Yuyan Gui

Bu-Shen-Ning-Xin decoction suppresses osteoclastogenesis via increasing dehydroepiandrosterone to prevent postmenopausal osteoporosis

Bu-Shen-Ning-Xin decoction (BSNXD), a traditional Chinese medicine, has been used to prevent and treat age-related diseases such as postmenopausal osteoporosis (PMO) for decades. This study sought to investigate the underlying mechanisms of BSNXD in terms of receptor activation of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro because of the critical roles of bone resorption in the development and progression of osteoporosis. In mice, serum levels of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), and 17-β-estradiol (E2) were evaluated with an enzyme immunoassay kit after ovariectomy. Levels of DHEA and DHEAS increased significantly following administration of BSNXD while the level of E2 did not. In addition, tartrate-resistance acid phosphatase staining showed that DHEA profoundly inhibited RANKL-induced osteoclastogenesis in vitro in a dose-dependent manner via estrogen receptor α (ERα) but not via estrogen receptor β or androgen receptors. Cytotoxicity was not detected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These data suggest that BSNXD prevents PMO by increasing DHEA via the ERαpathway to suppress osteoclastogenesis.

DHEA promotes osteoblast differentiation by regulating the expression of osteoblast-related genes and Foxp3 + regulatory T cells

Several studies have reported that dehydroepiandrosterone (DHEA) promotes osteoblast proliferation and inhibits osteoblast apoptosis and that DHEA inhibits osteoclast maturation. However, whether DHEA regulates osteoblast differentiation remains unclear. The present study first examined the effect of DHEA on bone morphology in vivo. DHEA was found to increase bone volume (BV), bone mineral density (BMD), and the number of trabeculae in bone (Th.N) and it was found to decrease trabecular spacing in bone (Th.sp) in ovariectomized (OVX) mice. Next, the effect of DHEA on osteoblast differentiation was examined in vitro and osteoblastogenesis-related marker genes, such as Runx2, Osterix, Collagen1, and Osteocalcin, were also detected. DHEA increased osteoblast production in mesenchymal stem cells (MSCs) cultured in osteoblastogenic medium, and DHEA increased the expression of Runx2 and osterix, thereby increasing the expression of osteocalcin and collagen1. Immune cells and bone interact, so changes in immune cells were detected in vivo. DHEA increased the number of Foxp3(+) regulatory T cells (Tregs) in the spleen but it did not affect CTLA-4 or IL-10. When MSCs were treated with DHEA in the presence of Tregs, alkaline phosphatase (ALP) activity increased. Osteoblasts and adipocytes are both generated by MSCs. If osteoblast differentiation increases, adipocyte differentiation will decrease, and the reverse also holds true. DHEA was found to increase the number of adipocytes in osteoblastogenic medium but it had no effect on the number of adipocytes and expression of PPARγ mRNA in adipogenic medium. This finding suggests that osteoblasts may be involved in adipocyte production. In conclusion, the current results suggest that DHEA can improve postmenopausal osteoporosis (PMO) by up-regulating osteoblast differentiation via the up-regulation of the expression of osteoblastogenesis-related genes and via an increase in Foxp3(+) Tregs.

DHEA prevents bone loss by suppressing the expansion of CD4+ T cells and TNFa production in the OVX-mouse model for postmenopausal osteoporosis

Recent studies have suggested that dehydroepiandrosterone (DHEA) might serve as a form of immunomodulatory therapy for postmenopausal osteoporosis (PMO). The current study investigated the effects of DHEA administration on ovariectomy (OVX)-induced bone loss and its corresponding immunological changes. Adult OVX mice were treated with DHEA or 17-β-estradiol (E2) for 12 weeks, with or without the aromatase inhibitor letrozole. DHEA improved bone mass after OVX and displayed action like that of E2 with regard to decreasing osteoclast-related parameters. DHEA also suppressed an OVX-induced increase in CD4(+) T cell subsets and TNF-α production. However, DHEA elevated serum E2 levels to a lesser extent than E2. Although letrozole decreased serum E2 levels in OVX mice treated with DHEA, it did not alter DHEAs effects on corresponding immunological changes due to OVX. In conclusion, DHEA may prevent bone loss by suppressing the OVX-induced expansion of CD4(+) T cells and TNF-α production in mice, independent of E2.

Bu-Shen-Ning-Xin Decoction ameliorated the osteoporotic phenotype of ovariectomized mice without affecting the serum estrogen concentration or uterus

Introduction Bu-Shen-Ning-Xin Decoction (BSNXD), a traditional Chinese medicinal composition, has been used as a remedy for postmenopausal osteoporosis, but its effects on bone metabolism and the uterus have not been reported. Purpose We aimed to determine the respective effects of BSNXD on the bones and the uterus of ovariectomized (OVX) mice to evaluate the efficacy and safety of this herbal formula. Materials and methods Postmenopausal osteoporosis animal models that were generated by ovariectomy were treated with BSNXD. Dual-energy X-ray absorptiometry was performed to analyze the bone mineral density, and histomorphometric analysis was performed to measure the parameters related to bone metabolism. Calcein labeling was performed to detect bone formation. The uteruses from the mice were weighed, and the histomorphometry was analyzed. Drug-derived serum was prepared to assess the 17-β-estradiol concentration via enzyme immunoassay. Results BSNXD administration ameliorated the osteoporotic phenotype of OVX mice, as evidenced by an increase in the bone mineral density and bone volume; these effects could not be abolished by the administration of the aromatase inhibitor letrozole. Moreover, BSNXD had no effect on the serum estrogen concentration or uterus. Conclusion These results suggest that BSNXD has ameliorating effects on bone loss due to estrogen deprivation without affecting the peripheral blood estrogen concentration or the uterus in OVX mice.

Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α.

Introduction Bu-Shen-Ning-Xin decoction (BSNXD) is a traditional Chinese medicinal composition that has been used as a remedy for postmenopausal osteoporosis, but the mechanisms affecting bone metabolism are not fully understood. Purpose We investigated the molecular mechanism and signaling pathway underlying the effect of BSNXD on osteoclastogenesis. Materials and methods A postmenopausal osteoporosis animal model generated by ovariectomy was administered BSNXD and drug-derived serum was prepared. An enzyme immunoassay was conducted to measure the 17-β-estradiol (E2) concentration in the drug-derived serum. Bone marrow-derived monocyte/macrophage precursor cells were treated with drug-derived serum, and tartrate-resistance acid phosphatase staining was conducted to observe osteoclastogenesis. A bone resorption assay was performed to analyze the effect on osteoclastic resorptive function. Real-time PCR, flow cytometry, Western blotting, transfection, and luciferase assays were conducted to explore the related mechanism. Results E2 was not elevated in BSNXD-derived serum. BSNXD-derived serum suppressed receptor activation of nuclear factor κB ligand (RANKL)-activated osteoclastogenesis in a dose-dependent manner; this effect could be reversed by estrogen receptor α antagonist methyl-piperidino-pyrazole. The serum suppressed RANKL-induced NF-κB transcription and inhibited the accumulation of nuclear factor of activated T-cells, cytoplasmic 1 in osteoclast precursor cells; the inhibitory effect was abolished by methyl-piperidino-pyrazole but not the estrogen receptor β antagonist or androgen receptor antagonist. Conclusion These results collectively suggest that administration of BSNXD presents inhibitory effects on osteoclast differentiation by abrogating the RANKL-induced nuclear factor of activated T-cells, cytoplasmic 1 and NF-κB signaling pathways downstream of estrogen receptor α, thereby contributing to the inhibitory effect on bone resorption.

The effect of DHEA on apoptosis and cohesin levels in oocytes in aged mice

Female fertility declines with age as the number of ovarian follicles decreases and aneuploidy increases. Degradation of the cohesin complex might be responsible for age-related aneuploidy. Dehydroepiandrosterone (DHEA) can improve the ovarian reserve and reduce the rate of aneuploidy, but the relationship between DHEA and cohesin levels in oocytes is still unknown. The aim of the current study was to evaluate the effect of the supplement DHEA on ovarian function, including the number of follicles and cohesin levels in oocytes. C57BL/6J mice at 3 weeks, 6 weeks, 12 weeks, 6 months, and 10 months of age were used to obtain a systematic view into follicle apoptosis and cohesin levels in oocytes. Nine-month-old C57BL/6J mice were administered saline (n = 5), 17β-estradiol (100 µg/kg per day, n = 5), or DHEA (5mg/Kg per day, n = 5). After 4 weeks, aged mice were weighed and sacrificed, and ovarian tissue samples were prepared. Anti-VASA staining and HE staining were used to count the number of follicles. Anti-γH2AX staining and TUNEL were used to measure follicle apoptosis and immunofluorescent staining was used to detect the levels of three oocyte cohesin subunits: REC8, SMC1β, and SMC3. Administration of the supplements 17β-estradiol and DHEA to aged mice increased the number of primordial and primary follicles and decreased the age-related apoptosis of follicles. Levels of the cohesin subunits REC8 and SMC1β declined with age, but DHEA and 17β-estradiol tended to delay that decline. The supplement DHEA increased the number of primordial and primary follicles in aged mice by inhibiting follicle apoptosis and tended to delay the decrease in cohesin levels in oocytes.

17-β-estradiol up-regulates apolipoprotein genes expression during osteoblast differentiation in vitro

Apolipoproteins are of great physiological importance and are associated with different diseases. Many independent studies of patterns of gene expression during osteoblast differentiation have been described, and some apolipoproteins have been induced during this process. 17-β-estradiol (E2) may enhance osteoblast physiological function. However, no studies have indicated whether E2 can modulate the expression of apolipoproteins during osteoblast differentiation in vitro. The aim of the current study was to observe the regulation of apolipoprotein mRNA expression by E2 during this process. Primary osteoblasts were collected from the calvaria of newborn mice and were subjected to osteoblast differentiation in vitro with serial concentrations of E2. RNA was isolated on days 0, 5, and 25 of differentiation. Real-time PCR was performed to analyze the levels of apolipoprotein mRNA. Results showed that during osteoblast differentiation all of the apolipoprotein genes were up-regulated by E2 in a dose-dependent manner. Moreover, only ApoE was strongly induced during the mineralization of cultured osteoblasts. This result suggests that ApoE might be involved in osteoblast differentiation. The hypothesis is that E2 promotes osteoblast differentiation by up-regulating ApoE gene expression, though further study is needed to confirm this hypothesis.

Dehydroepiandrosterone improves the ovarian reserve of women with diminished ovarian reserve and is a potential regulator of the immune response in the ovaries

Diminished ovarian reserve (DOR) has a high morbidity rate worldwide and has become a primary cause of infertility. DOR is a daunting obstacle in in vitro fertilization (IVF) and leads to poor ovarian response, high cancellation rates, poor IVF outcomes, and low pregnancy rates. Abnormal autoimmune function may also contribute to DOR. Dehydroepiandrosterone (DHEA) is a C19 androgenic steroid. DHEA is secreted mainly by the adrenal gland, and its secretion declines with age. DHEA has a pro-inflammatory immune function that opposes cortisol. The cortisol to DHEA ratio increases with age, which may lead to decreased immune function. DHEA supplementation helps improve this situation. A number of clinical case control studies and several prospective randomized clinical trials have observed a positive effect of DHEA supplementation in women with DOR. However, the underlying mechanism by which DHEA improves ovarian reserve remains unclear. DHEA functions as an immune regulator in many different tissues in mammals and may also play an important role in regulating the immune response in the ovaries. The conversion of DHEA to downstream sex steroids may allow it to regulate the immune response there. DHEA can also enhance the Th1 immune response and regulate the balance of the Th1/Th2 response. DHEA treatment can increase selective T lymphocyte infiltration in mice, resulting in a decline in the CD4+ T lymphocyte population and an upregulation of the CD8+ T lymphocyte population in ovarian tissue, thus regulating the balance of CD4+/CD8+ T cells. This review mainly focuses on how DHEA supplementation affects regulation of the immune response in the ovaries.

Heterozygous deletion of LRP5 gene in mice alters profile of immune cells and modulates differentiation of osteoblasts

Skeletal homeostasis is dynamically influenced by the immune system. Low density lipoprotein receptor-related protein-5 (LRP5) is a co-receptor of the Wnt signaling pathway, which modulates bone metabolism in humans and mice. Immune disorders can lead to abnormal bone metabolism. It is unclear whether and how LRP5 alters the balance of the immune system to modulate bone homeostasis. In this study, we used primary osteoblast to detect the differentiation of osteoblasts in vitro, the immune cells of spleen and bone marrow of 6-month old LRP5 heterozygote (HZ) and wild-type (WT) mice were analyzed by Flow cytometry. We found that LRP5+/- could influence the differentiation of osteoblasts by decreasing the mRNA level of Osterix, and increasing the mRNA level of Runx2 and the ratio of receptor activator for nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG). In the LRP5+/- mice, percentages of NK cells, CD3e+ cells, and CD8a+ T cells were increased in both spleen and bone marrow, and percentages of CD106+ cells and CD11c+ cells were increased in spleen while decreased in bone marrow, conversely, CD62L+ cells were decreased in spleen while increased in bone marrow compared to the WT mice. Percentages of CD4+ cells, CD14+ cells, and CD254+ cells were increased in the spleen, and CTLA4+ cells were increased in the bone marrow of the LRP5+/- mice. The mRNA level of Wnt signaling molecules such as β-catenin, and c-myc were decreased and APC was increased in spleen lymphocytes and bone marrow lymphocytes, and the mRNA level of Wnt3a was decreased in spleen lymphocytes while no change in bone marrow lymphocytes was seen with silencing LRP5 by specific small interfering RNA. In conclusion, heterozygous deletion of the LRP5 gene in mice could alter the profile of the immune cells, influence the balance of immune environment, and modulate bone homeostasis, which might present a potential mechanism to explore the Wnt signaling pathway in the modulation of the immune system.