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Kaempferol is the active ingredient of Er‐Xian decoction (EXD), a traditional Chinese medicine formula used clinically to treat ovarian dysfunction, but the mechanism of kaempferol relieving age‐related diminished ovarian reserve (AR‐DOR) is still unclear. In this study, 36 volunteers and 78 DOR patients (37 patients with EXD treatment) were enrolled in the clinical research. Meanwhile, 32‐week‐old female mice were used to establish the AR‐DOR model, and these model mice were intragastrically administered with 100 mg/kg kaempferol in the presence or absence of 200 mg/kg geranylgeranylacetone (GGA) or 1 mg/kg geldanamycin (GDA). The effects of kaempferol on serum hormone levels and oxidative stress‐related indexes were detected by enzyme‐linked immunosorbent assay. Antral follicle count (AFC) was determined by hematoxylin–eosin staining. The protein levels of HSP90 and nuclear factor erythroid 2‐related factor 2 (NRF2) were assayed by Western blot. This study displayed that the serum anti‐Mullerian hormone (AMH) level in DOR patients with EXD treatment was higher than that in DOR patients without EXD treatment. Kaempferol treatment reversed the low levels of AMH, estradiol (E2), AFC, superoxide dismutase (SOD), and catalase (CAT), as well as the high levels of follicle‐stimulating hormone (FSH), reactive oxygen species (ROS), and malonaldehyde (MDA). The results showed that HSP90 was predicted to have high affinity with kaempferol, and its expression was inhibited by kaempferol, while the expression of NRF2, the target of HSP90, was up‐regulated by kaempferol. However, the above effects of kaempferol were reversed by GGA. On the contrary, GDA enhanced the therapeutic effects of kaempferol on AR‐DOR mice. Moreover, the treatment of kaempferol resulted in a reduction in the phosphorylation level of heat shock factor 1 (HSF1), the transcription factor associated with HSP90, and an increase in the phosphorylation level of Src, a client protein of HSP90. In summary, kaempferol exerts an antioxidant effect on AR‐DOR by inhibiting HSP90 expression to up‐regulate NRF2 expression. This study provides a theoretical basis for the clinical application of kaempferol in AR‐DOR. [ABSTRACT FROM AUTHOR]

Osteoporosis (OP) is a systemic disease susceptible to fracture due to the decline of bone mineral density and bone mass, the destruction of bone tissue microstructure, and increased bone fragility. At present, the treatments of OP mainly include bisphosphonates, hormone therapy, and RANKL antibody therapy. However, these treatments have observable side effects and cannot fundamentally improve bone metabolism. Currently, the prescription of herbal medicine and their derived proprietary Chinese medicines are playing increasingly important roles in the treatment of OP due to their significant curative effects and few side effects. Among these prescriptions, Gushukang Granules (GSK), Xianling Gubao Capsules (XLGB), and Er-xian Decoction (EXD) are widely employed at the clinic on therapy of OP, which also is in line with the compatibility principle of “different treatments for the same disease” in herbal medicine. However, at present, the functional interpretation of “different treatments for the same disease” in herbal medicine still lacks systematic quantitative research, especially on the detection of key component groups and mechanisms. To solve this problem, we designed a new bioinformatics model based on random walk, optimized programming, and information gain to analyze the components and targets to figure out the Functional Response Motifs (FRMs) of different prescriptions for the therapy of OP. The distribution of high relevance score, the number of reported evidence, and coverage of enriched pathways were performed to verify the precision and reliability of FRMs. At the same time, the information gain and target influence of each component was calculated, and the key component groups in all FRMs of each prescription were screened to speculate the potential action mode of different prescriptions on the same disease. Results show that the relevance score and the number of reported evidence of high reliable genes in FRMs were higher than those of the pathogenic genes of OP. Furthermore, the gene enrichment pathways in FRMs could cover 79.6, 81, and 79.5% of the gene enrichment pathways in the component-target (C-T) network. Functional pathway enrichment analysis showed that GSK, XLGB, and EXD all treat OP through osteoclast differentiation (hsa04380), calcium signaling pathway (hsa04020), MAPK signaling pathway (hsa04010), and PI3K-Akt signaling pathway (hsa04151). Combined with experiments, the key component groups and the mechanism of “different treatments for the same disease” in the three prescriptions and proprietary Chinese medicines were verified. This study provides methodological references for the optimization and mechanism speculation of Chinese medicine prescriptions and proprietary Chinese medicines.

Asthenozoospermia (AZS) remains a significant clinical problem of male factor infertility. Er-Xian decoction (EXD) is a traditional Chinese medicine with potent antioxidant activity to treat AZS. To investigate the protective effects of EXD on sperm motility and deglycase (DJ)-1 expression in AZS model rats. Sixty mature male Sprague-Dawley rats (200 – 250 g) were randomized into five equally sized groups, including ornidazole (ORN)-induced AZS model group, or L-carnitine (0.1 g/kg) treated group or EXD group (7.5, 15, or 30 g crude drug/kg). Oxidative stress was assessed by measuring superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px). DJ-1 expression in testis and epididymis tissue was measured via qRT-PCR, Western blotting, and immunofluorescence staining. Hematoxylin and eosin staining was used to gauge morphological changes of testis and epididymis. Sperm motility was significantly reduced theAZS model group, while increased in the low-, intermediate-, and high-dose EXD treatment groups by 45.51%, 49.43%, and 58.31%, respectively (P < 0.001), which with a similar increase of 57.21% being observed in the L-carnitine treatment group. Relative to the control group, oxidative stress indices were significantly altered in AZS model rats, which exhibited significant reductions in SOD and GSH-Px levels and significantly increased MDA levels (49.44 ± 1.38 U/ml, 14.02 ± 0.70 U/ml, and 26.37 ± 1.03 nmol/ml, respectively). After EXD treatment, oxidative stress indexes were significantly improved relative to those in these model rats, with high-dose EXD yielding more significant improvements in these oxidative stress indices relative to L-carnitine treatment. While AZS model rats exhibited morphological abnormalities, tissue disorder, and reduced cell counts in the testis and epididymis, these were reversed by EXD treatment in a dose-dependent manner. EXD treatment was also associated with a significant increase in DJ-1 protein expression in testis and epididymis tissue samples relative to the levels observed in AZS model rats. EXD is firstly reported could significantly improve sperm motility in AZS rats and is more effective at higher dosage, even better than L-carnitine. The protective effect of EXD on sperm motility is based on the DJ-1 expression. [ABSTRACT FROM AUTHOR]

Abstract Background Er-Xian decoction (EXD), a formula of Chinese medicine, is often used to treat menopausal syndrome in China. The aim of the present study was to explore the potential cardioprotective mechanism of EXD against myocardial injury in an ovariectomy-induced menopausal rat model. Methods We divided the female Wistar rats into ovariectomy group and sham operation group (SHAM group). The ovariectomized (OVX) rats received treatment of vehicle (OVX group), EXD (EXD group) or 17β-estradiol (E2 group). After 12-week of treatment, the level of estradiol in serum was detected using an electrochemiluminescence immunoassay, and electrophysiologic changes in myocardial action potentials (AP) were evaluated using intracellular microelectrode technique. Changes in the histopathology of the left ventricle and the ultrastructure of the cardiomyocytes were observed by hematoxylin and eosin (HE) staining and transmission electronmicroscopy to assess myocardial injury. Microarrays were applied for the evaluation of gene expression profiles in ventricular muscle of the OVX and EXD rats. Further pathway analyses of the differential expression genes were carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG). And real-time quantitative RT-PCR (qRT-PCR) was used for verification of the key findings. Results The results from electrophysiological and histomorphological observations demonstrated that EXD had a substantial myocardial protective effect. The EXD-treated rats, in comparison with the OVX rats, demonstrated up-regulated expression of 28 genes yet down-regulated expression of 157 genes in the ventricular muscle. The qRT-PCR assay validated all selected differential expression genes. The KEGG pathway analysis showed that the down-regulated genes were relevant to cardiomyopathy and myocardial contractility. EXD could decrease the mRNA expressions of cardiac myosin (Myh7, Myl2) and integrin (Itgb5) in the ventricular myocardium. Conclusion EXD had a protective effect against myocardial injury in OVX rats, and this cardioprotective effect may be associated with modulation of the expression of cardiac myosin or integrin at the mRNA level.

Background: Er-Xian decoction (EXD), a formula of Chinese medicine, is often used to treat menopausal syndrome in China. The aim of the present study was to explore the potential cardioprotective mechanism of EXD against myocardial injury in an ovariectomy-induced menopausal rat model. Methods: We divided the female Wistar rats into ovariectomy group and sham operation group (SHAM group). The ovariectomized (OVX) rats received treatment of vehicle (OVX group), EXD (EXD group) or 17β-estradiol (E2 group). After 12-week of treatment, the level of estradiol in serum was detected using an electrochemiluminescence immunoassay, and electrophysiologic changes in myocardial action potentials (AP) were evaluated using intracellular microelectrode technique. Changes in the histopathology of the left ventricle and the ultrastructure of the cardiomyocytes were observed by hematoxylin and eosin (HE) staining and transmission electronmicroscopy to assess myocardial injury. Microarrays were applied for the evaluation of gene expression profiles in ventricular muscle of the OVX and EXD rats. Further pathway analyses of the differential expression genes were carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG). And real-time quantitative RT-PCR (qRT-PCR) was used for verification of the key findings. Results: The results from electrophysiological and histomorphological observations demonstrated that EXD had a substantial myocardial protective effect. The EXD-treated rats, in comparison with the OVX rats, demonstrated up-regulated expression of 28 genes yet down-regulated expression of 157 genes in the ventricular muscle. The qRT-PCR assay validated all selected differential expression genes. The KEGG pathway analysis showed that the down-regulated genes were relevant to cardiomyopathy and myocardial contractility. EXD could decrease the mRNA expressions of cardiac myosin (Myh7, Myl2) and integrin (Itgb5) in the ventricular myocardium. Conclusion: EXD had a protective effect against myocardial injury in OVX rats, and this cardioprotective effect may be associated with modulation of the expression of cardiac myosin or integrin at the mRNA level. [ABSTRACT FROM AUTHOR]

It is very important to rapidly discover and identify the multiple components of traditional Chinese medicine (TCM) formula. High performance liquid chromatography with high resolution tandem mass spectrometry (HPLC-HRMS/MS) has been widely used to analyze TCM formula and contains multiple-dimension data including retention time (RT), high resolution mass (HRMS), multiple-stage mass spectrometric (MS n ), and isotope intensity distribution (IID) data. So it is very necessary to exploit a useful strategy to utilize multiple-dimension data to rapidly probe structural information and identify chemical compounds. In this study, a new strategy to initiatively use the multiple-dimension LC–MS data has been developed to discover and identify unknown compounds of TCM in many styles. The strategy guarantees the fast discovery of candidate structural information and provides efficient structure clues for identification. The strategy contains four steps in sequence: (1) to discover potential compounds and obtain sub-structure information by the mass spectral tree similarity filter (MTSF) technique, based on HRMS and MS n data; (2) to classify potential compounds into known chemical classes by discriminant analysis (DA) on the basis of RT and HRMS data; (3) to hit the candidate structural information of compounds by intersection sub-structure between MTSF and DA (M,D-INSS); (4) to annotate and confirm candidate structures by IID data. This strategy allowed for the high exclusion efficiency (greater than 41%) of irrelevant ions in er-xian decoction (EXD) while providing accurate structural information of 553 potential compounds and identifying 66 candidates, therefore accelerating and simplifying the discovery and identification of unknown compounds in TCM formula. [ABSTRACT FROM AUTHOR]

A metabonomics method using 1H nuclear magnetic resonance spectroscopy (1HNMR) was applied to obtain a systematic view of the development and progression of postmenopausal osteoporosis. Using partial least squares discriminant analysis (PLS-DA), 26 and 34 characteristic resonances were found respectively in urine and plasma of ovariectomized rats (Variable importance, VIP value ≥1.0), and the significant altered metabolites identified in the plasma and urine were 10 and 9, respectively. Changes in these metabolites were related to the pathways of lipid, energy and amino acid metabolism, some of which involved the oxidative system. The described method was also used to analyze the therapeutic effects of Er-Xian Decoction (EXD), a traditional Chinese medicine widely used in the clinical treatment of osteoporosis in China. The results showed that EXD administration could provide satisfactory effects on osteoporosis through partially regulating the perturbed pathways of lipid, energy and amino acid metabolism and improving the anti-oxidative ability.

Er-Xian Decoction (EXD) has been used for the treatment of osteoporosis disorders, menopausal syndrome, and other aging diseases in the Chinese traditional healthcare system. However, the targets and mechanism of action have not been clarified. This study was designed to investigate the effects and possible target proteins of EXD on osteoblastic UMR-106 cells and osteoclasts induced from RAW264.7 cells using the proteomic analysis technique. We found that EXD at a concentration of 50–200 μg/mL significantly enhanced osteoblastic UMR-106 cell proliferation, alkaline phosphatase (ALP) activity and formation of bone nodules, and decreased tartrate-resistant acid phosphatase (TRAP) activity and the bone resorption action of osteoclasts induced from RAW 264.7 cells. In EXD-treated osteoblasts, there were increases in the expression of heat-shock protein 1, high mobility group protein (Hmgb1), acidic ribosomal phosphoprotein P0, histone 2, carbonyl reductase 1, ATP synthase, aldolase A, and Rho GDP dissociation inhibitor (GDI)-alpha; and reduction in the expression of carbonic anhydrase 3, prohibitin, hemiferrin, far upstream element (FUSE)-binding protein. In EXD-treated osteoclasts, there were increases in the expression of vimentin, protein disulfide isomerase associated 3 and alpha-fetoprotein; and reduction in the expression of calnexin. These results indicated that EXD modulates bone metabolism through regulation of osteoblastic proliferation, apoptosis, and cell activation, and osteoclastic protein folding and aggregation.

Er-xian Decoction (EXD) is a well-known prescription widely used to prevent and treat climacteric syndrome and osteoporosis in China. BK channel positively affects osteoblast bone formation in vitro. However, it is still unclear whether the effect of EXD on promoting osteoblasts osteogenic differentiation is related to BK channel. The study is aimed at determining whether the EXD-containing serum promotes the proliferation of osteoblasts and their differentiation through BK channel. The chemical compounds of EXD were analyzed by UPLC-Q-TOF/MS. An osteogenic induction medium (OM) was used to induce MC3T3-E1 cells' osteogenic differentiation. The effects of EXD-containing serum and tetraethylammonium (TEA) on the proliferation activity of Mc3t3-e1 cells were detected by CCK-8 assay. ALP activity was determined by an alkaline phosphatase kit. The protein expression (BMP2, OPG, and COL1) was analyzed by Western blot, and the mRNA expression (Runx2, OPG, and BMP2) was assessed by real-time PCR. Alizarin red was used to stain the mineralized region of osteoblasts. In addition, we analyzed the relationship between BK channel and its downstream PTEN/Akt/Foxo1 signaling pathway. 72 compounds were identified by UPLC-Q-TOF/MS analysis in EXD. Mangiferin, ferulic acid, berberine, and icariin were main active components of EXD. EXD-containing serum could enhance the cell viability of MC3T3-E1 cells by decreasing the expression of BKα protein. EXD-containing serum increased ALP activity and calcium nodule formation of Mc3t3-e1 cells, promoted the protein expression of BKα, COL1, BMP2, OPG, and the mRNA expression of RUNX2, OPG, and BMP2, however, these effects can be reversed after adding TEA. In addition, EXD-containing serum could upregulate phosphorylation of Akt and Foxo1 in osteogenic-induced Mc3t3-e1 cells, and lower the expression of PTEN. And these effects of EXD-containing serum could be reduced by TEA. The effect of EXD-containing serum on promoting cell proliferation and osteogenic differentiation of Mc3t3-e1 cells might be linked to the regulation of BK channel. [Display omitted] • 72 chemical components were identified in EXD by UPLC-Q-TOF/MS. • EXD-containing serum promotes Mc3t3-e1 cell proliferation by inhibiting BKα expression. • EXD-containing serum promotes osteoblasts osteogenic differentiation by increasing BKα expression. • EXD-containing serum activates PTEN/Akt/Foxo1 signaling pathway through BK channel to accelerate bone formation. [ABSTRACT FROM AUTHOR]

Er-Xian decoction (EXD) is a traditional Chinese medicine (TCM) formula used to treat osteoporosis (OP). However, the anti-OP mechanism of EXD has not yet been fully elucidated. The study aimed to verify the anti-OP effect of EXD and to explore its underlying mechanism. The anti-OP targets and mechanisms of EXD were predicted by network pharmacological analysis. Then, an ovariectomized (OVX) rat model was established to validate the key anti-OP mechanism of EXD. Firstly, the therapeutic effect of EXD on OP was confirmed using micro-CT bone analysis, pathological observation, and ELISA detection. Secondly, serum metabolites related to key biological processes were detected using an automatic biochemical analyzer and GC-MS. Finally, ELISA, qRT-PCR, and western blot were utilized to further explore the potential key anti-OP pathway of EXD. A total of 159 anti-OP targets of EXD were identified. Functional annotation revealed that OP treatment using EXD was associated with lipid metabolism, fatty acid (FA) metabolism, and PI3K/AKT signaling pathway. Experimental studies confirmed that EXD ameliorated ovariectomy-induced bone loss and bone microstructure deterioration. EXD treatment also upregulated the level of serum estrogen and downregulated the level of OC, PⅠNP, CTX-1, TC, and LDL-C. Besides, principal component analysis (PCA) and heat map of serum FAs distinguished OVX rats from the SHAM and EXD groups. Serum concentrations of important n-3 FAs, including C20:3N3, C20:5N3, and C22:5N3, were significantly increased in the EXD group. The increased stearoyl-CoA desaturase 1 (SCD1) index 1 and index 2 in the OVX group were reversed by EXD administration. Additionally, EXD reversed the decreased serum IGF1 level and tibia IGF1R, PI3K, and AKT expression in OVX rats. EXD ameliorated ovariectomy-induced bone loss by modulating lipid metabolism, FA metabolism, and IGF1/PI3K/AKT pathway. [Display omitted] • Er-Xian decoction can ameliorate ovariectomy-induced bone loss. • Er-Xian decoction ameliorated ovariectomy-induced bone loss by regulating lipid metabolism and fatty acid metabolism. • Er-Xian decoction ameliorated ovariectomy-induced bone loss by activating IGF1/PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Background: Er-Xian Decoction (EXD) is one of the traditional Chinese medicine (TCM) with unique effect on osteoporosis, menopausal syndrome and delayed puberty in China for many years. Objective: We aim to evaluate the potential activity of starting hypothalamic-pituitary-testicular (HPT) axis of male rats with delayed puberty. Materials and Methods: Delayed puberty model of male Sprague- Dawley (SD) rats were established with soy isoflavones (90 mg·kg-1) and were treated by EXD extract at doses of 5, 10 g·kg-1 or Testosterone undecanoate (TU) for 8 weeks. Body weight, body length, testis weight, T, E2 and luteinizing hormone (LH) in serum, gonadotropin releasing hormone (GnRH) in hypothalamus, follicle stimulating hormone (FSH) and LH in pituitary gland were determined by ELISA. Immunohistochemistry was used to detect LH in pituitary gland. Results: Soy isoflavones could significantly decrease body weight, body length, testicular organ coefficient T in serum, GnRH in hypothalamus, FSH and LH in pituitary gland. Both of EXD and TU could improve the condition. E2 and LH in serum of all groups were non-significance of difference (P > 0.05). The immunohistochemical results were well consistent with LH in pituitary gland. Conclusion: The results of the present research indicate that EXD extract is effective to start the HPT axis in puberty and can significantly improve sexual developmental inhibition caused by soy isoflavones. [ABSTRACT FROM AUTHOR]

Er-xian Decoction (EXD), containing Herba epimedii Maxim (HEP) and Curculigo orchioides Gaertn (XM) as principal drugs, is a traditional Chinese medicine (TCM) formula prescribed for the treatment of postmenopausal osteoporosis. In the present study, the in vivo anti-osteoporosis effects of EXD, HEP and XM on four-month-old ovariectomized (OVX) Sprague-Dawley rats were investigated. Micro-computed tomography analysis showed that EXD could significantly improve the micro-architectural parameters (BMD, BV/TV, Tb.N, Tb.Th, and Tb.Sp) of trabecular bone in the distal femur and proximal tibia in OVX rats (p < 0.05). The biomechanical parameters of the distal femur in rats treated with EXD were also improved significantly (p < 0.05 vs. OVX group). The in vivo efficacy of EXD was found to be superior to HEP or XM alone in improving the bone properties of OVX rats. Treatment of rat osteoblastic-like UMR-106 cells with EXD, HEP, and XM significantly promoted the cell proliferation rate (p < 0.05) with the most promising effects observed in cells treated with EXD (p < 0.001). The proliferative effect in UMR-106 cells induced by EXD, HEP, and XM were abolished in the presence of the estrogen antagonist, ICI182780, suggesting that their effects were mediated by estrogen receptor (ER). Additionally, EXD could activate ER-α and ER-β mediated estrogen-response element (ERE)-dependent luciferase activity as well as phosphorylate ER-α at serine 118 in UMR-106 cells. Taken together, EXD offered better osteoprotective effects than its single principal herb, and the beneficial effects of EXD in preventing bone deteriorations are, at least partially, through the ER signaling pathway. [ABSTRACT FROM AUTHOR]

A metabonomics method using 1H nuclear magnetic resonance spectroscopy (¹HNMR) was applied to obtain a systematic view of the development and progression of postmenopausal osteoporosis. Using partial least squares discriminant analysis (PLS-DA), 26 and 34 characteristic resonances were found respectively in urine and plasma of ovariectomized rats (Variable importance, VIP value ≥1.0), and the significant altered metabolites identified in the plasma and urine were 10 and 9, respectively. Changes in these metabolites were related to the pathways of lipid, energy and amino acid metabolism, some of which involved the oxidative system. The described method was also used to analyze the therapeutic effects of Er-Xian Decoction (EXD), a traditional Chinese medicine widely used in the clinical treatment of osteoporosis in China. The results showed that EXD administration could provide satisfactory effects on osteoporosis through partially regulating the perturbed pathways of lipid, energy and amino acid metabolism and improving the anti-oxidative ability. [ABSTRACT FROM AUTHOR]

Abstract: In this study, a sensitive and specific ultra-performance liquid chromatography–tandem mass method has been developed and validated for the identification and determination of 7 flavonoids in dog plasma for the first time: epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2″-O-rhamnosyl icariside II, and baohuoside I. Chromatographic separation was accomplished on an Agilent Zorbax-SB C18 column (50mm×2.1mm, 1.8μm) with a gradient elution system composed of 0.3% acetic acid and 0.3% acetic acid in acetonitrile at a flow rate of 0.4mL/min. Detection was based on a triple quadrupole mass spectrometer using a multiple reaction monitoring mode with an electrospray ionization source. All of the calibration curves showed good linearity (r >0.99) within the tested concentration ranges. The lower limits of quantification of the seven analytes were all lower than 0.0654ng/mL. The relative standard deviations for intra- and inter-batches of the seven analytes were less than 13.7% and 14.9%, respectively, at four concentration levels of quality control samples, and the recoveries were between 92.8% and 114.5%, respectively. In addition, the seven flavonoids were found to be stable in dog plasma samples under short- and long-term storage and processing conditions. The validated method was successfully applied to a bioequivalence study in dog plasma after the oral administration of extracts of Herba Epimedii and Er-Xian Decoction. [ABSTRACT FROM AUTHOR]

Er-Xian Decoction (EXD) has been used for the treatment of osteoporosis disorders, menopausal syndrome, and other aging diseases in the Chinese traditional healthcare system. However, the targets and mechanism of action have not been clarified. This study was designed to investigate the effects and possible target proteins of EXD on osteoblastic UMR-106 cells and osteoclasts induced from RAW264.7 cells using the proteomic analysis technique. We found that EXD at a concentration of 50-200 μg/mL significantly enhanced osteoblastic UMR-106 cell proliferation, alkaline phosphatase (ALP) activity and formation of bone nodules, and decreased tartrate-resistant acid phosphatase (TRAP) activity and the bone resorption action of osteoclasts induced from RAW 264.7 cells. In EXD-treated osteoblasts, there were increases in the expression of heat-shock protein 1, high mobility group protein (Hmgb1), acidic ribosomal phosphoprotein P0, histone 2, carbonyl reductase 1, ATP synthase, aldolase A, and Rho GDP dissociation inhibitor (GDI)-alpha; and reduction in the expression of carbonic anhydrase 3, prohibitin, hemiferrin, far upstream element (FUSE)-binding protein. In EXD-treated osteoclasts, there were increases in the expression of vimentin, protein disulfide isomerase associated 3 and alpha-fetoprotein; and reduction in the expression of calnexin. These results indicated that EXD modulates bone metabolism through regulation of osteoblastic proliferation, apoptosis, and cell activation, and osteoclastic protein folding and aggregation. [ABSTRACT FROM AUTHOR]

Abstract: Aim of the study: Er Xian Decoction (EXD), a traditional Chinese medicine formula, has long been used for the treatment of osteoporosis and menopausal syndrome in China. The present study was designed to investigate the antiosteoporotic constituents of EXD, and evaluate their antiosteoporotic effects in ovariectomized rats. Materials and methods: Osteoblasts in neonatal calvaria cultures and osteoclasts derived from rat marrow cells were used to bioactivity-guided screen the active constituents. The proliferation of osteoblast was assayed by MTT methods. The activity of ALP and TRAP was measured by p- nitrophenyl sodium phosphate assay. The antiosteoporotic effects of icariin (1), anemarsaponin B II (8) and berberine (6) were verified by using OVX rats model. The bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry using the small animal scan mode. The undecalcified longitudinal proximal tibial metaphysical (PTM) sections were cut and stained for the bone histomorphometric analysis. Results: Bioactivity-guided fractionation has led to the successful isolation of antiosteoporotic constituents, i.e., icariin (1), icariside I (2), baohuoside I (3), mangiferin (4), neomangiferin (5), berberine (6), anemarsaponin B (7), anemarsaponin BII (8), anemarsaponin C (9), anemarrhenasaponin I (10), rubiadin-1-methyl ether (11) and obaculactone (12) from EXD. Further study showed that icariin (1), anemarsaponin BII (8) and berberine (6) increased the BMD in ovariectomized rats, and icariin (1) not only increased the bone formation, but also inhibited bone resorption; anemarsaponin BII (8) mainly increased bone formation and berberine (6) only inhibited the bone resorption in ovariectomized rats. Conclusion: Our findings demonstrate that multiple ingredients are responsible for antiosteoporotic activity in traditional Chinese medicine formula Er-Xian decoction. [Copyright &y& Elsevier]

The purpose of this systemic review is to assess the efficacy of Er-xian decoction (EXD), a formula of Chinese medicine, in relieving menopausal symptoms. Seven databases were extensively retrieved. The Chinese electronic databases include VIP Information, CBMdisc, and CNKI. The English electronic databases include AMED, CINAHL, Cochrane Library, and MEDLINE. Randomized controlled trials using EXD as a main intervention were included in the study selection. The quality of studies was assessed by Jadad scale and the criteria referred in Cochrane reviewers' handbook. Two independent reviewers were responsible for data extraction and assessment. Discrepancies were rectified referring to the original articles. The efficacy of EXD treatment for menopausal symptoms was evaluated by meta-analysis. There were 154 articles retrieved according to the search strategy, 677 participants involved in the 5 studies that satisfied the selection criteria. Meta-analysis indicated that administration of EXD significantly relieved at least one menopausal symptom when compared to the control group at a 95% confidence interval (p < 0.01). The curing effect of EXD with all symptoms relieved was significant as compared with the control groups (p < 0.01). The results also indicated that the efficacy of EXD was better than the other non-menopausal hormone therapy (p < 0.01), while there was no significant difference between the EXD and menopausal hormone therapy groups. The EXD is effective in treating menopausal symptoms. However, owing to the low quality of the investigated studies, more randomized controlled trials are needed before evidence-based recommendation regarding the effectiveness of EXD in the management of menopausal symptoms can be provided. [ABSTRACT FROM AUTHOR]

Osteoporosis (OP) is a systemic disease susceptible to fracture due to the decline of bone mineral density and bone mass, the destruction of bone tissue microstructure, and increased bone fragility. At present, the treatments of OP mainly include bisphosphonates, hormone therapy, and RANKL antibody therapy. However, these treatments have observable side effects and cannot fundamentally improve bone metabolism. Currently, the prescription of herbal medicine and their derived proprietary Chinese medicines are playing increasingly important roles in the treatment of OP due to their significant curative effects and few side effects. Among these prescriptions, Gushukang Granules (GSK), Xianling Gubao Capsules (XLGB), and Er-xian Decoction (EXD) are widely employed at the clinic on therapy of OP, which also is in line with the compatibility principle of "different treatments for the same disease" in herbal medicine. However, at present, the functional interpretation of "different treatments for the same disease" in herbal medicine still lacks systematic quantitative research, especially on the detection of key component groups and mechanisms. To solve this problem, we designed a new bioinformatics model based on random walk, optimized programming, and information gain to analyze the components and targets to figure out the Functional Response Motifs (FRMs) of different prescriptions for the therapy of OP. The distribution of high relevance score, the number of reported evidence, and coverage of enriched pathways were performed to verify the precision and reliability of FRMs. At the same time, the information gain and target influence of each component was calculated, and the key component groups in all FRMs of each prescription were screened to speculate the potential action mode of different prescriptions on the same disease. Results show that the relevance score and the number of reported evidence of high reliable genes in FRMs were higher than those of the pathogenic genes of OP. Furthermore, the gene enrichment pathways in FRMs could cover 79.6, 81, and 79.5% of the gene enrichment pathways in the component-target (C-T) network. Functional pathway enrichment analysis showed that GSK, XLGB, and EXD all treat OP through osteoclast differentiation (hsa04380), calcium signaling pathway (hsa04020), MAPK signaling pathway (hsa04010), and PI3K-Akt signaling pathway (hsa04151). Combined with experiments, the key component groups and the mechanism of "different treatments for the same disease" in the three prescriptions and proprietary Chinese medicines were verified. This study provides methodological references for the optimization and mechanism speculation of Chinese medicine prescriptions and proprietary Chinese medicines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Liu, Yin, Li, Wu, Chen, Yu, Lu and Guan.)

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. The present study aimed to evaluate whether EXD selectively exerted estrogenic activities and interacted with Selective Estrogen Receptor Modulators (SERMs). 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. 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. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Objective: To investigate the therapeutic effect of Er-Xian decoction on the autoimmune premature ovarian failure (POF) in mice, and its regulatory mechanisms., Methods: Female BALB mice were treated with a single intraperitoneal injection of zona pellucida 3 (ZP3). One week later, mice received low (5 g/kg), moderate (10 g/kg) and high (20g/kg) doses of EXD by gastrogavage once daily for 90 d. Premarin (0.03 mg/kg) served as the positive control group. Serum levels of estradiol (E2), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured by enzyme-linked immunosorbent assay, lymphocyte subtypes were analyzed, and follicular structure differences were observed by hematoxylin and eosin staining. The main mechanisms of POF was investigated by immunohistochemical analysis., Results: Serum E2 levels in POF model mice were decreased, whereas FSH and LH levels were dramatically increased. Serum levels of LH and FSH were reduced in POF model mice treated with EXD (moderate and high doses) and premarin, while serum level of E2 were increased after POF model mice had been treated with EXD and premarin. The CD3+ T, CD4+ T, CD4+ T/CD8+ T ratio of mice in the positive control group and high and medium dose groups of EXD increased (P < 0.05), and the number of CD8 + T cells decreased significantly (P < 0.05) when compared with the model group. Bone morphogenetic protein 15 (BMP15) and Akt were repressed in autoimmune POF model mice, whereas high expression was observed in control mice and those treated with EXD (moderate and high doses) and premarin., Conclusion: EXD is effective in treating ZP3-induced POF in mice and increased expression of BMP-15, and Akt is represented in the mechanism accounting for this therapeutic effect.

To investigate the effect of Er-xian decoction on ovariectomy induced-learning and memory disability in rats based on the differences in hippocampus proteomic expression, the female rats were ovariectomized for perimenopausal status and then randomly divided into 3 groups, namely sham group, control group and Er-xian decoction group. The rats in sham group and control group were given normal saline by gavage everyday, the rats in Er-xian decoction group were given 12 g·kg⁻¹ of Er-xian decoction by gavage once a day. After treatment for 90 day, all of the animals were tested by Morris water maze, and all of the samples were collected for testing. The hippocampus proteins were detected by Nano-LC-LTQ-Orbitrap system, and identified by Protein Discovery software. The intensity of differential proteins were quantitated by SIEVE software，and the biological functions of protein were analyzed by DIVAD database. The results showed that Er-xian decoction could significantly improve the abilities of learning and memory. Compared with control group， 216 differential proteins with a consistent variation trend with the sham group were found in the Er-xian decoction group. And they mainly concentrated in calcium signaling pathway, Rab3A and Rab14 were key genes with the highest functional classification enrichment score in the gene group. Er-xian decoction could significantly improve the abilities of learning and memory in perimenopausal model rats. The invention effect of Er-xian decoction may be correlated with the calcium signaling pathway and Ras-related protein Rab-3A, and Ras-related protein Rab-14., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

This experiment was mainly aimed to investigate the effect of Er-xian decoction on osteoporosis and the femur proteomics in ovariectomized rats with osteoporosis. The female SD rats were randomly divided into sham operation group, model group, Alendronate group (1 mg•kg⁻¹), Er-xian decoction group (in dose of 8 g•kg⁻¹) according to their weight. The rats in sham operation group and model group were gavaged with normal saline; the rats in Alendronate group were gavaged with the Alendronate at the dose of 1 mg•kg⁻¹ and the rats in Er-xian decoction group were gavaged with Er-xian decoction at the dose of 8 g•kg⁻¹, once a day for continuous 90 days. Then the femoral bone mineral density (BMD) was detected. The femoral bone proteins were detected by NanoLC-LTQ-Orbitrap system, identified by Protein Discovery software, and the intensity of differentially expressed proteins were quantitated by SIEVE software. The results showed that Er-xian decoction could significantly improve femoral BMD in ovariectomized rats. As compared with model group, 41 differentially expressed proteins whose variation trend was consistent with the sham operation group, were found in Er-xian decoction group, mainly including biological oxidation related protein, signal transduction pathway related protein, proteins involved in aliphatic acid metabolism, cytoskeleton related protein, proteins involved in energy metabolism, and proteins involved in glucose metabolism etc. The osteoporosis could be prevented and cured by Er-xian decoction. The differentially expressed proteins such as carbonic anhydrase 2 and integrin β1 may be the action targets for Er-xian decoction., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Background: Er-Xian Decoction (EXD) is one of the traditional Chinese medicine (TCM) with unique effect on osteoporosis, menopausal syndrome and delayed puberty in China for many years., Objective: We aim to evaluate the potential activity of starting hypothalamic-pituitary-testicular (HPT) axis of male rats with delayed puberty., Materials and Methods: Delayed puberty model of male Sprague-Dawley (SD) rats were established with soy isoflavones (90 mg·kg(-1)) and were treated by EXD extract at doses of 5, 10 g·kg(-1) or Testosterone undecanoate (TU) for 8 weeks. Body weight, body length, testis weight, T, E2 and luteinizing hormone (LH) in serum, gonadotropin releasing hormone (GnRH) in hypothalamus, follicle stimulating hormone (FSH) and LH in pituitary gland were determined by ELISA. Immunohistochemistry was used to detect LH in pituitary gland., Results: Soy isoflavones could significantly decrease body weight, body length, testicular organ coefficient T in serum, GnRH in hypothalamus, FSH and LH in pituitary gland. Both of EXD and TU could improve the condition. E2 and LH in serum of all groups were non-significance of difference (P > 0.05). The immunohistochemical results were well consistent with LH in pituitary gland., Conclusion: The results of the present research indicate that EXD extract is effective to start the HPT axis in puberty and can significantly improve sexual developmental inhibition caused by soy isoflavones.

A metabonomics method using (1)H nuclear magnetic resonance spectroscopy ((1)HNMR) was applied to obtain a systematic view of the development and progression of postmenopausal osteoporosis. Using partial least squares discriminant analysis (PLS-DA), 26 and 34 characteristic resonances were found respectively in urine and plasma of ovariectomized rats (Variable importance, VIP value ≥1.0), and the significant altered metabolites identified in the plasma and urine were 10 and 9, respectively. Changes in these metabolites were related to the pathways of lipid, energy and amino acid metabolism, some of which involved the oxidative system. The described method was also used to analyze the therapeutic effects of Er-Xian Decoction (EXD), a traditional Chinese medicine widely used in the clinical treatment of osteoporosis in China. The results showed that EXD administration could provide satisfactory effects on osteoporosis through partially regulating the perturbed pathways of lipid, energy and amino acid metabolism and improving the anti-oxidative ability.