Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3ß/ß-catenin signaling.

Background: Eurycomanone (EN) is a diterpenoid compound isolated from the roots of Eurycoma longifolia (E. longifolia). Previous studies have confirmed that E. longifolia can enhance bone regeneration and bone strength. We previously isolated and identified ten quassinoids from E. longifolia, and the result displayed that five aqueous extracts have the effects on promotion of bone formation, among whom EN showed the strongest activity. However, the molecular mechanism of EN on bone formation was unknown, and we further investigated in this study. Methods: After the verification of purity of extracted EN, following experiments were conducted. Firstly, the pharmacologic action of EN on normal bone mineralization and the therapeutic effect of EN on Dex-induced bone loss using zebrafish larvae. The mineralization area and integral optical density (IOD) were evaluated using alizarin red staining. Then the vital signaling pathways of EN relevant to OP was identified through network pharmacology analysis. Eventually in vitro, the effect of EN on cell viability, osteogenesis activities were investigated in human bone marrow mesenchymal stem cells (hMSCs) and C3H10 cells, and the molecular mechanisms by which applying AKT inhibitor A-443654 in hMSCs. Results: In zebrafish larvae, the administration in medium of EN (0.2, 1, and 5 µM) dramatically enhanced the skull mineralization area and integral optical density (IOD), and increased mRNA expressions of osteoblast formation genes (ALP, RUNX2a, SP7, OCN). Meanwhile, exposure of EN remarkably alleviated the inhibition of bone formation induced by dexamethasone (Dex), prominently improved the mineralization, up-regulated osteoblast-specific genes and down-regulated osteoclast-related genes (CTSK, RANKL, NFATc1, TRAF6) in Dex-treated bone loss zebrafish larvae. Network pharmacology outcomes showed the MAPK and PI3K-AKT signaling pathways are closely associated with 10 hub genes (especially AKT1), and AKT/GSK-3ß/ß-catenin was selected as the candidate analysis pathway. In hMSCs and C3H10 cells, results showed that EN at appropriate concentrations of 0.008-5 µM effectively increased the cell proliferation. In addition, EN (0.04, 0.2, and 1 µM) significantly stimulated osteogenic differentiation and mineralization as well as significantly increased the protein phosphorylation of AKT and GSK-3ß, and expression of ß-catenin, evidencing by the results of ALP and ARS staining, qPCR and western blotting. Whereas opposite results were presented in hMSCs when treated with AKT inhibitor A-443654, which effectively inhibited the pro-osteogenesis effect induced by EN, suggesting EN represent powerful potential in promoting osteogenesis of hMSCs, which may be closely related to the AKT/GSK-3ß/ß-catenin signaling pathway. Conclusions: Altogether, our findings indicate that EN possesses remarkable effect on bone formation via activating AKT/GSK-3ß/ß-catenin signaling pathway in most tested concentrations. The translational potential of this article: This study demonstrates EN is a new effective monomer in promoting bone formation, which may be a promising anabolic agent for osteoporosis (OP) treatment.

Quassinoids from Eurycoma longifolia and their bone formation evaluation in zebrafish, C3H10 cells and silico.

Phytochemicals with bone formation potential in traditional medicines captured more and more attentions due to their advantages to bone loss and fewer side effects. As a famous aphrodisiac phytomedicine, Eurycoma longifolia (EL) has acquired general recognition in improving male sexual health, and thus been considered as traditional medicine for the treatment of androgen-deficient osteoporosis. Although the aqueous extract of EL had been proved to be beneficial to bone loss, the active constituents and the mechanisms underlying the effects are still obscure. The current study performed a chemical investigation on the roots of EL, which resulted in the isolation and identification of ten quassinoids (EL-1-EL-10), and then conducted their osteogenic activity evaluations in vivo zebrafish model with or without dexamethasone (Dex) and in vitro C3H10 cell model. The result displayed that most tested concentrations of EL-1-EL-5 could significantly increase the mineralization areas and integrated optical densities (IODs) of skull in both zebrafish model. The majority tested concentrations of EL-1-EL-5 could also improve the mRNA expression of early osteogenic associated genes ALPL, Runx2a, Sp7 in zebrafish model without Dex, but only a few could accelerate the mRNA expression of late osteogenic associated genes OCN. These results suggested the ability of EL-1-EL-5 to increase bone formation mainly by accelerating osteogenic differentiation at the early stage. The structure-based virtual screening based on the pharmacophores in ePharmaLib, as well as the molecular docking study, implied that the effects of the quassinoids (EL-1-EL-5) on the enhancement of bone formation might be related with improving the content and the activity of androgen through binding with CYP19A, SHBG and AKR1C2, and activating bone metabolism-related ANDR target genes and signal pathways by combining with ANDR directly. Although the assumptions are in silico model-based and further in vitro and in vivo validations are still necessary, we provided a new perspective to explore the potential of EL to be used as an alternative treatment for not only androgen-deficient osteoporosis, but also estrogen-deficient bone loss, by combining with SHBG.

Anti-Osteogenic Effect of Danshensu in Ankylosing Spondylitis: An in Vitro Study Based on Integrated Network Pharmacology.

Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by abnormal bone metabolism, with few effective treatments available. Danshensu [3-(3,4-dihydroxy-phenyl) lactic acid) is a bioactive compound from traditional Chinese medicine with a variety of pharmacologic effects. In the present study, we investigated the pharmacologic effect and molecular mechanism of Danshensu in AS. Potential targets of Danshensu were identified in four drugs-genes databases; and potential pharmacologic target genes in AS were identified in three diseases-genes databases. Differentially expressed genes related to AS were obtained from the Gene Expression Omnibus database. Overlapping targets of Danshensu and AS were determined and a disease-active ingredient-target interaction network was constructed with Cytoscape software. Enrichment analyses of the common targets were performed using Bioconductor. To test the validity of the constructed network, an in vitro model was established by treating osteoblasts from newborn rats with low concentrations of tumor necrosis factor (TNF)-α. Then, the in vitro model and AS fibroblasts were treated with Danshensu (1-10 µM). Osteogenesis was evaluated by alkaline phosphatase staining and activity assay, alizarin red staining, quantitative PCR, and western blotting. We identified 2944 AS-related genes and 406 Danshensu targets, including 47 that were common to both datasets. The main signaling pathways associated with the targets were the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways. A low concentration of TNF-α (0.01 ng/ml) promoted the differentiation of osteoblasts; this was inhibited by Danshensu, which had the same effect on AS fibroblasts but had the opposite effect on normal osteoblasts. Danshensu also decreased the phosphorylation of JNK and ERK in AS fibroblasts. There results provide evidence that Danshensu exerts an anti-osteogenic effect via suppression of JNK and ERK signaling, highlighting its therapeutic potential for the treatment of AS.

Pteisolic acid G, a novel ent-kaurane diterpenoid, inhibits viability and induces apoptosis in human colorectal carcinoma cells.

Human colorectal cancer (CRC) is a major cause of cancer morbidity and mortality, and its incidence rates are increasing in economical transitioning areas globally. To develop efficient chemotherapy drugs for CRC, the present study isolated and identified a novel ent-kaurane diterpenoid from Pteris semipinnata, termed pterisolic acid G (PAG). This ent-kaurane diterpenoid was demonstrated to significantly inhibit the growth of human CRC HCT116 cells in a time- and dose-dependent manner, determined using the Cell Counting Kit-8 assay. Additionally, western blot analysis, Hoechst 33342 staining and cytometry analysis revealed that PAG not only inhibited the viability of HCT116 cells by suppressing the dishevelled segment polarity protein 2/glycogen synthase kinase 3 ß/ß-catenin pathway, but also induced the apoptosis of HCT116 cells by downregulating nuclear factor-κB p65 activity, stimulating p53 expression and promoting the generation of intracellular reactive oxygen species. These results suggest that PAG, a novel inhibitor of the Wnt/ß-catenin pathway and inducer of apoptosis, should be investigated in more detail using in vivo experiments and comprehensive mechanistic studies in order to examine the potential use of PAG as a novel therapeutic agent for the treatment of CRC.

Dalbinol, a rotenoid from Amorpha fruticosa L., exerts anti-proliferative activity by facilitating ß-catenin degradation in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly malignant tumor, and the main cause of treatment failure is malignant proliferation. Aberrations in Wnt/ß-catenin signaling are associated with HCC development. Despite the improvements in overall survival made over the past decade from the advent of molecularly targeted therapies, these treatments do not have efficacy in all patients with different pathogeneses. Therefore, there is a demand for novel chemotherapeutic agents for HCC. To this end, we built a natural compound library and screened out a rotenoid named dalbinol from the seeds of Amorpha fruticosa L. Our data demonstrated that dalbinol inhibited the growth of HepG2, HepG2/ADM and Huh7 cells in a concentration-dependent manner. Pharmacological experiments also showed that dalbinol suppressed growth and induced apoptosis in these HCC cell lines in vitro. Furthermore, we found that dalbinol promoted ß-catenin degradation, which was mediated by the ubiquitin-proteasome pathway. To summarize, our results illustrate that dalbinol inhibited HCC cell growth by facilitating ß-catenin degradation through the ubiquitin-proteasome pathway. Hence, we propose that dalbinol will be a promising agent for the treatment of HCC subtypes with aberrant Wnt/ß-catenin pathway activation.

Cytotoxic rotenoid glycosides from the seeds of Amorpha fruticosa.

Four new rotenoid glycosides, namely amorphaside A-D (1-4), along with four known ones (5-8) were isolated from the seeds of Amorpha fruticosa. Their chemical structures and absolute configurations were elucidated by HRESIMS, NMR and CD spectra, as well as deduction from biosynthesis route. The sugar units were determined by acid hydrolysis, appropriate derivatization and HPLC analysis. The in vitro anti-proliferative activities of all compounds were evaluated against MCF-7 and HCT-116 cell lines. The results showed that compounds 1-3 had no effect on cell proliferation in the two cell lines even with the concentration of 50 µM, and compounds 4, 7 and 8 had selective cytotoxicity against MCF-7 with IC50 values of 3.90, 0.95 and 34.08 µM, respectively, while compounds 5 and 6 both showed significant cytotoxicity to the two cell lines with IC50 values less than 2.00 µM, even better than the positive control cisplatin. These preliminary results indicated that compounds 5 and 6 might be valuable to anticancer drug candidates.

[Study on sensitization and mechanism of CGA-BSA].

OBJECTIVE: To investigate the sensitization and mechanism of artificial antigen of chlorogenic acid (CGA-BSA). METHOD: Using intensive immunization to establish allergy animal model on guinea pig and preparing antiserum and tissue for further test. Using HE staining to observe pathology change of lungs, trachea, liver. Using passive mast cell (PMC) degranulation test to observe the immunogenicity of CGA-BSA and using ELISA to detect IgE and histamine in plasma. RESULT: There established allergy animal model on guinea pig, which include a increase cell degranulation by a ratio (63.58 +/- 10.23)% in PMC test, increase of specific antibody IgE and increase of histamine in plasma after provocation by ELISA. CONCLUSION: Allergen CGA-BSA could provoke allergenic response in guinea pig, and the allergic response belongs to type I allergy.

[Study of immunogenicity of chlorogenic acid-BSA with different number of CGA].

OBJECTIVE: To investigate the immunogenicity of chlorogenic acid-BSA(CGA-BSA) and the foundation for the allergization of CGA-BSA. METHOD: The CGA-BSA with different number of CGA was synthesized to allergize the BALB/c mice and SD rats to get antiserum. The level of IgE and histamine was analyzed by ELISA, and the passive cutaneous anaphylaxis (PCA) test was carried to analyze the antibody titer. RESULT: When the coupling rate of CGA-BSA is 20, the highest IgE and histamine level in BALB/c mice and the antibody titer in PCA are the highest. CONCLUSION: It has better immunogenicity when the coupling rate of CGA-BSA is between 10 to 20, while the CGA-BSA 20 with the best immunogenicity.

Antidepressant-like effects of 3,6'-disinapoyl sucrose on hippocampal neuronal plasticity and neurotrophic signal pathway in chronically mild stressed rats.

Recent studies suggest that the behavioral effects of chronic antidepressant treatment are mediated by stimulation of hippocampal neuronal plasticity and neurogenesis. The present study was designed to examine the effects of 3,6'-disinapoyl sucrose (DISS), a bioactive component of Polygala tenuifolia Willd, on the expressions of four plasticity-associated genes: cell adhesion molecule L1 (CAM-L1), laminin, cAMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in hippocampus, all of which are involved in neuronal plasticity and neurite outgrowth. We confirmed that chronic stress in rats caused a reduction in sensitivity to reward (sucrose consumption) and a decrease in mRNA levels of CAM-L1, laminin, and BDNF, together with a decrease in protein levels of phosphorylated CREB and BDNF. Repeated administration of DISS for 21 days at doses of 5, 10 and 20mg/kg reversed stress-induced alterations in sucrose consumption and these target mRNA and protein levels. In conclusion, increased expressions in the hippocampus of three noradrenergic-regulated plasticity genes and one neurotrophic factor may be one of the molecular and cellular mechanisms underlying the antidepressant action of DISS in chronic mild stress (CMS) rats.

[Effects of cembrane-type diterpenes on proliferation of PC12 cells and their antagonistic effects on neurotoxicity induced by glutamate].

OBJECTIVE: To investigate the effects of cembrane-type diterpenes extracted from Sinularia flexibilis on the proliferation of PC12 cells and their protective effects on PC12 cells exposed to glutamate. METHODS: Methyl thiazolyl tetrazolium (MTT) method was adopted to observe the effects of cembrane-type diterpenes (compound 1, compound 2 and compound 3) on the proliferation of PC12 cells. And the protective effects of the three compounds on PC12 cells exposed to glutamate were also detected by MTT. Furthermore, the influence of compound 1 on intracellular concentration of calcium in PC12 cells exposed to glutamate was detected by laser confocal microscopy. RESULTS: After 72-hour PC12 cell culture, OD values in the 2, 10 and 50 micromol/L compound 1 groups were significantly higher than that in the normal group (P<0.05, P<0.01). After 24-hour glutamate damage, OD values in the 0.4, 2 and 50 micromol/L compound 1 groups, the 0.4, 2 and 100 micromol/L compound 2 groups and the 2 micromol/L compound 3 group were obviously increased as compared with the untreated group (P<0.01, P<0.05). After 48-hour glutamate damage, OD values in the compound 1 group were approximate to those in the normal control and the positive control group while were significantly higher than that in the untreated group (P<0.01, P<0.05), but no dose-dependent effect was observed. Compound 1 of 0.4, 2, 50 micromol/L could significantly reduce the intracellular concentration of calcium in PC12 cells exposed to glutamate (P<0.05, P<0.01), which was also approximate to the effect of nimodipine (positive control drug). CONCLUSION: Cembrane-type diterpenes (compound 1, compound 2 and compound 3) extracted from Sinularia flexibilis have obvious protective effects on PC12 cells damaged by glutamate, and compound 1 has the best neuroprotective effect. The mechanism of the neuroprotective effect of compound 1 may lie in reducing the intracellular concentration of calcium in PC12 cells exposed to glutamate and relieving the calcium overload.

A bioactive compound from Polygala tenuifolia regulates efficiency of chronic stress on hypothalamic-pituitary-adrenal axis.

3,6'-Disinapoyl sucrose (DISS) is the active oligosaccharide ester component from roots of Polygala tenuifolia, and its antidepressant effects was found in the forced swimming test (FST) and tail suspension test (TST). We aimed to study the antidepressant effects of DISS in the chronic unpredictable mild stress (CMS) model in rats and explore the underlying mechanisms in the hypothalamic-pituitary-adrenal (HPA) axis. We found that when subjected to the chronic stress protocol for 28 days, animals showed reduced sensitivity to reward and abnormality in the HPA axis. DISS (10 or 20 mg/kg, i.g.) improved the reward reaction as measured by increasing sucrose consumption, remarkably reduced serum CORT, ACTH and CRH levels in the CMS-treated rats. In addition, DISS enhanced the expression of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA. These results indicated that the antidepressant effects of DISS in chronically stressed animals might relate to the modulating effects on the HPA axis, which might be an important mechanism for its antidepressant effect.

[Antidepressant-like effects of piperine and its neuroprotective mechanism].

OBJECTIVE: To observe the antidepressant effect of piperine and its neuroprotective mechanism. METHOD: The behavioral studies were performed in forced swimming test (FST) and tail suspension test (TST). To further explore the mechanisms underlying their antidepressant-like activities, CORT-induced neuroblastoma SH-SY5Y cells and isolated and cultured neural progenitor cells. By using MTT assay, the effect of piperine on neural cells proliferation was observed. RESULT: The research results indicated that after a week of administration, piperine (10, 20 mg x kg(-1)) could significantly reduce the duration of immobility in both FST and TST. Piperine has the protective effect on neuroblastoma cells and increased proliferation of hippocampus neural progenitor cells. CONCLUSION: In the present study, we demonstrated that the antidepressant-like effects of piperine and its mechanisms might be involved by up-regulation of the progenitor cell proliferation of hippocampus and cytoprotective activity.

[Antidepressant effects of piperine and its neuroprotective mechanism in rats].

OBJECTIVE: To study the antidepressant effects of piperine in chronic unpredictable mild stress (CUMS) rats and to explore the underlying mechanisms in the hypothalamic-pituitary-adrenal (HPA) axis. METHODS: Fifty rats were housed alone and exposed to an unpredicted sequence of mild stressors to induce CUMS, and then were divided into untreated group, desipramine group and low-, medium- and high-dose piperine groups. Another 10 normal rats were used as normal control. The behavior of the rats was detected by body weight and sucrose preference test, and the serum levels of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in different groups were observed by radioimmunoassay. RESULTS: The results indicated that after 21-day administration, piperine at concentrations of 10 and 20 mg/kg could significantly improve the behavioral disorder by increasing sucrose consumption. Piperione at concentrations of 5, 10 and 20 mg/kg could remarkably reduce serum ACTH and CRH levels in the CUMS rats, but had little effect on the content of CORT. CONCLUSION: Piperine can relieve depression in CUMS rats by modulating the function of HPA axis.

[Study on antidepressant components of sucrose ester from Polygala tenuifolia].

OBJECTIVE: To investigate the antidepressant components of Polygala tenuifolia. METHOD: The chromatographic method was used to isolate and purify the chemical constituents, their structures were identified by spectral analysis, MTT method was applied to investigate their cytotoxic activities. RESULT: Nine compounds were isolated from the roots of P. tenuifolia. Their structures were identified as sibiricose A, (1), sibiricose A5 (2), tenuifoliside A (3) and 3', 6-disinapoyl sucrose (4), sibiricose A6 (5), 3, 4, 5-trimethoxycinnamate (6), polygalaxanthone III (7), tenuifolioses A (8), tenuifolioses H (9) and some compounds' activities to PC12 were observed. CONCLUSION: Compound 1 was isolated from this plant for the first time. Compounds 2,3 could protect PC12 cells damage induced by P. tenuifolia.

Two correlation patterns as indicators for underlying dynamics of complex systems.

Two spatial correlation patterns, fixed-to-arbitrary and neighboring bin correlation patterns, are suggested. It is demonstrated that these patterns present scale-independent and distinguishable measures for correlated and uncorrelated random multiplicative cascade processes. Their application to very high multiplicity events in relativistic heavy ion collisions is discussed.

Optical nonlinearity of nanocrystalline Au/ZnO composite films.

The third-order nonlinear optical susceptibilities, chi(3), of composite films consisting of nanocrystalline Au and ZnO particles were investigated by use of a degenerate four-wave mixing scheme. The maximum value of chi(3), measured at a laser wavelength of 532 nm and a pulse duration of 70 ps, was approximately 2 x 10(-6) esu. Also, this chi(3) value was achieved with small absorption (the surface-plasmon peak was at the 615-nm wavelength). Our composite materials showed no discernible degradation after they were subjected to irradiation for a total of 3 x 10(7) high-intensity pulses (24 Mw/cm2, 70-ps pulse duration at 500 Hz) during 16 h of testing.