Mechanisms of electroacupuncture relieves uterine smooth muscle spasm in dysmenorrhea rats based on Rho/ROCK signaling pathway. / 基于Rho/ROCKä¿¡å·é€šè·¯æŽ¢è®¨ç”µé’ˆä»»è„‰ã€ç£è„‰ç»ç©´ç¼“è§£ç±»ç—›ç»æ¨¡åž‹å¤§é¼ å­å®«å¹³æ»‘è‚Œç—‰æŒ›çš„æœºåˆ¶.

OBJECTIVES: To investigate the effect of electroacupuncture (EA) on Rho/Rho-associated coiled-coil-forming kinases (ROCK) signaling pathway of uterus tissue in rats with dysmenorrhea, so as to explore the underlying mechanism of EA treating primary dysmenorrhea (PD) and uterine smooth muscle spasm, and to observe whether there is a difference in the effect of meridian acupoints in Conception Vessel (CV) and Governer Vessel (GV). METHODS: Sixty female SD rats were randomly divided into saline, model, CV, GV, and non-acupoint groups, with 12 rats in each group. The dysmenorrhea model was established by subcutaneous injection of estradiol diphenhydrate combined with intraperitoneal injection of oxytocin (OT). EA (2 Hz) was applied to "Qihai" (CV6) and "Zhongji" (CV3) for CV group, "Mingmen" (GV4) and "Yaoshu" (GV2) for GV group, "non-acupoint 1" and "non-acupoint 3" on the left side for non-acupoint group, and manual acupuncture was applied to "Guanyuan" (CV4) for CV group, "Yaoyangguan" (GV3) for GV group, "non-acupoint 2" on the left side for non-acupoint group. The treatment was conducted for 20 min each time, once daily for 10 days. The writhing score was evaluated. The smooth myoelectric signals of rats' uterus in vivo were recorded by multi-channel physiological recorder. The uterine histopathological changes were observed by HE staining. The contents of prostaglandin F2α (PGF2α), OT and calcium ion (Ca2+) in uterine tissue of rats were detected by ELISA. The protein and mRNA expression levels of smooth muscle 22-α (SM22-α), RhoA and ROCKⅡ in uterine tissue were detected by Western blot and fluorescence quantitative PCR, respectively. RESULTS: Compared with the saline group, the writhing score of rats in the model group was increased (P<0.01), the amplitude voltage of uterine smooth muscle in vivo was elevated (P<0.01), the contents of PGF2α, OT and Ca2+, the protein and mRNA expression of SM22-α, RhoA and ROCK Ⅱ in uterine tissue were all increased (P<0.01). Compared with the model and the non-acupoint groups, the writhing scores of the CV and the GV groups were decreased (P<0.01, P<0.05), the amplitude voltage of uterine smooth muscle was decreased (P<0.01), the contents of PGF2α, OT and Ca2+ in uterine tissue were decreased (P<0.01, P<0.05), and the protein expression and mRNA expression of SM22-α, RhoA and ROCKⅡ in uterine tissue were decreased (P<0.01, P<0.05). HE staining showed extensive exfoliation of uterine intima with severe edema and increased glandular secretion in the model group, which was alleviated in the CV and GV groups. CONCLUSIONS: EA at acupoints of CV and GV can significantly reduce the writhing score, uterine smooth muscle amplitude voltage, pathological injury degree of uterus, and relieve spasm of uterine smooth muscle in dysmenorrhea rats, which may be related to its effect in regulating PGF2α and OT contents, inhibiting the Rho/ROCK signaling pathway, and reducing the SM22-α, RhoA, ROCKⅡ protein and mRNA expression, and Ca2+ content in uterine tissue.

Nigella sativa oil restores hormonal levels, and endocrine signals among thyroid, ovarian, and uterine tissues of female Wistar rats following sodium fluoride toxicity.

The current study aimed to explore the possible prophylactic and therapeutic effect of Nigella sativa L. oil (NSO) against disruption of endocrine signals and injuries in the thyroid gland, ovary, and uterine tissues induced by sodium fluoride (NaF). Twenty-eight mature female Wistar rats were randomly allocated into four experimental groups (n = 7/group) as follows: control group; NaF group, orally received NaF (20 mg/kg b.wt.) daily; NSO/NaF, orally received NSO (300 mg/kg b.wt.) two weeks before being given NaF and continued throughout the experiment; and NSO+NaF group orally received NSO concurrently with NaF. Our results indicated that NSO restored hormonal balance and suppressed oxidative damage and inflammation. Moreover, the levels of triiodothyronine, thyroxine, thyroid peroxidase, estrogen (E2), progesterone, follicle-stimulating hormone, and luteinizing hormone were elevated, while prostaglandins F2-α and cortisol levels were decreased in NSO treated groups compared to NaF-intoxicated rats. As well, NSO significantly boosted levels of antioxidant molecules, and lowered lipid peroxidation of examined tissues, unlike NaF-treated group. NSO also up-regulated antioxidant enzymes, anti-apoptotic protein, zona pellucida sperm-binding protein, bone morphogenetic protein, and thyroid stimulating hormone, conversely down-regulated inflammatory cytokines, apoptotic proteins, estrogen receptor-α, estrogen receptor-ß, and thyroid stimulating hormone receptors compared to NaF-intoxicated group. Additionally, NSO ameliorated tissue damage of the thyroid gland, ovary, and uterus induced by NaF. -Overall, the prophylactic group (NSO/NaF) performed better antioxidant and anti-inflammatory activities than the treated group almost in all examined tissues, which is reflected by the improvement in the structure of the thyroid, ovarian, and uterine tissues.

Tissue-Specific RNA Localization in the Uterus During Implantation, Decidualization and Placentation: Technical Nuances of Various Labeling Approaches.

In situ hybridization (ISH) is a sensitive method used to localize a specific sequence of DNA or RNA in biological samples, including cells, tissue sections or whole organs. RNA ISH can be used to determine spatial gene expression using a single-stranded probe with a reverse-complementary sequence. Cell-specific gene expression has been studied using mRNA and protein levels. Signals produced by RNA probes are usually more specific than those produced by antibodies in immunostaining. Currently, ISH is the most widely used method to localize mRNA molecules. Traditionally, probes were labeled with radioactive isotopes, but the cumbersome procedures and potential health risk limit their acceptance. Recently, probes labeled with nonradioactive materials including digoxigenin, biotin and various fluorophores have been developed. The tyramide signal amplification system further enhances the sensitivity of detection. These methods have been applied in numerous studies in various tissues including reproductive organs. This article details three methods of RNA in situ hybridization: radioactive in situ hybridization, digoxigenin in situ hybridization, and digoxigenin-tyramide signal amplification fluorescein in situ hybridization. The pros and cons of each protocol are discussed. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Radioactive in situ hybridization (radioactive-ISH) Basic Protocol 2: Digoxigenin in situ hybridization (DIG-ISH) Basic Protocol 3: Digoxigenin-tyramide signal amplification fluorescein in situ hybridization (DIG-TSA-FISH).

Epirubicin toxicity in rat's ovary and uterus: A protective role of 3-Indolepropionic acid supplementation.

The "anthracycline, Epirubicin (EPI)," in managing breast cancer, is highly cytotoxic. Tryptophan-derived 3-indolepropionic acid (3-IPA) decreases oxidative damage, and its prospect of alleviating EPI-induced cytotoxicity was examined in rats' hypothalamus-ovary-uterus axis. Female rats: Control, EPI (2.5 mg/kg), 3-IPA alone (40 mg/kg), EPI+3-IPA (2.5 mg/kg + 20 mg/kg), EPI + 3-IPA2 (2.5 mg/kg + 40 mg/kg) were treated for 28 days. Subsequently, reproductive hormones, oxidative and inflammatory stress biomarkers, and tissue histology were examined. 3-IPA prevented EPI-induced decreases in the follicle-stimulating hormone, estradiol, progesterone and prolactin levels. EPI-mediated reduction in antioxidant enzymes, reduced glutathione and total sulfhydryl groups were partially counteracted by 3-IPA co-treatment. Increased oxidative and inflammatory stress biomarkers caused by treatment with EPI alone were lessened by 3-IPA co-treatment. Also, 3-IPA reduced histological damage in the examined tissues. Conclusively, 3-IPA ameliorated biochemical markers and tissue injury caused by EPI treatment alone via an antioxidative and anti-inflammatory mechanism while stabilising serum hormone dynamics.

Curcumol Attenuates Endometriosis by Inhibiting the JAK2/STAT3 Signaling Pathway.

BACKGROUND Curcumol is a hydrogenated austenitic compound with hemiketal. In this study we evaluated the effects of curcumol on local inflammatory response, cell proliferation, and metastasis in endometriosis, and elucidated the underlying mechanisms. MATERIAL AND METHODS Ectopic endometrial stromal cells were treated with increasing doses of curcumol. The MTT assay was used to assess cell viability. FITC-labeled annexin-V/PI double-staining method and flow cytometry were used to determine cell apoptosis. Cell migration was evaluated using a wound healing assay. ELISA kits were used to detect the levels of TNF-alpha, IL-6, and IL-1ß. Western blot assay was used to examine the phosphorylation degree of JAK2 and STAT3 and the expression of Bax, Bcl2, and caspase-3 proteins. Autologous endometrial transplantation was used to establish a rat model to assess the anti-EMS effect of curcumol in vivo. RESULTS Curcumol can inhibit the proliferation of ectopic endometrial stromal cells, promote cell apoptosis, and weaken cell migration ability. Curcumol can reduce the expression of Bax and caspase-3 protein and increase the expression of Bcl2 protein. Curcumol also can inhibit the secretion of inflammatory cytokines, including tumor necrosis cytokines (TNF)-alpha, interleukin (IL)-6, and IL-1ß, by ectopic endometrial stromal cells. In addition, curcumol can also inhibit the phosphorylation of JAK2 and STAT3. In vivo experiments also proved that curcumol could inhibit the growth of ectopic lesions in EMS model rats. CONCLUSIONS Curcumol can inhibit the JAK2/STAT3 pathway, reduce the inflammatory cytokines secreted by ectopic endometrial stromal cells, inhibit cell proliferation and migration, and reduce the volume of ectopic lesions.

Progesterone and interferon tau regulate expression of polyamine enzymes during the ovine peri-implantation period†.

Progesterone (P4) and interferon tau (IFNT) are important for establishment and maintenance of pregnancy in ruminants. Agmatine and polyamines (putrescine, spermidine, and spermine) have important roles in the survival, growth, and development of mammalian conceptuses. This study tested the hypothesis that P4 and/or IFNT stimulate the expression of genes and proteins involved in the metabolism and transport of polyamines in the ovine endometrium. Rambouillet ewes (n = 24) were surgically fitted with intrauterine catheters on Day 7 of the estrous cycle. They received daily intramuscular injections of 50 mg P4 in corn oil vehicle and/or 75-mg progesterone receptor antagonist (RU486) in corn oil vehicle from Days 8-15, and twice daily intrauterine injections (25 µg/uterine horn/day) of either control serum proteins (CX) or IFNT from Days 11-15, resulting in four treatment groups: (i) P4 + CX; (ii) P4 + IFNT; (iii) RU486 + P4 + CX; or (iv) RU486 + P4 + IFNT. On Day 16, ewes were hysterectomized. The total amounts of arginine, citrulline, ornithine, agmatine, and putrescine in uterine flushings were affected (P < 0.05) by P4 and/or IFNT. P4 increased endometrial expression of SLC22A2 (P < 0.01) and SLC22A3 (P < 0.05) mRNAs. IFNT affected endometrial expression of MAT2B (P < 0.001), SAT1 (P < 0.01), and SMOX (P < 0.05) mRNAs, independent of P4. IFNT increased the abundance of SRM protein in uterine luminal (LE), superficial glandular (sGE), and glandular epithelia (GE), as well as MAT2B protein in uterine LE and sGE. These results indicate that P4 and IFNT act synergistically to regulate the expression of key genes required for cell-specific metabolism and transport of polyamines in the ovine endometrium during the peri-implantation period of pregnancy.

Comparative study of estrogenic activities of phytoestrogens using OECD in vitro and in vivo testing methods.

With growing scientific interest in phytoestrogens, a number of studies have investigated the estrogenic potential of phytoestrogens in a wide variety of assay systems. However, evaluations of individual phytoestrogens with different assay systems make it difficult for predicting their relative estrogenic potency. The objective of this study was to compare estrogenic properties of fifteen known phytoestrogens using an estrogen receptor-α (ER-α) dimerization assay and Organization for Economic Cooperation and Development (OECD) standardized methods including in vitro estrogen receptor (ER) transactivation assay using VM7Luc4E2 cells and in vivo uterotrophic assay using an immature rat model. Human ER-α dimerization assay showed positive responses of eight test compounds and negative responses of seven compounds. These results were consistently found in luciferase reporter assay results for evaluating ER transactivation ability. Seven test compounds exhibiting relatively higher in vitro estrogenic activities were subjected to uterotrophic bioassays. Significant increases in uterine weights were only found after treatments with biochanin A, 8-prenylnaringenin, and coumestrol. Importantly, their uterotrophic effects were lost when animals were co-treated with antagonist of ER, indicating their ER-dependent effects in the uterus. In addition, analysis of estrogen responsive genes revealed that these phytoestrogens regulated uterine gene expressions differently compared to estrogens. Test methods used in this study provided a high consistency between in vitro and in vivo results. Thus, they could be used as effective screening tools for phytoestrogens, particularly focusing on their interactions with ER-α.

Protective role of Mas Cotek (Ficus deltoidea) against the toxic effects of bisphenol A on morphology and sex steroid receptor expression in the rat uterus.

BACKGROUND: Numerous scientific studies have found that young women are at a high risk of reproductive infertility due to their routine exposure to numerous bisphenol A (BPA) products. This risk is highly associated with the production of reactive oxygen species from BPA products. Ficus deltoidea, which has strong antioxidant properties, was selected as a potential protective agent to counter the detrimental effects of BPA in the rat uterus. METHODS: Female Sprague-Dawley rats were allocated into four groups (n = 8) as follows: (i) the Normal Control group (NC), (ii) the BPA-exposed group (PC), (iii) the group concurrently treated with BPA and F. deltoidea (FC) and (iv) the group treated with F. deltoidea alone (F). RESULTS: After 6 weeks of concurrent treatment with F. deltoidea, uterine abnormalities in the BPA-exposed rats showed a significant improvement. Specifically, the size of stromal cells increased; interstitial spaces between stromal cells expanded; the histology of the glandular epithelium and the myometrium appeared normal and mitotic figures were present. The suppressive effects of BPA on the expression levels of sex steroid receptors (ERα and ERß) and the immunity gene C3 were significantly normalised by F. deltoidea treatment. The role of F. deltoidea as an antioxidant agent was proven by the significant reduction in malondialdehyde level in BPA-exposed rats. Moreover, in BPA-exposed rats, concurrent treatment with F. deltoidea could normalise the level of the gonadotropin hormone, which could be associated with an increase in the percentage of rats with a normal oestrous cycle. CONCLUSION: F. deltoidea has the potential to counter the toxic effects of BPA on the female reproductive system. These protective effects might be due to the phytochemical properties of F. deltoidea. Therefore, future study is warranted to identify the bioactive components that contribute to the protective effects of F. deltoidea.

Estrogenic Effects of Extracts and Isolated Compounds from Belowground and Aerial Parts of Spartina anglica.

Menopause, caused by decreases in estrogen production, results in symptoms such as facial flushing, vaginal atrophy, and osteoporosis. Although hormone replacement therapy is utilized to treat menopausal symptoms, it is associated with a risk of breast cancer development. We aimed to evaluate the estrogenic activities of Spartina anglica (SA) and its compounds and identify potential candidates for the treatment of estrogen reduction without the risk of breast cancer. We evaluated the estrogenic and anti-proliferative effects of extracts of SA and its compounds in MCF-7 breast cancer cells. We performed an uterotrophic assay using an immature female rat model. Among extracts of SA, belowground part (SA-bg-E50) had potent estrogenic activity. In the immature female rat model, the administration of SA-bg-E50 increased uterine weight compared with that in the normal group. Among the compounds isolated from SA, 1,3-di-O-trans-feruloyl-(-)-quinic acid (1) had significant estrogenic activity and induced phosphorylation at serine residues of estrogen receptor (ER)α. All extracts and compounds from SA did not increase MCF-7 cell proliferation. Compound 1 is expected to act as an ERα ligand and have estrogenic effects, without side effects, such as breast cancer development.

Water extract of Er-xian decoction selectively exerts estrogenic activities and interacts with SERMs in estrogen-sensitive tissues.

ETHNOPHARMACOLOGICAL RELEVANCE: The increasing use of "kidney"-nourishing Traditional Chinese Medicine (TCM) like Er-xian decoction (EXD) for management of menopausal symptoms and osteoporosis has aroused concerns about their safety, and whether they interact with prescription drugs as both of them act via estrogen receptors (ERs) and regulate serum estradiol. AIM OF THE STUDY: The present study aimed to evaluate whether EXD selectively exerted estrogenic activities and interacted with Selective Estrogen Receptor Modulators (SERMs). MATERIALS AND METHODS: In vivo, mature ovariectomized (OVX) rats were administrated with EXD or combined treatment of EXD and SERMs for 12 weeks. The tissue-selective effect of EXD and its interaction of SERMs were studied in four estrogen sensitive tissues, bone, brain, breast and uterus. In vitro, the interaction of extracts of EXD-treated serum and SERMs in four ER-positive cell lines. RESULTS: In OVX rats, EXD selectively alleviated estrogen deficiency-induced changes in the bone and brain without inducing any estrogenic effects in the breast or uterus. Two-way ANOVA indicated the presence of interactions between EXD and SERMs in OVX rats but EXD did not significantly alter the tissue responses to SERMs in the bone, breast or brain. Indeed, the combined use of EXD and SERMs appeared to suppress the estrogenic effect of raloxifene and tamoxifen in the uterus. Extract of EXD-treated serum directly stimulated cell proliferation or differentiation in human osteosarcoma MG-63, neuroblastoma SHSY5Y, breast cancer MCF-7, and endometrial Ishikawa cells. Two-way ANOVA revealed that EXD-treated serum interacted with SERMs at various concentrations and altered the effects of tamoxifen in MG-63 and MCF-7 cells. CONCLUSIONS: EXD exerted estrogenic effects in a tissue-selective manner and interacted with SERMs. Combined treatment of EXD and SERMs did not hamper the beneficial effects of SERMs on the bone or brain but appeared to moderate the estrogenic effect of SERMs in the uterus.

Regulative effect of Taohong Siwu decoction on extracellular matrix of endometrium in drug-induced abortion.

OBJECTIVE: To explore the effects of Taohong Siwu decoction (, THSWD) on the extracellular matrix of endometrium in rats following drug-induced abortion. METHODS: Thirty-six pregnant female rats were administered mifepristone and misoprostol to induce abortion, and amounts of uterine bleeding were recorded. Pathological damage and collagen accumulation were detected by hematoxylin-eosin staining and Masson's trichrome staining in uterus, respectively. Myeloperoxidase was evaluated by immunohistochemistry.The expression levels of fibronectin, laminin, matrix metalloproteinase 9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) were quantified using western blotting. RESULTS: THSWD could promote endometrial protection in rats following drug-induced abortion. The contents of cellulose and myeloperoxidase were significantly decreased in uterine tissue of THSWD-treated groups. Moreover, THSWD significantly decreased the expression levels of fibronectin, laminin, and TIMP-1. THSWD also significantly increased MMP-9 expression and the MMP-9/TIMP1 ratio. CONCLUSION: THSWD plays a critical role in endometrial protection by reducing extracellular matrix deposition and uterine fibrosis. These effects may have been achieved by increasing MMP-9, reducing TIMP-1, and/or altering the ratio of MMP-9/TIMP-1.

Methionine supplementing effects on intestine, liver and uterus morphology, and on positivity and expression of Calbindin-D28k and TRPV6 epithelial calcium carriers in laying quail in thermoneutral conditions and under thermal stress.

This study aimed to provide the performance, localization and expression of the epithelial calcium transporter channels Calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quail under heat stress (HS), and with methionine supplementation (MS). This study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels in the kidneys, intestine and uterus of 504 laying quails under different MS (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under HS (32°C) had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb, and increased positivity in proximal CTs. Deleterious effects of HS were minimized with MS for: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney (P≤0.05). Epithelial calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the MS, increasing egg production in HS and quality in termoneutrality (P≤0.05). MS further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity of Calb, and increase Calb and TRPV6 expression in the kidney (P≤0.001) under thermoneutrality. It was concluded that the use of MS (120%) is justifiable in order to partially reverse the deleterious effects of HS on the production, in the epithelial calcium carriers, and in the digestory and reproductive morphology of laying quail.

Augmentation of the Female Reproductive System Using Honey: A Mini Systematic Review.

Phytochemical contents of honey are presumed to be beneficial to the female reproductive system (FRS). However, the biological effects of honey supplementation (HS) in vivo on the FRS remain unclear. This review aims to investigate the current literature on the effects of HS on the FRS, particularly on the sex hormone profile and reproductive organs (uterus and vagina). A systematic literature search using Scopus, MEDLINE via Ovid and Cochrane Library databases was conducted. Records were screened and identified for preclinical and clinical studies addressing the effects of HS on the FRS. Data on populations, interventions, outcomes and methodological quality were extracted. Studies were synthesised using tables and written summaries. Of the 198 identified records, six fulfilled the inclusion criteria. All six records were used for data extraction: two experimental studies using rats as the model organism and four human clinical studies of honey on female reproductive health. HS elevated the progesterone levels, restrained body weight increase, prevented uterine and vaginal atrophies in ovariectomised rats, attenuated symptoms of candidiasis and improved oxidative status in patients. Current evidence shows that short-term HS following surgical or physiological menopause exerts an oestrogenic, antioxidant and anti-inflammatory effect on the FRS. However, insufficient long-term studies preclude any definitive conclusions.

Uterotonic Effects of Aqueous and Methanolic Extracts of Lannea acida in Wistar Rats: An In Vitro Study.

Lannea acida (Anacardiaceae), commonly called Kikié in the Noun division (West-Cameroon), is a tree whose bark is used locally to facilitate delivery. This study was aimed at evaluating the in vitro uterotonic effects of aqueous and methanol extracts of L. acida in Wistar rats. Uterine strips isolated from rats pretreated with 5 µg estradiol (48 h) were mounted in a single-organ bath containing aerated and thermostated De Jalon solution (37 °C). After equilibration, non-cumulative effects of L. acida extracts were recorded after application. The effect of the methanol extract (the most active extract) was monitored in the presence of atosiban (a competitive antagonist of oxytocin receptors), atropine (a specific type 3 muscarinic receptor antagonist), nifedipine (an L-type calcium channel antagonist), and 2-Aminoethoxydiphenyl borate (2-ADB, a specific antagonist of inositol 1,4,5-triphosphate receptors type 1), and in calcium-free medium containing EGTA to elucidate its mechanism of action. L. acida induced uterine contraction in a concentration-dependent manner with the methanol extract (1.506 ± 0.032 gf) being the most effective. Administration of atosiban (2 µmol/L) and atropine (1 µmol/L) reduced the contractile effect of L. acida. Complete inhibition was observed with nifedipine, 2-APB, and calcium-free medium containing EGTA. These results suggest that L. acida possesses uterotonic effects mediated through oxytocin receptors with mobilization of extracellular calcium.

The ameliorating effects of tokishakuyakusan in a rat model of implantation failure involves endometrial gland leukemia inhibitory factor and decidualization.

ETHNOPHARMACOLOGICAL RELEVANCE: Tokishakuyakusan (TSS) is a Kampo medicine that is prescribed for the treatment of infertility in Japan. However, its precise mechanism of action remains unclear. AIM OF THE STUDY: Leukemia inhibitory factor (LIF) in the endometrium plays an indispensable role in embryo implantation and is linked to infertility or implantation failure. Previously, we demonstrated that TSS ameliorated implantation failure induced by mifepristone (RU-486), an antagonist of progesterone, in rats. Herein, we aimed to clarify whether the ameliorating effect of TSS on implantation failure in the rat model involves endometrial LIF. Additionally, we determined whether decidualization, the dysfunction of which is linked to infertility or implantation failure similar to LIF, progesterone, and other implantation-related factors, are involved in the effect of TSS. MATERIALS AND METHODS: The implantation failure rat model was developed via the subcutaneous administration of RU-486 (7 mg/kg) on day 3 post-coitus. Sesame oil was administered as the vehicle control. Rats were fed a diet containing 1% or 3% TSS or a control diet from day 13 pre-coitus. Subsequently, the implantation sites were assessed, and plasma progesterone levels were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on day 8 post-coitus. The LIF mRNA of the endometrial gland, which was segmented via laser-microdissection from the endometrial tissue, was measured, and endometrial LIF immunostaining was carried out on day 5. The gene expression of different factors related to implantation, including decidualization and progesterone-responsiveness on days 5 and 6, were measured. The human endometrial Ishikawa cell line derived from human adenocarcinoma was treated with TSS (30-300 µg/mL) for 24 h, and the LIF concentrations in the cell culture supernatants were measured. RESULTS: RU-486 decreased the number of implantation sites in the uterus of rats; however, the decrease was significantly alleviated by TSS (3%-diet), which tended to increase plasma progesterone. In rats with RU-486-induced implantation failure, endometrial gland LIF mRNA and endometrial LIF protein were markedly decreased while the gene expression of both decidualization-related factors such as interleukin-11, insulin-like growth factor binding protein-1, and cyclooxygenase-2, and progesterone responsive-related factors such as FK506 binding protein 5, were significantly decreased. These changes in the uterus of rats with implantation failure were significantly alleviated by TSS (3%-diet). Additionally, TSS significantly enhanced LIF protein production and LIF mRNA in Ishikawa cells. CONCLUSIONS: The mechanism whereby TSS ameliorates RU-486-induced implantation failure in rats may involve the alleviation of decreased LIF production derived from the endometrial gland, and a dysfunction of decidualization, including lower progesterone responsiveness in the model. These findings may partly contribute to the interpretation of the beneficial effects of TSS on infertility.

Effects of soy isoflavones extract on the lipid profile and uterus in ovariectomized rats.

AIM: Although soy isoflavones (ISO) have been shown to relief postmenopausal symptoms, it remains inconclusive whether ISO can improve lipid-profile without uterotrophic effects under estrogen-deficiency. Thus, we investigated the effects of ISO on lipid-profile and uterus of ovariectomized (Ovx) rats. MATERIALS AND METHODS: Twenty-five adult rats were Ovx or Sham-operated (Sham) and assigned into five groups: Sham and Ovx groups, administered with vehicle solutions; Ovx-E, treated with 10 µg/kg of 17ß-Estradiol; Ovx-ISO, treated with 200 mg/kg of ISO; Ovx-E + ISO, treated with estradiol + ISO combined. After fifty days of treatments, rats were euthanized and uterine horns were processed for histomorphometry or to collagen fibers and glycosaminoglycans evaluations. Blood samples were collected to evaluate levels of triglycerides, total cholesterol (TC) and its fractions (HDL/VLDL). Data were subjected to statistical analysis (p < .05). RESULTS: Uterus weight was lower in Ovx group than the Sham and Ovx-E groups, whereas it was similar between Ovx and Ovx-ISO groups. Histomorphometry showed atrophic uterus in Ovx and Ovx-ISO groups, whereas uterotrophic effects were noticed in Ovx-E and Ovx-E + ISO groups. Collagen fibers-birefringence was higher in Sham, Ovx, and Ovx-ISO groups than in Ovx-E and Ovx-E + ISO groups. Sulfated glycosaminoglycans content was similar among Sham, Ovx, and Ovx-ISO groups, while it was higher in estrogen-treated groups; total glycosaminoglycans content was similar among groups. TC and HDL was higher in Ovx-ISO group, whereas VLDL and triglycerides levels was higher in Ovx-E + ISO group and similar among other groups. CONCLUSION: Soy isoflavones at 200 mg/kg have slight beneficial effects on lipid-profile without uterotrophic effects in Ovx rats.

Effect of flaxseed oil on biochemical parameters, hormonal indexes and stereological changes in ovariectomized rats.

The ovariectomized rat is a widely used preclinical model for studying postmenopausal and its complications. In this study, the therapeutic effect of flaxseed oil on the ovariectomized adult rats was investigated. Our results showed that biochemical parameters including calcium, oestrogen and progesterone levels increase 8 weeks after ovariectomy in rats. Also, the amount of alkaline phosphatase decreased significantly after 8 weeks compared with the OVX rat. The healing potential of flaxseed oil was proven by successfully recovering the affected tissue and preventing the unpleasant symptoms of ovariectomized rats. The biological effects of flaxseed oil may be due to high amounts of fatty acids, phytoestrogens and an array of antioxidants. The results suggest that flaxseed oil can mimic the action of oestrogen and can be a potential treatment for hormone replacement therapy (HRT).

Effect of Gloriosa superba linn (EEGS) on mPT and monosodium glutamate-induced proliferative disorder using rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperplasia, Tumors and cancers are various forms of proliferative disorders affecting humans. Surgery is the main treatment approach while other options are also associated with adverse effects. There is therefore a need for the development of better alternative therapy that is cost effective and readily available with little or no adverse effect. Some bioactive agents in medicinal plants exhibit their anti-proliferative potential by induction of mitochondrial permeability transition pore (mPT) opening. Gloriosa superba, a medicinal plant, is folklorically used in the treatment of tumors and cancers. AIM OF THE STUDY: This study therefore aimed at investigating the effect of ethanol leaf extract of Gloriosa superba (EEGS) on mPT and monosodium glutamate-induced proliferative disorder in some specific tissues using rat model. MATERIALS AND METHODS: Isolated rat liver mitochondria were exposed to different concentrations (10, 30, 50, 70 and 90 µg/ml) of EEGS. The mPT pore opening, cytochrome c release, mitochondrial ATPase activity and lipid peroxidation were assessed spectrophotometrically. Caspases 9 and 3 activities were carried out using ELISA technique. Histological assessment of the liver, prostate and uterus of normal and monosodium glutamate (MSG)-treated rats were carried out. RESULTS: The results showed significant induction of mPT pore opening, release of cytochrome c, enhancement of mitochondrial ATPase activity, inhibition of lipid peroxidation and activation of caspases 9 and 3 activities by EEGS. The histological assessment revealed the presence of MSG-induced hepato-cellular damage, benign prostate hyperplasia and uterine hyperplasia which were ameliorated by EEGS co-administration. CONCLUSIONS: These findings suggest that EEGS contains putative agents that can induce apoptosis via induction of mPT pore opening and as well protect against MSG-induced hepato-cellular damage and proliferative disorder in prostate and uterus.

Amino Acids in Reproductive Nutrition and Health.

Amino acids are not only the building blocks of proteins, an indispensable component of cells, but also play versatile roles in regulating cell metabolism, proliferation, differentiation and growth by themselves or through their derivatives. At the whole body level, the bioavailability and metabolism of amino acids, interacting with other macronutrients, is critical for the physiological processes of reproduction including gametogenesis, fertilization, implantation, placentation, fetal growth and development. In fertilization and early pregnancy, histotroph in oviductal and uterine secretions provides nutrients and microenvironment for conceptus (embryo and extraembryonic membranes) development. These nutrients include select amino acids in histotroph (arginine, leucine and glutamine of particular interest) that stimulate conceptus growth and development, as well as interactions between maternal uterus and the conceptus, thus impacting maintenance of pregnancy, placental growth, development and functions, fetal growth and development, and consequential pregnancy outcomes. Gestational protein undernutrition causes fetal growth restriction and predisposes cardiovascular, metabolic diseases and others in offspring via multiple mechanisms, whereas the supplementation of glycine, leucine and taurine during pregnancy partially rescues growth restriction and beneficially modulates fetal programming. Thus, amino acids are essential for the fertility of humans and all animals.

Revealing active ingredients, potential targets, and action mechanism of Ermiao fang for treating endometritis based on network pharmacology strategy.

ETHNOPHARMACOLOGICAL RELEVANCE: Ermiao fang (EMF) is a traditional Chinese medicinal herbal formula from ancient times and recorded in the pharmacopeia of the People's Republic of China. It is composed of two typical Chinese herbal medicines, Cortex Phellodendri (Huangbai), the bark of Phellodendron chinensis Schneid. (Rutaceae), and Rhizoma Atractylodis (Cangzhu), the rhizome of Atractylodes lancea (Thunb.) DC. (Compositae). EMF has been clinically used for the treatment of endometritis for many years in China. AIM OF THE STUDY: This study was aimed to identify the active ingredients, potential targets, and mechanism of action of EMF for the treatment of endometritis. MATERIALS AND METHODS: In this research, the pharmacological effects of EMF on endometritis were first evaluated by establishing a rat model of endometritis. A network pharmacology-based analytical strategy was then used to predict its targets and signaling pathways. An endometritis-related protein target and compound database was built for EMF. The compounds in EMF and those absorbed into the blood were identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). High-throughput virtual screening and molecule docking methods were used to predict the protein targets of EMF. The surface plasmon resonance analysis (SPR) method was used to validate the affinity between the compound and proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to predict the related pathways. Western blotting analysis was used to evaluate the expression of key proteins in the related pathways. RESULTS: The animal study showed that EMF could reduce uterine inflammation in rats with endometritis. Then, an ingredient database including 187 compounds and a protein target database including 836 proteins were constructed. Twenty-four compounds in EMF were identified by UHPLC-Q-TOF/MS, among which eight compounds were present in rat plasma after an oral administration of EMF. Afterward, 39 potential target proteins were predicted by the high-throughput screening method, and 20 of them were selected after further screening using molecular docking. Subsequently, an ingredient-target network was constructed, and the target proteins were classified into the NF-κB and MAPK signal pathways by KEGG pathway enrichment analysis. Finally, the affinity between the active ingredients and the target proteins was verified by SPR. The Western blotting analysis showed that EMF significantly inhibited the elevated NF-κB and MAPK pathway proteins in rats with endometritis. CONCLUSIONS: EMF exhibited a significant pharmacological effect on rats with endometritis. Network pharmacology analysis revealed that eight compounds were absorbed into the blood after oral administration and interacted with 20 targets. Western blotting analysis indicated that EMF exerted anti-inflammatory effects by inhibiting the NF-κB and MAPK signaling pathway proteins in the treatment of endometritis.