Kaempferol exerts antioxidant effects in age-related diminished ovarian reserve by regulating the HSP90/NRF2 pathway.

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.

Er-Xian decoction attenuates ovariectomy-induced osteoporosis by modulating fatty acid metabolism and IGF1/PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: The study aimed to verify the anti-OP effect of EXD and to explore its underlying mechanism. METHODS: 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. RESULTS: 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. CONCLUSION: EXD ameliorated ovariectomy-induced bone loss by modulating lipid metabolism, FA metabolism, and IGF1/PI3K/AKT pathway.

Er-xian decoction drug-containing serum promotes Mc3t3-e1 cell proliferation and osteogenic differentiation via regulating BK channel.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: The study is aimed at determining whether the EXD-containing serum promotes the proliferation of osteoblasts and their differentiation through BK channel. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Uncovering Hidden Mechanisms of Different Prescriptions Treatment for Osteoporosis via Novel Bioinformatics Model and Experiment Validation.

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.

Effect of Er-Xian decoction on autoimmune premature ovarian failure in mice.

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.

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 mechanism of Er-xian Decoction in regulating lipid metabolism disorders on bilateral ovariectomized rats based on metabolomics study / 药学学报

Studies have shown that women's menopause caused by permanent cessation of ovarian function is closely related to lipid metabolism disorders. Er-xian Decoction has been used in the clinical treatment for gynecological diseases and has a good effect on diseases related to reduced sex hormone function. In this study, metabolomics was performed on bilateral ovariectomized model rats within 12 weeks after modeling to mimic the physiological state of menopausal women in different menopausal stages and Er-xian Decoction dosed model rats. The results of liver oil red O staining sections showed lipid metabolic disorder of bilateral ovariectomized model rats and the regulating effects of Er-xian Decoction. 46 potential biomarkers (6 steroid hormones, 3 sphingolipids, 11 phospholipids and 26 glycerides) in plasma and 32 potential biomarkers (1 steroid hormones, 20 phospholipids and 11 glycerides) in liver were obtained based on lipidomics analysis. Then, we analyzed the differential metabolic pathways and construct the lipid metabolism network significantly regulated by Er-xian Decoction. The results provided valuable information for in-depth understanding of the gradual changes on lipid metabolism disorders under menopausal conditions and the characteristics and mechanisms of compound Er-xian Decoction's regulatory effects. The study complied with the procedures established by the Animal Experiment Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and passed the animal experiment ethics examine (No. 00000918).

The protective effect of Er-Xian decoction against myocardial injury in menopausal rat model.

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 17ß-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.

[Hippocampus proteomics for effect of Er-xian decoction on ovariectomy induced-learning and memory disabilities in rats].

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.

Effect of antiosteoporotic active fraction from Er-Xian Decoction on retinoic acid-induced bone loss in rats / 第二军医大学学报

Objective To explore the effect of antiosteoporotic active fraction from Er-Xian Decoction (EXD) on retinoic acid-induced osteoporosis in rats. Methods Sixty female SD rats were randomly divided into normal control group, model group, estradiol valerate (0.2 mg/kg) group and antiosteoporotic active fraction from EXD low-, middle- and high-dose groups (50, 100, 200 mg/kg). Except for the rats of normal group, the others were intragastrically treated with 70 mg/(kg • d) of retinoic acid for 14 days to induce osteoporosis. Then the rats were treated with estradiol valeratse or antiosteoporotic active fraction from EXD (10 mL/kg) for 28 days. The biochemical parameters in serum and urine of rats were detected by ELISA. The bone mineral density and trabecular bone microstructure of distal femur were analyzed with micro-CT. Results The antiosteoporotic active fraction from EXD significantly decreased calcium content in urine and activities of alkaline phosphatase and tartratse-resistant acid phosphatase in the sera of retinoic acid-induced osteoporotic rats (P0.05, P0.01). High-dose of antiosteoporotic active fraction from EXD significantly increased bone mineral density of distal femur (P0.05). And the antiosteoporotic active fraction from EXD improved the trabecular bone microstructure of distal femur in retinoic acid-induced osteoporotic rats. Conclusion Antiosteoporotic active fraction from EXD can reduce bone loss and deterioration of bone microstructure in retinoic acid-induced osteoporotic rats.

Hippocampus proteomics for effect of Er-xian decoction on ovariectomy induced-learning and memory disabilities in rats / 中国中药杂志

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.

A multiple-dimension liquid chromatography coupled with mass spectrometry data strategy for the rapid discovery and identification of unknown compounds from a Chinese herbal formula (Er-xian decoction).

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 (MSn), 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 MSn 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.

[Effect of Er-xian decoction on femur proteomics in ovariectomized osteoporosis rats].

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 ß1 may be the action targets for Er-xian decoction.

Effect of Cupping plus Medication on Quality of Life and Estrogen Level in Perimenopausal Syndrome Patients / 上海针灸杂志

Objective To observe the effect of cupping plus medication on the quality of life and estrogen level in perimenopausal syndrome patients. Method A total of 40 eligible perimenopausal syndrome patients were randomized into a cupping-medication group and a Chinese medication group, 20 cases in each group. The Chinese medication group was intervened by Er Xian decoction, the cupping-medication group was intervened by moving cupping on the back in addition to the treatment given to the Chinese medication group, once a week. Each item of the Menopause-specific Quality of Life (MENQOL), the levels of estradiol (E2), follicle-stimulating hormone (FSH) and luteotropic hormone (LH) in serum were observed before and after the treatment, and the results were analyzed. Result After two treatment courses, the vasomotor symptoms, mental state, physiological state, and sexual life scores in the two groups were significantly improved compared to the scores before the treatment (P0.05). Conclusion Moving cupping on the back plus Er Xian decoction can effectively improve the quality of life of perimenopausal syndrome patients and significantly increase the serum E2 level, superior to using Er Xian decoction alone.

Effect of Er-xian decoction on femur proteomics in ovariectomized osteoporosis rats / 中国中药杂志

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.

Studies on Effects of Er-Xian Decoction in Follicular Granulosa Cell Apoptosis in Premature Ovarian Failure Rats / 世界科学技术-中医药现代化

This study was aimed to investigate effects of Er-Xian (EX) decoction in follicular granulosa cell apoptosis in chemotherapy-induced premature ovarian failure (POF) of rats. SD rats were randomly divided into the normal group, model group, positive group and EX decoction group. Intraperitoneal injection of cisplatin was used in the establishment of chemotherapy-induced POF rat model. Intragastric administration of corresponding medication was give to each group for four weeks. The general conditions of rats were observed. The serum contents of E2, FSH, LH and Pro were determined. The ovarian tissue morphology was observed. Granulosa cell apoptosis was analyzed by TUNEL. The expressions of BAX, Bcl-2 and VEGF protein were detected by immunohistochemistry. The results showed that in the model group, the body weight decreased with disordered estrous cycle. After treatment, the weight gained, and the estrous cycle recovered. Compared with the normal group, in the model group, E2 decreased, FSH and LH levels increased. There was no significant difference in the content of progesterone among different groups. Detection of TUNEL showed that the positive expression in the model group was increased compared with the normal group (P < 0.05). The positive expression of EX decoction group was reduced compared to the positive group (P < 0.05). Compared with the normal group, the expression of BAX was obviously increased and Bcl-2 was decreased in the model group. After EX decoction treatment, the expression of BAX was decreased and the Bcl-2 was increased compared to the model group. The expression of VEGF protein in the model group was obviously less than that in the normal group. It was concluded that cisplatin can induce follicular granulosa cell apoptosis, and cause premature ovarian function recession. EX decoction can adjust the content of different hormones in serum, alter expression of apoptosis related protein to reduce the damage of cisplatin on ovarian follicular development, in order to promote follicular development, improve and enhance the ovarian function.

Er-Xian Decoction, a traditional Chinese herbal formula, intervening early in hypothalamic-pituitary axis of male rats with delayed puberty.

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 HNMR-based metabonomics study of postmenopausal osteoporosis and intervention effects of Er-Xian Decoction in ovariectomized rats.

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.

Protective effect of Er-xian decoction on ovariectomy-induced osteoporosis in rats / 第二军医大学学报

Objective: To investigate the protective effects of Er-xian decoction on ovariectomy-induced osteoporosis in rats. Methods: The osteoporosis model was induced by ovariectomy in rats. Twelve weeks later, the undecalcified longitudinal proximal tibial metaphysical sections were made and stained for the bone histomorphometric analysis. Bone density of femur metaphysis was determined by dual-energy X-ray absorptiometry. Results: Er-xian decoction significantly increased trabecular area and trabecular thickness (P<0.01) and decreased trabecular separation (P<0.05). The parameters of bone formation of rats, such as MAR, BFR /BS, BFR/BV, and BFR/TV, were increased significantly after Er-xian decoction treatment (P< 0.05, P<0.01). Moreover, Er-xian decoction obviously reduced osteoclast number (P<0.01) and enhanced bone density of femur metaphysis in ovariectomized rats (P < 0.05). Conclusion: Er-xian decoction has antiosteoporotic effects on ovariectomized rats by promoting bone formation, increasing bone density, and inhibiting bone resorption.

Effects of Er-xian Decoction and Its Component Herbs on Estrogen Receptors in Adrenal Development Rats / 中国中医药信息杂志

Objective To explore the possible functional mechanism of Er-xian Decoction and its component herbs on the expression of extrogen receptor(ER) in adrenal development rats.Methods Fifty-four development female SD rats were randomly divided into nine groups:normal control group(A),positive control group(B),Er-xian Decoction group(C),Herba Epimedii group(D),Rhizoma Curculiginis group(E),Radix Morindae Officinalis group(F),Radix Angelicae Sinensis group(G),Cortex Phellodendri group(H),Rhizoma Anemarrhenae group(I).After the rats were administrated for six days,serum were got to measure testosterone(T).Bilateral adrenal gland was weighed and embedded by paraffin.Mean optical density(MOD) were studied by the immunohistochemical method and image analyzing system.Results The adrenal coefficient was increased in group B and C,and the level of T was decreased in group C,D,E and I.ER? was mainly observed in the cytoplasm of zona glomerulosa and zona fasciculate.MOD in group C was increased while declined in group B.ER? was mainly observed in nucleus of zona glomerulosa and zona fasciculate.MOD in group C,D,E and I was less than group B.Conclusion Er-xian Decoction and its component herbs of warming Shen(Herba Epimedii,Rhizoma Curculiginis) and nourishing Yin(Rhizoma Anemarrhenae) play a phytoestrogen role by influencing the expression of ER? and ER ? in the adrenal cortex.