Icariin, a phytoestrogen, exerts rapid estrogenic actions through crosstalk of estrogen receptors in osteoblasts.

Icariin, a flavonoid glycoside derived from Epimedium brevicornum Maxim, exerts bone protective effects via estrogen receptors (ERs). This study aimed to investigate the role of ER-α66, ER-α36, and GPER in bone metabolism in osteoblasts following treatment with icariin. Human osteoblastic MG-63 cells and osteoblast-specific ER-α66 knockout mice were employed. The ERs crosstalk in the estrogenic action of icariin was evaluated in ER-α66-negative human embryonic kidney HEK293 cells. Icariin, like E2, regulated ER-α36 and GPER protein expression in osteoblasts by downregulating them and upregulating ER-α66. ER-α36 and GPER suppressed the actions of icariin and E2 in bone metabolism. However, the in vivo administration of E2 (2 mg/kg/day) or icariin (300 mg/kg/day) restored bone conditions in KO osteoblasts. ER-α36 and GPER expression increased significantly and rapidly activated and translocated in KO osteoblasts after treatment with E2 or icariin. ER-α36 overexpression in KO osteoblasts further promoted the OPG/RANKL ratio induced by E2 or icariin treatment. This study showed icariin and E2 elicit rapid estrogenic responses in bone through recruiting ER-α66, ER-α36, and GPER. Notably, in osteoblasts lacking ER-α66, ER-α36, and GPER mediate the estrogenic effects of icariin and E2, while in intact osteoblasts, ER-α36 and GPER act as negative regulators of ER-α66.

Structural characterization and protective effect against renal fibrosis of polysaccharide from Ligustrum lucidum Ait.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait., is a traditional Chinese medicine that has been used for tonifying the kidney and liver for decades. AIM OF THE STUDY: This study aimed to explore and identify polysaccharides from FLL and elucidate its protective effect against renal fibrosis. MATERIALS AND METHODS: Polysaccharides were extracted and isolated from FLL. The purified fraction was identified by serial phytochemical work, such as gel-permeation chromatography, ion chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance. Mice with unilateral ureteral obstruction (UUO) were applied as a renal fibrosis model. The male UUO mice were pretreated with heteropolysaccharide (Poly) 1 week prior to surgery and continuously treated for 7 days after the operation. Renal fibrosis was assessed by Periodic Acid-Schiff (PAS) staining and Masson's trichrome staining in paraffin-embedded slides. The murine mesangial cells SV40-MES13 upon angiotensin II (Ang II) treatment were developed as an in vitro fibrotic model. The cells were treated by Poly in the presence of Ang II. Molecular expression was detected by RT-PCR, immunoblotting, and immunofluorescence staining. RESULTS: We identified a heteropolysaccharide composed of arabinose and galactose (molar ratio, 0.73:0.27) with a predicted chemical structure characterized by a backbone composed of 1,5-α-Araf, 1,3,5-α-Araf, 1,6-α-Galp, and 1,3,6-ß-Galp and side chains comprised of T-α-Araf, T-α-Arap, and 1,3-α-Araf. Pretreatment of UUO mice with Poly effectively alleviated glomerulosclerosis and tubulointerstitial fibrosis. Moreover, Poly pretreatment down-regulated the expression of extracellular matrix (ECM) protein fibronectin (FN), profibrotic factor VEGF, proinflammatory cytokines MCP-1 and Rantes in the obstructed kidney. Similarly, the incubation of SV40-MES13 cells with Poly significantly inhibited Ang II-induced elevation in accumulation and expression level of FN and attenuated Ang II-evoked up-regulation in protein expression of MCP-1 and Rantes. CONCLUSIONS: Our study isolated and identified a naturally occurring heteropolysaccharide in FLL and revealed its potential in protecting the kidneys from fibrosis.

Vitamin D Supplementation Improves Handgrip Strength in Postmenopausal Women: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

Introduction: In postmenopausal women, vitamin D deficiency (as defined by the circulating level of 25(OH)D being below 20 ng/ml (50 nmol/L)) is a regular occurrence. The effect of vitamin D supplementation on the muscle function of postmenopausal women has been controversial. This systematic review and meta-analysis of randomized controlled trials (RCTs) examines and summarizes the effects of vitamin D supplementation on the muscular strength and mobility of postmenopausal women. Methods: RCTs that met the inclusion criteria for this study were identified by searching PubMed, EMBASE, and the Cochrane Library. Postmenopausal women who were included in the study were exposed to RCTs assessing the effectiveness of vitamin D supplements. Meta-analysis data were extracted by two independent reviewers and screened for methodological quality. RCTs that did not meet the minimum requirement for assessment were excluded. In the meta-analysis, the effect size (weighted mean differences, WMD) of handgrip strength (HGS) and timed-up and go test (TUG) with a 95% confidence interval (CI) was obtained to compare reported results across the included RCTs. Results: A total of 19 trials were included in this systematic review, among which 13 trials were eligible for the meta-analysis. In the 13 included studies, supplementing with vitamin D produced a weighted mean difference of 0.876 kg (95% CI = 0.180 to 1.571, P = 0.014, I2 = 68.5%) for HGS, a measurement of muscle strength. However, an insignificant decrease of 0.044 s was observed after analyzing the TUG (95% CI = -0.979 to 0.892, P = 0.927, I2 = 95%). According to subgroup analysis, vitamin D supplementation increased HGS in patients over the age of 60 (P = 0.001), in those without calcium supplementation (P = 0.032), and in those whose baseline vitamin D level was greater than 75 nmol/L (30 ng/ml) (P = 0.003). Conclusions: Taking into account the studies in this systematic review, vitamin D supplementation improved muscle strength in postmenopausal women. However, an insignificant result was demonstrated in terms of mobility after vitamin D supplementation.

Water Extract of Rhizoma Drynaria Selectively Exerts Estrogenic Activities in Ovariectomized Rats and Estrogen Receptor-Positive Cells.

Our previous study demonstrated that the bone protective actions of herbal medicine Rhizoma Drynariae (Gusuibu, RD) were mainly mediated by flavonoid phytoestrogens via estrogen receptors, raising concerns about the safety of using RD as it may induce estrogen-like risk-benefit profile and interact with other ER ligands, such as selective estrogen receptor modulators (SERMs), when coadministered. The present study evaluated the estrogenic activities of RD and its potential interaction with tamoxifen, a SERM, in estrogen-sensitive tissues by using mature ovariectomized (OVX) rats and ER-positive cells. Similar to but weaker than tamoxifen, RD at its clinical dose dramatically ameliorated OVX-induced changes in bone and dopamine metabolism-related markers in OVX rats. However, tamoxifen, but not RD, induced uterotrophic effects. No significant alteration in mammary gland was observed in OVX rats treated with RD, which was different from the inhibitory actions of tamoxifen. The two-way ANOVA results indicated the interactions between RD and tamoxifen in the bone, brain, and uterus of OVX rats while RD did not alter their responses to tamoxifen. Our results demonstrate that RD selectively exerts estrogenic actions in a different manner from tamoxifen. Moreover, RD interacts with tamoxifen without altering its effects in OVX rats.

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.

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.

A standardized extract of Danggui Buxue Tang decoction selectively exerts estrogenic activities distinctly from tamoxifen.

More and more menopausal women use Danggui Buxue Tang (DBT) for relieving their symptoms. Concerns for its safety have been raised as it contains phytoestrogen and acts via estrogen receptors (ERs). Our study aimed to determine whether DBT could selectively exert estrogenic activities and interact with tamoxifen in bone, brain, uterus, and breast by using ovariectomized (OVX) rats and ER-positive cells. In OVX rats, DBT induced a 31.4% increase in bone mineral density and restored the mRNA expression of dopamine biomarker in striatum, 3.32-fold for tyrosine hydrolase (p < .001) and 0.21-fold for dopamine transporter (p < .001), which was similar to tamoxifen; tamoxifen, but not DBT, increased uterus weight and Complement component 3 expression by more than twofold (p < .001); unlike tamoxifen, DBT induced mild proliferation in mammary gland. Two-way ANOVA indicated the interactions between them in OVX rats (p < .05) but DBT did not alter the responses to tamoxifen. DBT stimulated proliferation or differentiation and estrogen response element in MCF-7, MG-63, Ishikawa, and SHSY5Y cells and altered the effects of tamoxifen. In summary, DBT exerted estrogenic effects in tissue-selective manner, which was different from tamoxifen. DBT interacted with tamoxifen but did not significantly alter its effects in OVX rats.

Icariin ameliorates estrogen-deficiency induced bone loss by enhancing IGF-I signaling via its crosstalk with non-genomic ERα signaling.

BACKGROUND: Rapid, non-genomic estrogen receptor (ER) signaling plays an integral role in mediating the tissue selective properties of ER modulators. Icariin, a bone bioactive flavonoid, has been reported to selectively activate non-genomic ERα signaling in in vitro and in vivo studies. PURPOSE: The mechanisms underlying the estrogen-like bone protective effects of icariin are not fully understood, especially those that are related to insulin-like growth factor I (IGF-1) signaling. The bone protective effects of icariin were investigated in female mature ovariectomized (OVX) rats and the signaling of IGF-IR- ERα cross-talk was determined in osteoblastic cells. STUDY DESIGN AND METHODS: Icariin at 3 different dosages (50, 500 and 3000 ppm) were orally administrated to rats for 3 months through daily intake of phytoestrogen-free animal diets containing icariin. Bone marrow stromal cells (BMSCs) and osteoclast precursors from femurs were harvested for experiments and RNA-sequencing. The interactions between IGF-IR and non-genomic ERα signaling were examined in pre-osteoblastic MC3T3-E1 cells and mature osteoblasts differentiated from BMSCs. RESULTS: Our results show that chronic administration of icariin to OVX rats significantly protected them against bone loss at the long bone and lumbar spine without inducing any uterotrophic effects. Ex vivo studies using BMSCs and osteoclast precursors confirmed the stimulatory effects of icariin on osteoblastogenesis and its inhibitory effects on osteoclastogenesis, respectively. RNA-sequencing analysis of mRNA from BMSCs revealed that icariin at 500 ppm significantly altered IGF-1 signaling as well as PI3K-Akt pathways. Our results demonstrated for the first time the rapid induction of interactions between IGF-IR and ERα as well as IGF-IR signaling and the downstream Akt phosphorylation by icariin in MC3T3-E1 cells. The activation of ERα and Akt phosphorylation by icariin in MC3T3-E1 cells and the osteogenic effects of icariin on ALP activity in mature osteoblasts were shown to be IGF-IR-dependent. CONCLUSION: Our findings reveal that icariin activates both ERα and Akt via enhancing rapid induction of IGF-1 signaling in osteoblastic cells for osteogenesis and might be regarded as a novel pathway-selective phytoestrogen for management of postmenopausal osteoporosis.

Selective Estrogen Receptor Modulator-Like Activities of Herba epimedii Extract and its Interactions With Tamoxifen and Raloxifene in Bone Cells and Tissues.

Herba epimedii (HEP), a kidney-tonifying herb, has been commonly used alone or in formula for strengthening kidney function and treating bone disorders. Its bone protective activity has been demonstrated to be via estrogen receptor (ERs). HEP activates the phosphorylation of ERα in an estrogen response element- (ERE-) dependent manner. We examined the bone protective effects of HEP and its potential interactions with Selective Estrogen Receptor Modulators (SERMs, such as tamoxifen and raloxifene) as they act via the same ERs. Six-month-old mature Sprague Dawley sham-operated (Sham) or ovariectomized (OVX) rats were treated with either vehicle, 17ß-estradiol (1.0 mg/kg.day), tamoxifen (Tamo, 1.0 mg/kg.day), raloxifene (Ralo, 3.0 mg/kg.day), HEP (0.16 g/kg.day), or its combinations with respective SERMs (HEP + Tamo; HEP + Ralo) for 12 weeks. HEP and SERMs as well as their combinations significantly restored changes in bone mineral density (BMD), trabecular bone properties, and bone turnover biomarkers induced by ovarian sex hormone deficiency in ovariectomized rats. Besides the increase in serum estradiol, inhibition on follicle stimulating hormone (FSH) might also be involved in the osteoprotective activities of HEP and SERMs. HEP interacted with SERMs to protect bones from ovarian sex hormone deficiency without altering SERMs' bone protective activities. HEP neither induced changes in uterus weight nor altered the uterotrophic activity of SERMs in OVX rats. In human osteosarcoma MG-63 cells, HEP-treated serum (HEP-Ts) significantly promoted alkaline phosphatase (ALP) activity like the crude HEP extract did but did not stimulate ERE activity. Our study also reported that biologically activated HEP interacted with SERMs to promote ALP activity without altering the action of SERMs at most of the concentrations tested in MG-63 cells. HEP exerted bone protective activity and the use of HEP did not alter the bone protective activities of SERMs when they were used simultaneously in an estrogen-deficient rat model.

Natural Products as Potential Bone Therapies.

Demands for natural products, in the form of botanicals, dietary supplements, and herbal medicine, for management of chronic diseases are increasing globally. Natural products might be an alternative for the management of bone health to meet the demands of a growing aging population. Different types of natural products, including Chinese herbal medicine decoctions, herbs, and isolated phytochemicals, have been demonstrated to exert bone protective effects. The most common types of bone protective bioactives are flavonoids, stilbene, triterpenoids, coumestans, lignans, and phenolic acid. The actions of natural products can be mediated by acting systemically on the hormonal axis or locally via their direct or indirect effects on osteogenesis, osteoclastogenesis, as well as adipogenesis. Furthermore, with the use of metabolomic and microbiome approaches to understand the actions of natural products, novel mechanisms that involve gut-brain-bone axis are also revealed. These studies provide evidence to support the use of natural products as bone therapeutics as well as identify new biological targets for novel drug development.

Evaluation of anti-oxidant capacity of root of Scutellaria baicalensis Georgi, in comparison with roots of Polygonum multiflorum Thunb and Panax ginseng CA Meyer.

In Chinese communities, regular consumption of Chinese-medicated diets (CMD) (usually in the form of soup) is a traditional practice to promote health and prevent disease development. The overall improvement of health conditions is believed to be correlated with the anti-oxidant potentials of these herbs. Huangqin, roots of Scutellaria baicalensis Georgi (Lamiaceae), is one of the herbs commonly used in CMD. In this study, the anti-oxidant capacities of Huangqin extracts (water, ethanol and ether extracts) were evaluated and compared to commonly used CMD herbs, Heshouwu, roots of Polygonum multiflorum Thunb (Polygonaceae) and Renshen (or Ginseng), roots of Panax ginseng CA Meyer (Araliaceae). The anti-oxidant capacities were measured by using both cell-free assay [ferric reducing/anti-oxidant power (FRAP)] and biological methods [2,2'-azobis-(2-amidinopropane) (AAPH)-induced haemolysis assay and H(2)O(2)-induced cell damage on H9C2 cells]. Additionally, the total phenolic content was measured using Folin-Ciocalteu methods. Water extract of Huangqin has the highest anti-oxidant activities compared to the ethanol and ether extracts. A positive relationship between the anti-oxidant effects and total phenolic contents of extracts was demonstrated. This shows that Huangqin could be an effective dietary anti-oxidant that can be consumed regularly as a functional food for the prevention of oxidant/free radical-related diseases.

Folic acid consumption reduces resistin level and restores blunted acetylcholine-induced aortic relaxation in obese/diabetic mice.

Folic acid supplementation provides beneficial effects on endothelial functions in patients with hyperhomocysteinemia. However, its effects on vascular functions under diabetic conditions are largely unknown. Therefore, the effect(s) of folic acid (5.7 and 71 microg/kg/day for 4 weeks) on aortic relaxation was investigated using obese/diabetic (+db/+db) mice and lean littermate (+db/+m) mice. Acetylcholine-induced relaxation in +db/+db mice was less than that observed in +db/+m mice. The reduced relaxation in +db/+db mice was restored by consumption of 71 microg/kg folic acid. Acetylcholine-induced relaxation (with and without folic acid treatment) was sensitive to N(G)-nitro-L-arginine methyl ester, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, geldanamycin and triciribine. In addition, acetylcholine-induced relaxation was attenuated by resistin. The plasma level of resistin in +db/+db mice was sevenfold higher than that measured in +db/+m mice, and the elevated plasma level of resistin in +db/+db mice was reduced by 25% after treatment with 71 microg/kg folic acid. Folic acid slightly increased the ratio of reduced glutathione to oxidized glutathione in +db/+db mice. Moreover, folic acid caused a reduction in PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression, an increase in the phosphorylation of endothelial nitric oxide synthase (eNOS(Ser1177)) and Akt(Ser473), and an enhanced interaction of heat shock protein 90 (HSP90) with eNOS in both strains, with greater magnitude observed in +db/+db mice. In conclusion, folic acid consumption improved blunted acetylcholine-induced relaxation in +db/+db mice. The mechanism may be, at least partly, attributed to enhancement of PI3K/HSP90/eNOS/Akt cascade, reduction in plasma resistin level, down-regulation of PTEN and slight modification of oxidative state.

Folic acid supplementation modifies beta-adrenoceptor-mediated in vitro lipolysis of obese/diabetic (+db/+db) mice.

The effects of folic acid (5.7 and 71 microg/kg, 4 weeks) consumption on the beta-adrenoceptors (beta-ARs)-elicited lipolysis in vitro of the abdominal adipocytes of lean/control (+m/+db) and obese/diabetic (+db/+db) mice (female) were investigated. beta-AR agonists (salbutamol, a beta(2)-AR agonist; BRL 37344 and CGP 12177, beta(3)-AR agonists; adrenaline, a beta-AR agonist)-mediated lipolysis, beta(2)-, and beta(3)-ARs protein expression of the adipose tissues after folic acid consumption were evaluated. Our results demonstrate that a smaller magnitude of the basal (spontaneous) and the beta-AR agonists-triggered lipolysis was observed in +db/+db mice, and folic acid supplementation (71 microg/kg) resulted in an improvement of both the baseline and the beta-ARs-mediated lipolysis. In controls, a lower beta(2)-and beta(3)-ARs protein expression of the adipose tissues was detected in +db/+db mice, compared to +m/+db mice. In both strains fed with folic acid (71 microg/kg), a reduction of beta(2)-AR protein expression was observed compared to the respective controls. In +db/+db mice, folic acid (5.7 and 71 microg/kg) consumption caused a dose-dependent increase of beta(3)-AR protein expression compared to controls. We demonstrate that lipolysis elicited by beta-AR (beta(2)- and beta(3)-ARs) agonists was blunted in +db/+db mice. Folic acid consumption has significant modulatory effects on beta-ARs protein expression and lipolysis.