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.

Dynamic Evolution of Cardiac Function and Glucose and Lipid Metabolism in Ovariectomized Rats and the Intervention Effect of Erxian Decoction.

Aims: Abnormal changes in cardiac function have been reported in menopausal women, but there are few clinical studies on this topic. Erxian decoction (EXD) is a classic prescription that is widely used in the treatment of female menopausal diseases. The purpose of this study was to investigate the dynamic evolution of cardiac function and glucose and lipid metabolism in ovariectomized (OVX) rats and the intervention effect of EXD. Materials and Methods: The OVX climacteric rat model was established by bilateral ovariectomy. After successful modeling, the rats were randomly divided into four groups: the sham operation (SHAM) group (equal volumes of purified water), OVX group (equal volumes of purified water), estradiol (E2) group (1.8 × 10-4 g/kg), and EXD group (9 g/kg). Each group of rats was treated for 16 weeks. At the 4th, 8th, 12th, and 16th weeks after treatment, the cardiac function of the rats in each group was evaluated by ultrasound. The coaxial method was used to measure blood pressure (BP). Serum endothelin-1 (ET-1) and angiotensin-2 (Ang II) levels were determined by the enzyme-linked immunosorbent assay (ELISA). The strip method was used to measure fasting blood glucose (FBG). The serum total cholesterol (TC) and triglyceride (TG) levels of rats were measured with the oxidase method. Direct methods were used to measure serum high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) levels. At week 16 of dosing, serum E2 levels were determined by E2 radioimmunoassay. The myocardium and uterus of the rats in each group were stained with HE (hematoxylin-eosin). The ultrastructure of the rat myocardium was observed by electron microscopy. Results: After the 16th week of treatment, the serum E2 level decreased (P < 0.05), and the uterus was atrophied in OVX rats. The cardiac ejection fraction (EF%) decreased at 4 weeks after treatment, and systolic and diastolic function decreased after 12 weeks. After the 16th week, the EF%, which reflects the "pump" function of the heart, decreased significantly (P < 0.05 or P < 0.01). At the 4th, 8th, 12th, and 16th weeks of treatment, the systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean pressure (MBP) of the rats in the OVX group increased with time (P < 0.05 or P < 0.01). The serum ET-1 and Ang II levels of rats in the OVX group increased (P < 0.05 or P < 0.01). In the OVX group, FBG was increased (P < 0.05 or P < 0.01), and blood lipids, especially LDL-C, were significantly increased (P < 0.05 or P < 0.01). After the 16th week of treatment, the myocardial tissue of OVX rats showed obvious pathological changes. EXD significantly increased serum E2 levels (P < 0.01), decreased ET-1 and Ang II levels (P < 0.01), reduced the cardiac function risk factors BP, FBG, and blood lipids, and significantly improved cardiac function and structural changes in OVX rats (P < 0.05 or P < 0.01). Conclusions: EXD can improve abnormal cardiac structure and function in OVX rats.

Monascin abrogates RANKL-mediated osteoclastogenesis in RAW264.7 cells via regulating MAPKs signaling pathways.

Osteoclasts (OCs) are multinucleated cells that play a major role in osteolytic diseases such as osteoporosis. Monascin (Ms) is one of the active substances in the traditional Chinese medicine red yeast rice. Studies have found that red yeast rice can maintain bone health. In this study, the anti-osteoclastogenesis effects of Ms on RANKL-induced RAW264.7 cells were assessed, and the underlying mechanism was investigated. Ms exhibited inhibitory effects on OC differentiation and formation in a dose-dependent manner and suppressed the bone-resorbing activity of mature OCs. Ms blocked OCs-typical genes (c-Fos, NFATc1, CSTK, MMP-9, TRAP, ITG-ß3, OSCAR and DC-STAMP). Furthermore, Ms treatment considerably inhibited the activation of MAPKs, JNK and p38. Taken together, Ms suppresses RANKL-induced osteoclastogenesis of RAW264.7 cells by restraining MAPKs signaling pathways and is a potential therapeutic option as a novel OC inhibitor to mitigate bone erosion.

Exploring the Potential Mechanism of Artemisinin and Its Derivatives in the Treatment of Osteoporosis Based on Network Pharmacology and Molecular Docking.

Objective: This study is aimed at predicting and contrasting the mechanisms of artemisinin (ARS), dihydroartemisinin (DHA), artesunate (ART), artemether (ARM), and arteether (ARE) in the treatment of osteoporosis (OP) using network pharmacology and molecular docking. Methods: The targets of ARS, DHA, ART, ARM, and ARE were obtained from the SwissTargetPrediction. The targets related to OP were obtained from the TTD, DrugBank, Genecards, and DisGeNet databases. Then, the anti-OP targets of ARS, DHA, ART, ARM, and ARE were obtained and compared using the Venn diagram. Afterward, the protein-protein interaction (PPI) networks were built using the STRING database, and Cytoscape was used to select hub targets. Moreover, molecular docking validated the binding association between five molecules and hub targets. Finally, GO enrichment and KEGG pathway enrichment were conducted using the DAVID database. The common pathways of five molecules were analysed. Results: A total of 28, 37, 36, 27, and 33 anti-OP targets of ARS, DHA, ART, ARM, and ARE were acquired. EGFR, EGFR, CASP3, MAPK8, and CASP3 act as the top 1 anti-OP targets of ARS, DHA, ART, ARM, and ARE, respectively. MAPK14 is the common target of five molecules. All five molecules can bind well with these hubs and common targets. Meanwhile, functional annotation showed that MAPK, Serotonergic synapse, AMPK, prolactin, and prolactin signaling pathways are the top 1 anti-OP pathway of ARS, DHA, ART, ARM, and ARE, respectively. IL-17 signaling pathway and prolactin signaling pathway are common anti-OP pathways of five molecules. Besides, GO enrichment showed five biological processes and three molecular functions are common anti-OP mechanisms of five molecules. Conclusion: ARS, DHA, ART, ARM and ARE can treat OP through multi-targets and multi pathways, respectively. All five molecules can treat OP by targeting MAPK14 and acting on the IL-17 and prolactin signaling pathways.

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.

Protective effect of Rhizoma Dioscoreae extract against alveolar bone loss in ovariectomized rats via regulation of IL-6/STAT3 signaling.

The aim of the present study was to assess the effectiveness of Rhizoma Dioscoreae extract (RDE) on preventing rat alveolar bone loss induced by ovariectomy (OVX), and to determine the role of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in this effect. Female Wistar rats were subjected to OVX or sham surgery. The rats that had undergone OVX were treated with RDE (RDE group), vehicle (OVX group) or 17ß-estradiol subcutaneous injection (E2 group). Subsequently, bone metabolic activity was assessed by analyzing 3-D alveolar bone construction, bone mineral density, as well as the plasma biomarkers of bone turnover. The gene expression of alveolar bone in the OVX and RDE groups was evaluated by IL-6/STAT3 signaling pathway polymerase chain reaction (PCR) arrays, and differentially expressed genes were determined through reverse transcription-quantitative PCR. The inhibitory effect of RDE on alveolar bone loss in the OVX group was demonstrated in the study. In comparison with the OVX group, the RDE group exhibited 19 downregulated genes and 1 upregulated gene associated with the IL-6/STAT3 signaling pathway in alveolar bone. Thus, RDE was shown to relieve OVX-induced alveolar bone loss in rats, an effect which was likely associated with decreased abnormal bone remodeling via regulation of the IL-6/STAT3 signaling pathway.

Rhizoma Dioscoreae extract protects against alveolar bone loss by regulating the cell cycle: A predictive study based on the protein­protein interaction network.

Rhizoma Dioscoreae extract (RDE) exhibits a protective effect on alveolar bone loss in ovariectomized (OVX) rats. The aim of this study was to predict the pathways or targets that are regulated by RDE, by re­assessing our previously reported data and conducting a protein­protein interaction (PPI) network analysis. In total, 383 differentially expressed genes (≥3­fold) between alveolar bone samples from the RDE and OVX group rats were identified, and a PPI network was constructed based on these genes. Furthermore, four molecular clusters (A­D) in the PPI network with the smallest P­values were detected by molecular complex detection (MCODE) algorithm. Using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) tools, two molecular clusters (A and B) were enriched for biological process in Gene Ontology (GO). Only cluster A was associated with biological pathways in the IPA database. GO and pathway analysis results showed that cluster A, associated with cell cycle regulation, was the most important molecular cluster in the PPI network. In addition, cyclin­dependent kinase 1 (CDK1) may be a key molecule achieving the cell­cycle­regulatory function of cluster A. From the PPI network analysis, it was predicted that delayed cell cycle progression in excessive alveolar bone remodeling via downregulation of CDK1 may be another mechanism underling the anti­osteopenic effect of RDE on alveolar bone.

Rhizoma Dioscoreae extract protects against alveolar bone loss in ovariectomized rats via microRNAs regulation.

The aim of this study was to evaluate the osteoprotective effect of aqueous Rhizoma Dioscoreae extract (RDE) on the alveolar bone of rats with ovariectomy-induced bone loss. Female Wistar rats underwent either ovariectomy or sham operation (SHAM). The ovariectomized (OVX) rats were treated with vehicle (OVX), estradiol valerate (EV), or RDE. After treatments, the bone mineral density (BMD) and the three-dimensional microarchitecture of the alveolar bone were analyzed to assess bone mass. Microarrays were used to evaluate microRNA expression profiles in alveolar bone from RDE-treated and OVX rats. The differential expression of microRNAs was validated using real-time quantitative RT-PCR (qRT-PCR), and the target genes of validated microRNAs were predicted and further analyzed using Ingenuity Pathway Analysis (IPA). The key findings were verified using qRT-PCR. Our results show that RDE inhibits alveolar bone loss in OVX rats. Compared to the OVX rats, the RDE-treated rats showed upregulated expression levels of 8 microRNAs and downregulated expression levels of 8 microRNAs in the alveolar bone in the microarray analysis. qRT-PCR helped validate 13 of 16 differentially expressed microRNAs, and 114 putative target genes of the validated microRNAs were retrieved. The IPA showed that these putative target genes had the potential to code for proteins that were involved in the transforming growth factor (TGF)-ß/bone morphogenetic proteins (BMPs)/Smad signaling pathway (Tgfbr2/Bmpr2, Smad3/4/5, and Bcl-2) and interleukin (IL)-6/oncostatin M (OSM)/Jak1/STAT3 signaling pathway (Jak1, STAT3, and Il6r). These experiments revealed that RDE could inhibit ovariectomy-induced alveolar bone loss in rats. The mechanism of this anti-osteopenic effect in alveolar bone may involve the simultaneous inhibition of bone formation and bone resorption, which is associated with modulation of the TGF-ß/BMPs/Smad and the IL-6/OSM/Jak1/STAT3 signaling pathways via microRNA regulation.

[Effects of Shenmai injection on afterdepolarization and triggered activities in left ventricular papillary muscle in rat cardiac hypertrophy].

This study is to evaluate the effects of Shenmai injection on the temporal alterations of action potential (AP), early afterdepolarization (EAD) and delayed afterdepolarization (DAD) in papillary muscles. The action potentials were recorded by a glass electrode. APD at 90% repolarization (APD9 ) was measured, and spontaneous EAD and DAD were observed. The results show APD90 was significantly prolonged in model group compared with sham-operated group, whereas it was remained unchanged in Shenmai injec- tion treatment group and amiodarone group. The spontaneous EADs and DADs were frequently visible in model group. In conclusion, EAD, DAD and trigger activities increase gradually during pathological progression of rat cardiac hypertrophy, and Shenmai injection could improve the action potential change in rat cardiac hypertrophy.

[Effects of guizhi tang on inflammatory cytokines in myocardial ischemia and hyperlipidemia rats].

OBJECTIVE: To explore the effects of Guizhi Tang on the inflammatory cytokines in myocardial ischemia and hyperlipidemia rats. METHOD: The early changes of hyperlipid and atherosclerosis are caused by utilizing multiple factors including feeding hyperlipid and propylthiouracil and high doses of vitamin D3 for 12 weeks. Sixty male SD rats were randomly divided in to 5 groups: control group, model group, simvastatin group, low-dosage Guizhi Tang group, high-dosage Guizhi Tang group. At the end of six weeks treatment, pituitrin(pit) is abdominal cavity injected every 24 hours for a total of three times. Detecting the serum levels of SES, CRP, NO, SOD, MDA and the content of cardiac muscle tissue SOD, MDA, The expression of TNF-alpha in cardiac muscle tissue was detected by immunohistochemistry. RESULT: Guizhi Tang significantly decreased levels of SES, CRP and MAD, increased levels of NO and SOD, Guizhi Tang markedly decreased the level of protein expression of TNF-alpha in cardiac muscle tissue. CONCLUSION: Guizhi Tang may inhibit the proinflammatory factors and oxidation in myocardial ischemia and hyperlipidemia rats.

Ferulic acid promotes endothelial cells proliferation through up-regulating cyclin D1 and VEGF.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelicae Sinensis is a well known and commonly used traditional Chinese herbal medicine with many therapeutic effects such as neuroprotection, the promotion of hematopoiesis and the treatment of tumors. AIM OF THE STUDY: Ferulic acid (FA) is the main active component in Angelicae Sinensis. Previous research has demonstrated that Angelicae Sinensis is able to induce angiogenesis in vivo. This study investigated the effects of FA on the proliferation of a human umbilical vein endothelial cell line (ECV304) with regard to the modulation of endothelial cells, which is a key step of angiogenesis. MATERIALS AND METHODS: ECV304 cells were incubated with FA at different dosages (0.1 µg/mL, 1 µg/mL and 10 µg/mL). A series of assays was used to detect the effects of FA on: (i) cell proliferation; (ii) DNA synthesis; (iii) cell-cycle distribution; and (iv) mRNA expression of cyclin D1 and vascular endothelial growth factor (VEGF). These were assessed using the Cell Counting Kit-8 (CCK-8), bromodeoxyuridine-enzyme-linked immunosorbent assay (BrdU-ELISA), flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS AND DISCUSSION: The results showed that FA at a range of concentrations from 0.1 µg/mL to 10 µg/mL could markedly improve cell proliferation and DNA synthesis in a dose-dependent manner. Flow cytometry showed a significant decrease in the percentage of cells in the G(0)/G(1) phase and a significant increase in the percentage of cells in the S phase. Furthermore, we found that FA enhanced cyclin D1 and VEGF mRNA expression in ECV304 cells. CONCLUSION: FA was able to promote ECV304 cells proliferation in vitro. This effect might be observed through the modulation of cyclin D1 and VEGF.

[Effects of Panax notoginseng saponins on rat cardiomyocytes apoptosis induced by angiotengin II in vitro].

OBJECTIVE: To study the protective effects of Panax notoginseng saponins (PNS) on angiotensin II (Ang II)-induced rat cardiomyocyte apoptosis in vitro and the probable mechanism. METHOD: Cultured cardiomyocytes from neonatal rats were stimulated with Ang II. Cell viability was measured by MTT. Apoptosis was evaluated using Acridine Orange (AO) fluorescent dye staining and flow cytometry; Fluo-3 AM was used to test the change of intracellular free calcium. RESULT: It was found that incubating with Ang II (10(-7) mol x L(-1)) for 48 h increased cardiomyocyte apoptosis, PNS (25, 100 mg x mL(-1)) increased myocyte viability. PNS (50 mg x mL(-1)) significantly decreased this Ang II-induced rat cardiomyocyte apoptosis (P < 0.05) and decreased fluorescent intensity of intracellular calcium. CONCLUSION: PNS has a significant effect on Ang II-induced rat cardiomyocytes apoptosis in vitro by alleviating intracellular calcium overload.