as limiting that of coactivators in ESCs. CONCLUSIONS: Our data suggest that puerarin could suppress the proliferation of ESCs and could be a potential therapeutic agent for the treatment of endometriosis.

Puerarin suppresses proliferation of endometriotic stromal cells partly via the MAPK signaling pathway induced by 17ß-estradiol-BSA.

BACKGROUND: Puerarin is a major isoflavonoid compound extracted from Radix puerariae. It has a weak estrogenic action by binding to estrogen receptors (ERs). In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae. The genomic and non-genomic effects of puerarin were studied in our Lab. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the non-genomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E(2)-BSA). METHODOLOGY: ESCs were successfully established. Binding of puerarin to ERs was assessed by a radioactive competitive binding assay in ESCs. Activation of the signaling pathway was screened by human phospho-kinase array, and was further confirmed by western blot. Cell proliferation was analyzed according to the protocol of CCK-8. The mRNA and protein levels of cyclin D1, Cox-2 and Cyp19 were determined by real-time PCR and western blotting. Inhibitor of MEK1/2 or ER antagonist was used to confirm the involved signal pathway. PRINCIPAL FINDINGS: Our data demonstrated that the total binding ability of puerarin to ERs on viable cells is around 1/3 that of 17ß-estradiol (E(2)). E(2)-BSA was able to trigger a rapid, non-genomic, membrane-mediated activation of ERK1/2 in ESCs and this phenomenon was associated with an increased proliferation of ESCs. Treating ESCs with puerarin abrogated the phosphorylation of ERK and significantly decreased cell proliferation, as well as related gene expression levels enhanced by E(2)-BSA. CONCLUSIONS/SIGNIFICANCE: Puerarin suppresses proliferation of ESCs induced by E(2)-BSA partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of Cyclin D1, Cox-2 and Cyp19 are involved in the process. Our data further show that puerarin may be a new candidate to treat endometriosis.

[Effects of Chinese herbal medicine Neiyi Recipe-medicated serum on angiopoiesis of endometriosis in the chick chorioallantoic membrane model].

OBJECTIVE: To compare angiopoiesis ability of eutopic and ectopic endometrial tissue isolated from women with endometriosis and endometrium isolated from women without endometriosis (control), and to explore the inhibitory effects of medicated serum of Neiyi Recipe, a compound traditional Chinese herbal medicine. METHODS: Chick chorioallantoic membrane (CAM) model of endometriosis was established by transplanting endometrium onto CAM. The CAMs were then hatched with blank serum or medicated serum of danazol or Neiyi Recipe, which were prepared in rats by orally administering. The sizes of the transplanted tissue and new vessels around the transplanted tissue were measured. Expression of vascular endothelial growth factor (VEGF) was detected by immunohistochemical method. RESULTS: There was no difference in the sizes of transplanted tissue among CAM models of ectopic and eutopic endometrial tissue isolated from women with endometriosis or control (P>0.05), and more new vessels were found around the ectopic and eutopic endometrial tissue than the endometrial tissue of control (P<0.05). Compared with the controls, the size of the transplanted tissue and positive area of new vessels were significantly inhibited by Neiyi Recipe-medicated serum (P<0.01, P<0.05), and similar changes happened in the danazol groups, except for the size of transplanted tissue from ectopic endometrial tissue (P>0.05). Expression of VEGF was significantly higher in eutopic and ectopic endometrial tissue than in the control (P<0.01); the level of VEGF obviously reduced in the Neiyi Recipe and danazol groups (P<0.01), but no significant difference was detected between them. CONCLUSION: Endometrium from women with endometriosis stimulates the formation of new vessels by increase the expression of VEGF. Neiyi Recipe-medicated serum significantly decreases the expression of VEGF in eutopic and ectopic endometrial tissues and thus restrains the formation of new vessels, reduces the blood supply and inhibits growth of ectopic endometrial tissue, which are similar to danazol, but has greater efficacy in suppressing the expression of VEGF.

Endometriotic implants regress in rat models treated with puerarin by decreasing estradiol level.

Phytoestrogens, which have a weak estrogenic effect, bind to estrogen receptors (ERs), thereby competing with estradiol, have an antiestrogenic effect on women of reproductive age with high estrogenic level. Herein, we examined the ability of the phytoestrogen Puerarin to treat endometriosis in rat models of endometriosis. In total, 75 adult, mature female Sprague-Dawley rats in which endometriotic implants were successfully induced by transplanting autologous endometrial tissue to ectopic sites were used in this study. Oral gavage of Puerarin (at doses of 600, 200, or 60 mg/kg per day) or Danazol (80 mg/kg per day) started 4 weeks after implantation. Control model rats received vehicle alone. After administration for 4 weeks, the weight of the ectopic implants, estradiol concentration, as well as ER-α and Aromatase P450 (P450arom) expression in different groups of rat tissues were evaluated after treatment. The endometriotic tissue weight and serum estrogen levels were significantly lower in high, medium, low dose of Puerarin and Danazol treatment groups than that in control group (P < .05 or P < .01). Low-dose Puerarin inhibited P450arom expression and significantly reduced estrogen levels in endometriotic tissue (P < .01). Three doses of Puerarin had no adverse effects on liver, kidney, and ovary, whereas high-dose Puerarin administration caused thinner bone trabecula with distortion and breakage and Danazol administration caused mild or moderate hepatic cell damage. These data demonstrate that Puerarin was able to effectively suppress the growth and development of ectopic endometrium in the rat endometriosis model, even at low doses, suggesting it may be an effective treatment for endometriosis.