Parathyroid hormone (1-34) retards the lumbar facet joint degeneration and activates Wnt/ß-catenin signaling pathway in ovariectomized rats.

PURPOSE: Facet joint degeneration (FJD) is a major cause of low back pain. Parathyroid hormone (PTH) (1-34) is commonly used to treat osteoporosis. However, little is known about its effects on FJD induced by estrogen deficiency. This study aims to investigate the effects of PTH (1-34) on FJD induced by estrogen deficiency and the underlying pathogenesis of the disease. METHODS: Forty 3-month-old female Sprague-Dawley rats were randomly divided into four groups: 30 received bilateral ovariectomy (OVX) followed by 12 weeks of treatment with normal saline, PTH (1-34) or 17ß-estradiol (E2), and 10 received sham surgery followed by administration of normal saline. Status and Wnt/ß-catenin signaling activity in the cartilage and subchondral bone of the L4-L5 FJs and serum biomarkers were analyzed. RESULTS: Administration of PTH (1-34) and E2 ameliorated cartilage lesions, and significantly decreased MMP-13 and caspase-3 levels and chondrocyte apoptosis. PTH (1-34) but not E2 significantly increased cartilage thickness, number of chondrocytes, and the expression of aggrecan. PTH (1-34) significantly improved microarchitecture parameters of subchondral bone, increased the expression of collagen I and osteocalcin, and decreased RANKL/OPG ratio. E2 treatment significantly increased the OPG level and decreased the RANKL/OPG ratio in the subchondral bone of ovariectomized rats, but it did not significantly improve the microarchitecture parameters of subchondral bone. Wnt3a and ß-catenin expression was significantly reduced in the articular cartilage and subchondral bone in OVX rats, but PTH (1-34) could increase the expression of these proteins. E2 significantly increased the activity of Wnt/ß-catenin pathway only in cartilage, but not in subchondral bone. The restoration of Wnt/ß-catenin signaling had an obvious correlation with the improvement of some parameters associated with the FJs status. CONCLUSION: Wnt/ß-catenin signaling may be a potential therapeutic target for FJD induced by estrogen deficiency. PTH (1-34) is effective in treating this disease with better efficacy than 17ß-estradiol, and the efficacy may be attributed to its restoration of Wnt/ß-catenin signaling.

In vitro and in vivo studies on exogenous polyamines and α-difluoromethylornithine to enhance bone formation and suppress osteoclast differentiation.

Exogenous polyamines, including putrescine (PUT), spermidine (SPD), and spermine (SPM), and the irreversible inhibitor of the rate-limiting enzyme ornithine decarboxylase (ODC) of polyamine biosynthesis, α-difluoromethylornithine (DFMO), are implicated as stimulants for bone formation. We demonstrate in this study the osteogenic potential of exogenous polyamines and DFMO in human osteoblasts (hOBs), murine monocyte cell line RAW 264.7, and an ovariectomized rat model. The effect of polyamines and DFMO on hOBs and RAW 264.7 cells was studied by analyzing gene expression, alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) activity, and matrix mineralization. Ovariectomized rats were treated with polyamines and DFMO and analyzed by micro computed tomography (micro CT). The mRNA level of the early onset genes of osteogenic differentiation, Runt-related transcription factor 2 (Runx2) and ALP, was significantly elevated in hOBs under osteogenic conditions, while both ALP activity and matrix mineralization were enhanced by exogenous polyamines and DFMO. Under osteoclastogenic conditions, the gene expression of both receptor activator of nuclear factor-κB (RANK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) was reduced, and TRAP activity was suppressed by exogenous polyamines and DFMO in RAW 264.7 cells. In an osteoporotic animal model of ovariectomized rats, SPM and DFMO were found to improve bone volume in rat femurs, while trabecular thickness was increased in all treatment groups. Results from this study provide in vitro and in vivo evidence indicating that polyamines and DFMO act as stimulants for bone formation, and their osteogenic effect may be associated with the suppression of osteoclastogenesis.

Ethanolic extract of Persea americana Mill. (Lauraceae) seeds induced antiestrogenic effects in young female Wistar rats.

OBJECTIVES: The ethanol extract of Persea americana seeds was found to inhibit the development of estrogen-dependent conditions in female Wistar rats, suggesting the ability of its secondary metabolites to interact with estrogen receptors (ERs), either as partial agonists or as antagonists. To test this hypothesis, the abovementioned extract was assessed for its ability to mimic and/or antagonize estradiol effects. METHODS: Two experiments were conducted in ovariectomized (OVX) rats: (1) animals were treated with estradiol valerate (E2V; 1 mg/kg) or P. americana at doses of 25 and 50 mg/kg; (2) animals were treated with E2V alone (0.75 mg/kg) or in combination with P. americana at the abovementioned doses. Treatments were given orally for 3 days and animals were sacrificed for biochemical and histological analyses of the uterus and vagina. RESULTS: When administered alone, P. americana did not change the histomorphology of both organs (uterus and vagina). In combination with E2V, P. americana decreased uterine weight [30 % decrease (p<0.001) at 25 mg/kg and 24 % (p<0.01) at 50 mg/kg] and epithelium height (37 % decrease). This was associated with decreased estradiol levels (at least 86 % decrease, p<0.001) in the uterus. Similarly, vagina epithelium height decreased by at least 34 % (p<0.05) when E2V was co-administered with P. americana. CONCLUSIONS: The seed extract of P. americana contains ER antagonist secondary metabolites accounting for its ability to inhibit the development of estrogen-dependent conditions in female rats.

UPLC-Triple-TOF-MS-based serum metabonomic revealed the alleviating effect of QingYan Formula on perimenopausal syndrome rats.

The study aimed to investigate the relieving effect of QingYan Formula (QYF) in treating perimenopausal syndrome. A combination of metabonomic analysis and in vitro pharmacodynamic experiments was employed to achieve this objective.Over a period of 12 weeks, ovariectomized (OVX) rats were orally administered QYF's 70 % ethanol extract or estradiol valerate (EV). The results demonstrate that QYF restored the estrous cycle of ovariectomized rats and exhibited significant estrogenic activity, as indicated by reversal of uterine and vagina atrophy, improvement of serum estradiol level and decrease of serum luteinizing hormone(LH) level. Additionally, QYF administration effectively reduced high bone turnover and repaired trabecular microstructure damage. Metabonomic analysis of the OVX rats treated with QYF revealed the identification of 55 different metabolites in the serum, out of which 35 may be potential biomarkers. QYF could regulate the disturbed metabolic pathways including the Biosynthesis of unsaturated fatty acids, arachidonic acid metabolism, bile secretion, and steroid hormone biosynthesis. PI3KCA, SRC, and MAPK3 are potential therapeutic targets for QYF therapeutic effects. These findings support the efficacy of QYF in alleviating perimenopausal syndrome and regulating lipid metabolic disorders in OVX rats.

Oxyresveratrol attenuates bone resorption by inhibiting the mitogen-activated protein kinase pathway in ovariectomized rats.

BACKGROUND: Bone is continuously produced by osteoblasts and resorbed by osteoclasts to maintain homeostasis. Impaired bone resorption by osteoclasts causes bone diseases such as osteoporosis and arthritis. Most pharmacological treatment of osteoporosis focuses on inhibiting osteoclast differentiation, often to restore osteoclast/osteoclast balance. However, recent osteoporosis treatments have various side effects. According to a recent study, resveratrol, known as a stilbenoid family, is known to increase bone density, and the osteoclast inhibitory effect was confirmed using oxyresveratrol, a stilbenoid family. Here, we investigated the effect of oxyresveratrol on osteoclast differentiation and an ovariectomized mouse model. METHODS: Mouse leukemia monocyte/macrophage cell line RAW 264.7 was treated with oxyresveratrol, and cell cytotoxicity was confirmed by measuring MTT assay. Tartrate-resistant acid phosphatase (TRAP), an enzyme marker for osteoclasts, was confirmed by staining. In addition, osteoclast differentiation markers and MAPK-related markers were confirmed at the mRNA level and protein expression. The effect of oxyresveratrol was confirmed using ovariectomized mice. Deoxypyridinoline (DPD) was measured using mouse urine and TRAP activity was observed using serum. Bone mineral density was also measured using Micro-CT. RESULTS: The polyphenol oxyresveratrol inhibited receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclast differentiation of RAW 264.7 cells. Furthermore, oxyresveratrol inhibited TRAP activity and actin-ring formation. Moreover, oxyresveratrol suppressed the phosphorylation of the RANKL-induced mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK and significantly reduced the expression of bone differentiation markers (NFATc1, cathepsin K, and TRAP). CONCLUSION: Oxyresveratrol inhibits osteoclast differentiation via MAPK and increases bone density in ovariectomized rats, suggesting it has therapeutic potential for bone diseases such as osteoporosis. We confirmed the osteoporosis prevention effect of OR in Raw 264.7 cells, and future studies should confirm the effect of OR using rat bone marrow-derived cells.

Advantage of ostarine over raloxifene and their combined treatments for muscle of estrogen-deficient rats.

PURPOSE: Selective androgen (ostarine, OST) and estrogen (raloxifene, RAL) receptor modulators with improved tissue selectivity have been developed as alternatives to hormone replacement therapy. We investigated the combined effects of OST and RAL on muscle tissue in an estrogen-deficient rat model of postmenopausal conditions. METHODS: Three-month-old Sprague Dawley rats were divided into groups: (1) untreated non-ovariectomized rats (Non-OVX), (2) untreated ovariectomized rats (OVX), (3) OVX rats treated with OST, (4) OVX rats treated with RAL, (5) OVX rats treated with OST and RAL. Both compounds were administered in the diet. The average dose received was 0.6 ± 0.1 mg for OST and 11.1 ± 1.2 mg for RAL per kg body weight/day. After thirteen weeks, rat activity, muscle weight, structure, gene expression, and serum markers were analyzed. RESULTS: OST increased muscle weight, capillary ratio, insulin-like growth factor 1 (Igf-1) expression, serum phosphorus, uterine weight. RAL decreased muscle weight, capillary ratio, food intake, serum calcium and increased Igf-1 and Myostatin expression, serum follicle stimulating hormone (FSH). OST + RAL increased muscle nucleus ratio, uterine weight, serum phosphorus, FSH and luteinizing hormone and decreased body and muscle weight, serum calcium. Neither treatment changed muscle fiber size. OVX increased body and muscle weight, decreased uterine weight, serum calcium and magnesium. CONCLUSION: OST had beneficial effects on muscle in OVX rats. Side effects of OST on the uterus and serum electrolytes should be considered before using it for therapeutic purposes. RAL and RAL + OST had less effect on muscle and showed endocrinological side effects on pituitary-gonadal axis.

Osteogenic Effects of KY-054, a Novel Coumarin Derivative on Femur Cortical Bone in Ovariectomized Female Rats.

Osteoporosis is treated with oral and parenteral bone resorption inhibitors such as bisphosphonates, and parenteral osteogenic drugs including parathyroid hormone (PTH) analogues and anti-sclerostin antibodies. In the present study, we synthesized KY-054, a 4,6-substituted coumarin derivative, and found that it potently promoted osteoblast differentiation with an increase in alkaline phosphatase (ALP) activity at 0.01-1 µM in mouse-derived mesenchymal stem cells (ST2 cells) and rat bone marrow-derived mesenchymal stem cells (BMSCs). In the ovariectomized (OVX) rats, KY-054 (10 mg/kg/d, 8 weeks) increased plasma bone-type ALP activity, suggesting in vivo promoting effects on osteoblast differentiation and/or activation. In dual-energy X-ray absorption (DEXA) scanning, KY-054 significantly increased the distal and diaphyseal femurs areal bone mineral density (aBMD) that was decreased by ovariectomy, indicating its beneficial effects on bone mineral contents (BMC) and/or bone volume (BV). In micro-computed tomography (micro-CT) scanning, KY-054 had no effect on metaphysis trabecular bone loss and microarchitecture parameters weakened by ovariectomy, but instead increased metaphysis and diaphysis cortical bone volume (Ct.BV) and cortical BMC (Ct.BMC) without reducing medullary volume (Med.V), resulting in increased bone strength parameters. It is concluded that KY-054 preferentially promotes metaphysis and diaphysis cortical bone osteogenesis with little effect on metaphysis trabecular bone resorption, and is a potential orally active osteogenic anti-osteoporosis drug candidate.

Anti-osteoporosis mechanism of resistance exercise in ovariectomized rats based on transcriptome analysis: a pilot study.

Postmenopausal osteoporosis is the main cause of fractures in women. Resistance exercise has a positive effect on bone mineral density in postmenopausal osteoporosis patients, but its mechanism is unclear. The purpose of this study was to explore the mechanism of resistance exercise in improving ovariectomized osteoporotic rats based on the transcriptome sequencing technique. Eighteen female Sprague-Dawley rats were randomly divided into the sham-operated group, the non-exercise group, and the resistance exercise group. The rat model of postmenopausal osteoporosis was established by bilateral ovariectomy. Ten weeks after the operation, the resistance exercise group received 2 weeks of adaptive training, and 12 weeks of resistance exercise began in the 13th week. The rats were trained 5 days per week, in 4 sets of 3 repetitions per day. After the intervention, all rats were sacrificed, and the body weight, bone mineral density, trabecular bone microarchitecture, and bone biomechanics were examined. At the same time, RNA-seq and enrichment analysis of gene ontology and Kyoto Encyclopedia of Genes and Genomes were performed on the left tibias, followed by Elisa and RT-qPCR verification. It had been found that resistance exercise can effectively counteract the weight gain of ovariectomized osteoporotic rats, and has a good effect on bone mineral density and trabecular bone microarchitecture. Enrichment analysis showed that regulation of gene expression and osteoclast differentiation is the most closely related biological process and signaling pathway shared by RE/Ovx and NE/Ovx groups. Our results revealed that resistance exercise can play a role in inhibiting osteoclast activation and preventing the enhancement of osteoclast bone resorption function in ovariectomized osteoporotic rats by inhibiting Fos/Fosb-regulated TRAP activation and relieving Calcr inhibition, which has important application value in preventing bone loss caused by estrogen deficiency.

The Dose-Related Efficacy of Human Placenta-Derived Mesenchymal Stem Cell Transplantation on Antioxidant Effects in a Rat Model with Ovariectomy.

Oxidative stress initiates various degenerative diseases, and it is caused by excessive reactive oxygen species (ROS) production. Oxidative stress is a key factor that causes infertility by inducing ovarian dysfunction, characterized by irregular hormone levels, lower quality of mature follicles, and loss of follicles. Hence, stem cell therapy has been actively studied as an approach to overcome the side effects of hormone replacement therapy (HRT) on ovarian dysfunction. However, there is a lack of evidence about the appropriate number of cells required for stem cell therapy. Therefore, based on the antioxidant effects investigated in this study, we focused on determining the appropriate dose of stem cells for transplantation in an animal model with ovarian dysfunction. One week after half-ovariectomy, placenta-derived mesenchymal stem cells (PD-MSCs, 1 × 105 cells, 5 × 105 cells, or 2.5 × 106 cells) were injected intravenously into the Tx groups through the tail vein. As a result, the mRNA expression of hAlu gradually increased as the transplanted cell concentration increased. Compared with no transplantation (NTx), the transplantation of PD-MSCs improved folliculogenesis, including the levels of secreted hormones and numbers of follicles, by exerting antioxidant effects. Also, the levels of oxidized glutathione in the serum of animal models after transplantation were significantly increased (* p < 0.05). These results indicated that PD-MSC transplantation improved ovarian function in half-ovariectomized rats by exerting antioxidant effects. According to our data, increasing the number of transplanted cells did not proportionally increase the effectiveness of the treatment. We suggest that low-dose PD-MSC transplantation has the same therapeutic effect as described in previous studies. These findings provide new insights for further understanding reproductive systems and provide evidence for related clinical trials.

Investigation of taurine and aqueous garlic extract diet supplementation effect on the healing of rat osteoporotic fractures.

BACKGROUND: We have evaluated the effects of taurine and aqueous garlic extract (AGE) as a dietary supplement on osteoporotic fracture (OPF) healing in the ovariectomized rat femur fracture model. METHODS: In this experimental animal study,twenty-four osteoporosis-remodeled female Wistar albino rats were randomly divided into 3 groups (n: 8) according to their supplemented diet; control, taurine, and AGE groups. Unilateral femur middiaphysis mini-open osteotomy was stabilized with Kirschner wires. Six weeks after osteotomy, the rats were sacrificed before the femurs were harvested and OPF healing was evaluated with biochemical, histologic, microcomputed-tomography, and scintigraphic methods. RESULTS: As an indicator of the antiosteoporotic effect, the calcium levels of the taurine group were significantly lower than the AGE and control groups in biochemical analyzes (p < 0.01). In histological studies, the new bone diameter and new bone volume values of the taurine group were significantly higher than the control group (p = 0.002 and p = 0.032, respectively), while higher trabecular-compact callus was observed in the taurine and AGE groups, respectively, compared to the control group. In morphological analyses, taurine and AGE groups had significantly higher bone volume/tissue volume, trabecular number, bone surface density, and lower trabecular separation than the control group (p < 0.05). The scintigraphic imaging showed a significant increase in osteoblastic activity of the taurine group compared to the control group (p = 0.005). DISCUSSION: Taurine and AGE have positive anabolic effects, respectively, on the healing of OPFs, demonstrated by biochemical, histological, morphological, and scintigraphic methods.

α-Linolenic Acid Inhibits RANKL-Induced Osteoclastogenesis In Vitro and Prevents Inflammation In Vivo.

Inflammation is an important risk factor for bone-destroying diseases. Our preliminary research found that Zanthoxylum bungeanum seed oil (ZBSO) is abundant in unsaturated fatty acids and could inhibit osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells. However, the key constituents in ZBSO in the prevention of osteoclastogenesis and its possible mechanism related to inflammation are still unclear. Therefore, in this study, oleic acid (OA), linoleic acid (LA), palmitoleic acid (PLA), and alpha-linolenic acid (ALA) in ZBSO, havingthe strongest effect on RANKL-induced osteoclastogenesis, were selected by a tartrate-resistant acid phosphatase (TRAP) staining method. Furthermore, the effects of the selected fatty acids on anti-inflammation and anti-osteoclastogenesis in vitro and in vivo were assessed using RT-qPCR. Among the four major unsaturated fatty acids we tested, ALA displayed the strongest inhibitory effect on osteoclastogenesis. The increased expression of free fatty acid receptor 4 (FFAR4) and ß-arrestin2 (ßarr2), as well as the decreased expression of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), nuclear factor of activated T-cells c1 (NFATc1), and tartrate-resistant acid phosphatase (TRAP) in RAW264.7 cells after ALA treatment were observed. Moreover, in ovariectomized osteoporotic rats with ALA preventive intervention, we found that the expression of TNF-α, interleukin-6 (IL-6), interleukin-1ß (IL-1ß), NFATc1, and TRAP were decreased, while with the ALA therapeutic intervention, downregulated expression of NF-κB, NFATc1, TRAP, and transforming growth factor beta-activated kinase 1 (TAK1) were noticed. These results indicate that ALA, as the major unsaturated fatty acid in ZBSO, could inhibit RANKL-induced osteoclastogenesis via the FFAR4/ßarr2 signaling pathway and could prevent inflammation, suggesting that ZBSO may be a promising potential natural product of unsaturated fatty acids and a dietary supplement for the prevention of osteoclastogenesis and inflammatory diseases.

Postmenopausal osteoporosis: a bioinformatics-integrated experimental study the pathogenesis.

Postmenopausal osteoporosis (PMOP) is a chronic bone metabolic disease, which often causes fractures and various complications, it causes a great social and economic burden, and it is urgent to use modern research techniques to elucidate the pathogenesis of PMOP. At the same time, because of the complex physiological and pathological interaction mechanism between osteoporosis and sarcopenia, the correlation research has become a hot topic. Ovary removal is a commonly used experimental method to study the endocrine system of female animals, and it is also the best animal model to study PMOP. In this study, the preparation of the ovariectomized rat was confirmed through the detection of vaginal smear, the level of bone formation markers, and the analysis of bone tissue morphology. Transcriptome sequencing was used to analyze the molecular mechanism of PMOP in ovariectomized rats, qRT-PCR was used to verify the key targets. Results of Micro-CT and scanning electron microscopy (SEM) showed that the trabecular structure was disorganized and the symptoms of osteoporosis appeared, this indicating that the ovariectomized rats model was successfully prepared. Transcriptional sequencing results of femur tissue showed that 452 differentially expressed genes (DEGs) were screened. Bioinformatics analysis results showed that the osteoporosis caused by ovariectomized rats was mainly related to muscle contraction, calcium signaling pathway, etc. Results of qRT-PCR were consistent with transcriptome analysis. These results reveal the pathogenesis of PMOP in ovariectomized rats and also offer a possibility for elucidating the relevance of action between PMOP and sarcopenia.

Estradiol replacement improves high-fat diet-induced insulin resistance in ovariectomized rats.

The role of 17ß-estradiol (E2) in high-fat diet (HFD)-induced alteration of the protein kinase B (Akt) signaling pathway in ovariectomized (OVX) rats is unclear. Therefore, we examined whether chronic estrogen replacement restores HFD-induced impairment in insulin sensitivity by its effects concomitant with alterations in the Akt isoform 2 (Akt2) and Akt substrate of 160 kDa (AS160) phosphorylation in muscles of OVX rats. Nine-week-old female Wistar rats underwent ovariectomy under anesthesia; after 4 weeks, subcutaneous implantation of either E2 or placebo (PL) pellets was performed, and HFD feeding was initiated. Intravenous glucose tolerance tests were performed to assess insulin sensitivity. Following insulin injection into rats' portal vein, the liver and gastrocnemius muscle were dissected for insulin signaling analysis. We observed that HFD increased energy intake and body weight in the PL group; however, it was temporarily decreased in the E2 group. Adipose tissue accumulation was larger in HFD-fed rats than in normal chow diet (NCD)-fed rats in the PL group; however, this difference was not observed in the E2 group. HFD reduced insulin sensitivity in the PL group only. In vivo insulin stimulation increased Akt2 phosphorylation in the muscles of NCD-fed rats in both groups. In contrast, HFD affected insulin-stimulated phosphorylation of Akt2 and AS160 in the muscles of rats in the PL group but not in the E2 group. Our data suggest that E2 replacement improves HFD-induced insulin resistance, and this effect is accompanied by the alterations in the Akt2 and AS160 phosphorylation in insulin-stimulated muscles of OVX rats.

Efficacy of Yougui pill combined with Buzhong Yiqi decoction in alleviating the sexual dysfunction in female rats through modulation of the gut microbiota.

CONTEXT: Yougui pill combined with Buzhong Yiqi decoction (YPBYD) is used to relieve sexual dysfunction in clinical practice. OBJECTIVE: To investigate changes in microbial composition caused by sexual dysfunction and identify dominant bacteria related to YPBYD treatment. MATERIALS AND METHODS: Female Sprague-Dawley rats were randomly divided into four groups (n = 6): one group underwent Sham operation (Sham group), while three groups underwent ovariectomy (one model and two treatment groups). The ovariectomized (OVX) rats received oestradiol benzoate (250 µg/kg/week) or YPBYD (3.6 mL/d) via oral gavage for 4 weeks. Vaginal smear assay was performed; the serum levels of cyclic adenosine monophosphate (cAMP) and oestradiol (E2) were measured, followed by collection of stool samples for 16S rRNA sequencing. RESULTS: After YPBYD treatment, the levels of E2 and cAMP in OVX rats significantly increased (E2: from 20.45 ± 1.60 ng/L to 24.38 ± 1.70 ng/L; cAMP: from 261.41 ± 9.21 pg/mL to 373.75 ± 17.37 pg/mL). OVX treatment decreased diversity of gut microbiota and YPBYD treatment restored gut microbiota composition. Compared with Sham group, the abundance of Romboutsia significantly increased, while those of Proteobacteria and Staphylococcus markedly decreased in OVX group (all p < 0.05); meanwhile, the abundance of these microbes showed an opposite trend after YPBYD treatment. These microbiotas were involved in tyrosine and tryptophan biosynthesis and fatty acid metabolism. DISCUSSION AND CONCLUSIONS: These findings are the first to indicate YPBYD can alleviate female sexual dysfunction by modulating gut microbiota in OVX rats, which will help enhance the understanding on potential mechanism of YPBYD against sexual dysfunction.

Induction of P-glycoprotein expression and activity by prolactin in female rat liver.

AIM: P-glycoprotein (P-gp) plays a critical role in the excretion of xenobiotics into bile. Previous studies have demonstrated that prolactin (PRL) regulates biotransformation and bile salt transport. Here we investigate whether the capability of the liver to transport xenobiotics into bile is altered in hyperprolactinemic states studying the modulation of hepatic P-gp by PRL. METHODS: We used lactating post-partum rats (PP), as a model of physiological hyperprolactinemia (15 and 21 days after delivery: PP15 and PP21, respectively), and ovariectomized rats treated with PRL (300 µg/day, 7 days, via osmotic minipumps, OVX + PRL). Hepatic P-gp expression and activity were evaluated by western blotting and using rhodamine 123 as substrate in vivo, respectively. Since P-gp is encoded by Mdr1a and Mdr1b in rodents, we quantified their expression by qPCR in primary hepatocyte cultures exposed to 0.1 µg/ml of PRL after 12 h. To further study the mechanism of hepatic P-gp modulation by PRL, hepatocytes were pretreated with actinomycin D and then exposed to PRL (0.1 µg/ml) for 12 h. KEY FINDINGS: We found increased hepatic P-gp protein expression and activity in PP15 and OVX + PRL. Also, a significant increase in Mdr1a and Mdr1b mRNA levels was observed in primary hepatocyte cultures exposed to PRL, pointing out the hormone direct action. Actinomycin D prevented these increases, confirming a transcriptional up-regulation of P-gp by PRL. SIGNIFICANCE: These findings suggest the possibility of an increased biliary excretion of xenobiotics substrates of P-gp, including therapeutic agents, affecting their pharmaco/toxicokinetics in hyperprolactinemic situations.

Iron accumulation deteriorated bone loss in estrogen-deficient rats.

BACKGROUND: Postmenopausal osteoporosis is characterized by an imbalance of bone resorption exceeding bone formation, resulting in a net loss of bone mass. Whether a menopause-related excess of iron contributes to the development of postmenopausal osteoporosis has remained unresolved due to a lack of an appropriate animal model. This study aimed to explore the effects of iron accumulation in bone mass in estrogen-deficient rats. METHODS: In the present study, ovariectomy (OVX) was performed in female rats and the changes of iron metabolism and some related modulated genes were detected. Ferric ammonium citrate (FAC) was used as a donor of iron for OVX rats. Moreover, micro-CT was performed to assess the bone microarchitecture in sham group, OVX, and FAC groups. Histological detection of iron in liver was assessed by Perl's staining. The expressions of ß-CTX and osteocalcin were assessed by ELISA. RESULTS: It was found that serum iron decreased after OVX. It was found that the expressions of Hepcidin in liver and Fpn, DMT-1 in duodenum significantly decreased at transcriptional level in OVX group than sham group. However, no difference existed in the expression of DMT-1. Then, ferric ammonium citrate (FAC) was used as a donor of iron for OVX rats. The FAC group manifested significant iron accumulation by increased serum iron and hepatic iron content. In addition, FAC treatment accelerated bone loss and decreased BMD and biomechanics in OVX rats. Moreover, bone biomarker ß-CTX rather than osteocalcin increased significantly in FAC groups than OVX group. CONCLUSIONS: In conclusion, no iron accumulation occurred in OVX rats. Furthermore, iron accumulation could further deteriorate osteopenia through enhanced bone resorption.

Identification of the Active Ingredient and Beneficial Effects of Vitex rotundifolia Fruits on Menopausal Symptoms in Ovariectomized Rats.

Estrogen replacement therapy is a treatment to relieve the symptoms of menopause. Many studies suggest that natural bioactive ingredients from plants resemble estrogen in structure and biological functions and can relieve symptoms of menopause. The fruit of V. rotundifolia, called "Man HyungJa" in Korean, is a traditional medicine used to treat headache, migraine, eye pain, neuralgia, and premenstrual syndrome in Korea and China. The aim of the present study was to confirm that V. rotundifolia fruit extract (VFE) exerts biological functions similar to those of estrogen in menopausal syndrome. We investigated its in vitro effects on MCF-7 cells and in vivo estrogen-like effects on weight gain and uterine contraction in ovariectomized rats. Using the polar extract, the active constituents of VFE (artemetin, vitexicarpin, hesperidin, luteolin, vitexin, and vanillic acid) with estrogen-like activity were identified in MCF-7 cells. In animal experiments, the efficacy of VFE in ameliorating body weight gain was similar to that of estrogen, as evidenced from improvements in uterine atrophy. Vitexin and vitexicarpin are suggested as the active constituents of V. rotundifolia fruits.

Exploring the bone sparing effects of postbiotics in the post-menopausal rat model.

BACKGROUND: Post-menopausal osteoporosis is a concern of health organizations, and current treatments do not seem enough. Postbiotics as bioactive compounds produced by probiotics may be an attractive alternative for bone health. In this study, we prepared, formulated, and compared the effects of cell lysate and supernatant of five native probiotic strains (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Bifidobacterium longum, and Bacillus coagulans) in ovariectomized (OVX) rats. METHODS: The probiotic strains were isolated, and their cell-free supernatants and biomasses as postbiotics were extracted and formulated using standard microbial processes. The Sprague-Dawley rats were fed by 1 × 109 CFU/ml/day postbiotic preparations for 4 weeks immediately after ovariectomy. Dual-energy X-ray absorptiometry (DEXA) scans were accomplished to evaluate femur, spine, and tibia BMD. The serum biochemical markers [calcium, phosphorus, and alkaline phosphatase] were assessed. RESULTS: Postbiotics could considerably improve the global and femur area in OVX rats. In the case of global bone mineral density (BMD), Lactobacillus casei lysate and supernatant, Bacillus coagulans lysate and supernatant, lysate of Bifidobacterium longum and Lactobacillus acidophilus, and Lactobacillus reuteri supernatant significantly increased BMD. We found Bacillus coagulans supernatant meaningfully enriched tibia BMD. CONCLUSION: Postbiotic could ameliorate bone loss resulting from estrogen deficiency. Also, the effects of postbiotics on different bone sites are strain-dependent. More clinical studies need to explore the optimal administrative dose and duration of the specific postbiotics in protecting bone loss.

The lignan-rich fraction from Sambucus Williamsii Hance ameliorates dyslipidemia and insulin resistance and modulates gut microbiota composition in ovariectomized rats.

Menopausal women are susceptible to have high risk of cardiovascular diseases, type II diabetes and osteoporosis due to the metabolic disorder caused by estrogen deficiency. Accumulating evidence supports that gut microbiota is a key regulator of metabolic diseases. Our previous metabolomics study interestingly demonstrated that the anti-osteoporotic effects of lignan-rich fraction (SWCA) from Sambucus wialliamsii Hance were related to the restoration of a series of lipid and glucose metabolites. This study aims to investigate how SWCA modulates lipid and glucose metabolism and the underlying mechanism. Our results show that oral administration of SWCA (140 mg/kg and 280 mg/kg) for 10 weeks alleviated dyslipidemia, improved liver functions, prevented glucose tolerance and insulin actions, attenuated system inflammation and improved intestinal barrier in OVX rats. It also induced a high abundance of Actinobacteria, and restored microbial composition. We are the first to report the protective effects of the lignan-rich fraction from S. williamsii on dyslipidemia and insulin resistance. Our findings provide strong evidence for the application of this lignan-rich fraction to treat menopausal lipid disorder and insulin resistance-related diseases.

Blackcurrant (Ribes nigrum L.) Extract Exerts Potential Vasculoprotective Effects in Ovariectomized Rats, Including Prevention of Elastin Degradation and Pathological Vascular Remodeling.

Estrogen exerts cardioprotective effects in menopausal women. Phytoestrogens are plant-derived substances exhibiting estrogenic activity that could beneficially affect vascular health. We previously demonstrated that blackcurrant (Ribes nigrum L.) extract (BCE) treatment exerted beneficial effects on vascular health via phytoestrogenic activity in ovariectomized (OVX) rats, which are widely used as menopausal animal models. Here, we examined whether BCE treatment reduced elastin degradation and prevented pathological vascular remodeling in OVX rats fed a regular diet (OVX Control) or a 3% BCE-supplemented diet (OVX BCE), compared with sham surgery rats fed a regular diet (Sham) for 3 months. The results indicated a lower staining intensity of elastic fibers, greater elastin fragmentation, and higher α-smooth muscle actin protein expression in OVX Control rats than in OVX BCE and Sham rats. Pathological vascular remodeling was only observed in OVX Control rats. Additionally, we investigated matrix metalloproteinase (MMP)-12 mRNA expression levels to elucidate the mechanism underlying elastin degradation, revealing significantly upregulated MMP-12 mRNA expression in OVX Control rats compared with that in Sham and OVX BCE rats. Together, we identify BCE as exerting a vascular protective effect through reduced MMP-12 expression and vascular smooth muscle cell proliferation. To our knowledge, this is the first report indicating that BCE might protect against elastin degradation and pathological vascular remodeling during menopause.