High exposure compared with standard exposure to metoclopramide associated with a higher risk of parkinsonism: a nationwide population-based cohort study.

AIMS: We conducted a cohort study utilizing a nationwide health insurance database to assess the European Medicines Agency's restrictions on using metoclopramide and its association with the risk of parkinsonism. METHODS: New oral metoclopramide users aged ≥20 years, and age- and gender-matched non-users were recruited between 2001 and 2011. Users were divided into high-exposure (dose >30 mg day-1 and/or duration >5 days) and standard-exposure (dose ≤30 mg day-1 and duration ≤5 days) groups. The adjusted hazard ratio (aHR) with 95% confidence interval (CI) estimated the risk of parkinsonism. RESULTS: During a 1-year period, 122 of 218 931 (0.06%) users of metoclopramide vs. 56 of 218 931 (0.03%) non-users developed parkinsonism (P < 0.001). Among the 122 cases of parkinsonism in users, 64 (0.04%) were from 168 566 standard-exposure users and 58 (0.12%) from 50 365 high-exposure users. Compared with non-users, the risk of parkinsonism was higher in users (aHR 2.16; 95% CI 1.54, 3.02), including standard-exposure (aHR 1.73; 95% CI 1.11, 2.70), and high-exposure (aHR 3.15; 95% CI 1.78, 5.57) users. High-exposure users had a higher risk of parkinsonism than standard-exposure users (aHR 1.83; 95% CI 1.28, 2.63). Within the high-exposure group, 45 233 of 50 365 (89.81%) users and 55 of 58 (94.83%) parkinsonism were from long-duration exposure; 5 132 of 50 365 (10.19%) users and 3 of 58 (5.17%) parkinsonism were from high-dose exposure and long-duration + high-dose exposure. CONCLUSIONS: The risk of parkinsonism in metoclopramide users, although extremely low (0.06%), is 2.16-fold greater than in non-users. High-exposure users have a 1.83-fold higher risk than standard-exposure users. As users in high-exposure group had a higher risk of parkinsonism than in standard-exposure group, and the majority of users and parkinsonism in high-exposure group were from long-duration exposure; thus, physician are advised to avoid prescribing metoclopramide for >5 days, even if the daily dose is ≤30 mg.

Increased Fall Risk in Patients Receiving Androgen Deprivation Therapy for Prostate Cancer.

OBJECTIVE: To examine the relationship between the use of androgen deprivation therapy (ADT) and the subsequent risk of falls in men with prostate cancer (PC) by employing a population-based dataset. METHODS: We retrieved the study sample from the Taiwan Longitudinal Health Insurance Database 2005. We included 886 patients with PC who had received ADT as the study group, whereas 862 patients with PC who had not received ADT served as the comparison group. We then individually tracked each study patient for a 3-year period to identify those who subsequently received a diagnosis of a fall. We performed Cox proportional hazard regressions to calculate the hazard ratio (HR) and its corresponding 95% confidence interval (CI) for a fall during the 3-year follow-up period between these 2 groups. RESULTS: The incidence rates of falls per 1000 person-years were 13.37 (95% CI: 9.15~18.88) and 6.44 (95% CI: 3.61~10.63), respectively, for patients with PC who received ADT and those who did not receive ADT. Furthermore, the hazard ratio for a fall during the 3-year follow-up period for patients with PC who had received ADT was 1.95 (95% CI: 1.04~3.66, P = .037) compared to those who had not received ADT after censoring sampled patients who died during the 3-year follow-up period and adjusting for age, geographical location, monthly income, urbanization level, hypertension, diabetes, hyperlipidemia, coronary heart disease, Parkinson's disease, epilepsy, stroke, and mental illness. CONCLUSION: The present findings suggest that patients with PC who had received ADT had an increased risk of falls.

Synthesis of Canthardin Sulfanilamides and Their Acid Anhydride Analogues via a Ring-Opening Reaction of Activated Aziridines and Their Associated Pharmacological Effects.

The cantharidinimide derivatives, 5a-h, including sulfanilamides containing pyrimidyl, pyrazinyl, hydrogen, thiazolyl, and oxazolyl groups were synthesized. Modification of cantharidinimide by means of the reaction of activated aziridine ring opening led to the discovery of a novel class of antitumor compounds. The analogues 10i-k, 11l-n, 12o-p, and 16q-s were obtained from treating cantharidinimide 6 and analogues (7, 8, and 13) with activated aziridines, which produced a series of ring-opened products including normal and abnormal types. Some of these compounds showed cytotoxic effects in vitro against HL-60, Hep3B, MCF7, and MDA-MB-231 cancer cells. The most potent cytostatic compound, N-cantharidinimido-sulfamethazine (5a), exhibited anti-HL-60 and anti-Hep3B cell activities. Two compounds 5g and 5h displayed slight effects on the Hep3B cell line, while the other compounds produced no response in these four cell lines.

Radix Scrophulariae extracts (harpagoside) suppresses hypoxia-induced microglial activation and neurotoxicity.

BACKGROUND: Hypoxia could lead to microglia activation and inflammatory mediators' overproduction. These inflammatory molecules could amplify the neuroinflammatory process and exacerbate neuronal injury. The aim of this study is to find out whether harpagoside could reduce hypoxia-induced microglia activation. METHODS: In this study, primary microglia cells harvested from neonatal ICR mice were activated by exposure to hypoxia (1 % O2 for 3 h). Harpagoside had been shown to be no cytotoxicity on microglia cells by MTT assay. The scavenger effect of harpagoside on hypoxia-enhanced microglial cells proliferation, associated inflammatory genes expression (COX-II, IL-1ß and IL-6 genes) and NO synthesis were also examined. RESULTS: Hypoxia enhances active proliferation of microglial cells, while harpagoside can scavenge this effect. We find that harpagoside could scavenge hypoxia-enhanced inflammatory genes expression (COX-2, IL-1ß and IL-6 genes) and NO synthesis of microglial cells. Under 3 h' hypoxic stimulation, the nuclear contents of p65 and hypoxia inducible factor-1α (HIF-1α) significantly increase, while the cytosol IκB-α content decreases; these effects can be reversed by 1 h's pre-incubation of 10(-8) M harpagoside. Harpagoside could decrease IκB-α protein phosphorylation and inhibit p65 protein translocation from the cytosol to the nucleus, thus suppress NF-κB activation and reduce the HIF-1α generation. CONCLUSION: These results suggested that the anti-inflammatory mechanism of harpagoside might be associated with the NF-κB signaling pathway. Harpagoside protect against hypoxia-induced toxicity on microglial cells through HIF-α pathway.

Establishment of a two-dimensional chiral HPLC system for the simultaneous detection of lactate and 3-hydroxybutyrate enantiomers in human clinical samples.

A two-dimensional chiral high-performance liquid chromatography system was established for simultaneous detection of lactate (LA) and 3-hydroxybutyrate (3HB) enantiomers in human clinical samples. d-LA is increased upon kidney damage but 3HB protected against kidney injury. Therefore, determining the concentrations of D,L-LA and D,L-3HB simultaneously would be useful for evaluating pathological conditions. LA and 3HB were pre-column-derivatized with the fluorescent reagent 4-(N-chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) at 60 °C for 15 min and separated in the first dimension with a capillary monolithic octadecylsilane column. The mobile phase consisted of 13% acetonitrile and 0.05% tirfluoroacetic acid in water. Chiralpak QD-AX and KSAACSP-001S enantioselective columns were used in the second dimension to separate LA and 3HB enantiomers, respectively. Mobile phases were mixed solutions of methanol and acetonitrile containing formic acid. The separation factors were 1.14 and 1.08, respectively. The detection limit of LA and 3HB enantiomers was 10 fmol/injection. This method was applied to human clinical samples; intra- and inter-day relative standard deviations of LA and 3HB enantiomers were, respectively, 1.04-3.25% and 1.61-5.12% in plasma, 9.19-11.2% and 4.60-5.89% in urine, and 7.12-8.90% and 2.86-6.97% in saliva. This novel analytical method is a powerful tool for investigating variations in LA and 3HB enantiomers under disease conditions.

Arthropod steroid hormone (20-Hydroxyecdysone) suppresses IL-1ß-induced catabolic gene expression in cartilage.

BACKGROUND: In osteoarthritis (OA), the imbalance of chondrocytes' anabolic and catabolic factors can induce cartilage destruction. Interleukin-1 beta (IL-1ß) is a potent pro-inflammatory cytokine that is capable of inducing chondrocytes and synovial cells to synthesize MMPs. The hypoxia-inducible factor-2alpha (HIF-2alpha, encoded by Epas1) is the catabolic transcription factor in the osteoarthritic process. The purpose of this study is to validate the effects of ecdysteroids (Ecd) on IL-1ß-induced cartilage catabolism and the possible role of Ecd in treatment or prevention of early OA. METHODS: Chondrocytes and articular cartilage was harvested from newborn ICR mice. Ecd effect on chondrocytes viability was tested and the optimal concentration was determined by MTT assay. The effect of HIF-2α (EPAS1) in cartilage catabolism simulated by IL-1ß (5 ng/ml) was evaluated by articular cartilage explants culture. The effects of Ecd on IL-1ß-induced inflammatory conditions and their related catabolic genes expression were analyzed. RESULTS: Interleukin-1ß (IL-1ß) treatment on primary mouse articular cartilage explants enhanced their Epas1, matrix metalloproteinases (MMP-3, MMP-13) and ADAMTS-5 genes expression and down-regulated collagen type II (Col2a1) gene expression. With the pre-treatment of 10(-8) M Ecd, the catabolic effects of IL-1ß on articular cartilage were scavenged. CONCLUSION: In conclusions, Ecd can reduce the IL-1ß-induced inflammatory effect of the cartilage. Ecd may suppress IL-1ß-induced cartilage catabolism via HIF-2α pathway.

Osteoporosis is associated with antiepileptic drugs: a population-based study.

Controversy remains regarding the risk of bone abnormalities due to enzyme-inducing antiepileptic drugs (EIAEDs) and non-enzyme-inducing antiepileptic drugs (NEIAEDs). This case-control study aimed to investigate the possible association between osteoporosis and epilepsy disease and AEDs therapy using a population-based dataset in Taiwan. We first identified 48,102 cases, ≥ 18 years of age, who received a first-time diagnosis of osteoporosis, and then randomly selected 144,306 controls. We used conditional logistic regression analyses to compute the odds ratio (OR) and corresponding 95% confidence interval (CI) to compare a previous diagnosis of epilepsy between cases and controls. We found that of the 192,408 sampled subjects, epilepsy was found in 117 (0.24%) cases and 240 (0.17%) controls (p<0.001). Cases were found to be more likely to have previously been diagnosed with epilepsy than controls (OR: 1.41, 95% CI: 1.11 ≈ 1.78, p<0.01), after taking confounders into consideration. Furthermore, we found that, compared to controls, the adjusted OR of cases in which enzyme-inducing AEDs had been prescribed was 2.06 (95% CI: 1.43 ≈ 2.95). A higher proportion of cases with prescribed NEIAED was also found (OR: 2.09, 95% CI: 1.49 ≈ 2.92) compared to controls. This study demonstrates that patients with osteoporosis were more likely to have epilepsy and receive EIAED or NEIAED treatment. For patients with epilepsy who take AEDs, attention should be paid to the adverse effects of osteoporosis.

Biotransformation of dihydroisosteviol and the effects of transformed products on steroidogenic gene expressions.

The biotransformation of dihydroisosteviol with Absidia pseudocylindrospora ATCC 24169, Streptomyces griseus ATCC 10137, Mucor recurvatus MR36, and Aspergillus niger BCRC 31130 yielded 15 metabolites, eight of which were previously unknown. Structures of metabolites were established by 2D NMR techniques and HRMS data, two of which were further corroborated by chemical means, and another via single-crystal X-ray diffraction analysis. Subsequently, two steroidogenic cell lines (Y-1 mouse adrenal tumor and MA-10 mouse Leydig tumor cells) were used in a reverse transcription-PCR analysis to assess the effects of all compounds on steroidogenic gene expressions using forskolin as a positive control. The tested gene expressions included steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) enzyme. Gene expression profiles showed that ten of the tested compounds effectively suppressed P450SCC mRNA expression in both Y-1 and MA-10 cells. Several induced SF-1 gene expression and two enhanced StAR gene expression in Y-1 cells. By contrast, in MA-10 cells, one compound effectively suppressed StAR mRNA expression, whereas for others effectively suppressed SF-1 gene expression. The results suggest that analogs of dihydroisosteviol can be potential modulators to alter steroidogenic gene expressions and subsequent enzyme activities.

Biological characterization of oxidized hyaluronic acid/resveratrol hydrogel for cartilage tissue engineering.

Osteoarthritis is a type of arthritis that is caused by breakdown of cartilage, with eventual loss of the cartilage of the joints. The ability of self-repair in damaged cartilage tissue is limited; the aim of this work is to fabricate and characterize an oxidized hyaluronic acid/resveratrol (Oxi-HA/Res) hydrogel for future applications in cartilage tissue engineering. Under physiological conditions, the Oxi-HA/Res hydrogel was prepared by chemical crosslinking of Oxi-HA with resveratrol solution and characterized by Fourier transform infrared spectrometry assay; the biocompatibility and gene expression of chondrocytes within the Oxi-HA-Res hydrogel then analyzed. The cell viability and cytotoxicity assays showed that the Oxi-HA/Res hydrogel has good biocompatibility. Oxi-HA/Res hydrogel can upregulate expression of type II collagen, aggrecan, and Sox-9 genes; while down-regulating IL-1ß, MMP-1, MMP-3, MMP13 gene expression. It can also reduce LPS-induced inflammation and chondrocyte damage. The results of this study showed that the Oxi-HA/Res hydrogel is biocompatible with chondrocytes, allows for extracellular matrix synthesis, and also reduce LPS-induced inflammation and damage. These results suggest that Oxi-HA/Res hydrogel may be a potential suitable cell carrier for chondrocyte cells in the treatment of cartilage defect. However, further in vivo study is mandatory for future possible clinical applications.

A simple procedure for preparation of N-thiazol, thiadiazol, pyridyl and sulfanylamidocantharidinimines analogues and evaluation of their cytotoxicities against human HL-60, MCF7, Neuro-2a and A549 carcinoma cell.

The lab made an effort to prepare some biological active cantharidinimines by heating the reactant 1 and 2a-g, 5h-i and 7j-r amines to suitable temperature with ethanol to provide 18 N-thiazolyl-, sulfanyl-, aminopyridyl-, bromopyridyl-, alkylpyridyl- and hydroxypyridylcantharidinimines 3a-g, 4a-c, 6h-i and 8j-r in yield of 4-77% (Chart 1). These cantharidinimine derivatives were tested for their capabilities to suppress growth of the human carcinoma cell lines, HL-60, MCF7, Neuro-2a and A549, because the incidence rate is more prominent in Asian countries than western countries. Compounds 3c-d and 6h-i were found to have some antitumor activity in HL-60 but less activity in MCF cell and compounds 8j-l displayed some inhibition effects to A549 cell line, but less effect to Neuro-2a cell line. Compounds 8m-r had no cytotoxic effect against both cell lines. The cytotoxic effects of these cantharidinimine compounds seemed to be better than the cantharidinimide compounds which we had mentioned several years ago.

Stimulatory effect of puerarin on bone formation through co-activation of nitric oxide and bone morphogenetic protein-2/mitogen-activated protein kinases pathways in mice.

BACKGROUND: Estrogen deficiency results in loss of bone mass. Phytoestrogens are plant-derived non-steroidal compounds with estrogen-like activity that bind to estrogen receptors. The main aim of this study was to investigate the effect of the phytoestrogen puerarin on adult mouse osteoblasts. METHODS: Osteoblast cells were harvested from 8-month old female imprinting control region (ICR) mice. The effects of puerarin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The production of nitric oxide (NO) and the expression of bone morphogenetic protein-2 (BMP-2), SMAD4, mitogen-activated protein kinases (MAPK), core binding factor α1/ runt-related transcription factor 2 (Cbfa1/Runx2), osteoprotegerin (OPG), and receptor activator of NF-κB ligand (RANKL) genes were analyzed. The activation of signal pathways was further confirmed by specific pathway inhibitors. RESULTS: The osteoblast viability reached its maximum at 10(-8) mol/L puerarin. At this concentration, puerarin increases the proliferation and matrix mineralization of osteoblasts and promotes NO synthesis. With 10(-8) mol/L puerarin treatment, BMP-2, SMAD4, Cbfa1/Runx2, and OPG gene expression were up-regulated, while the RANKL gene expression is down-regulated. Concurrent treatment involving the (bone morphogenetic protein) BMP antagonist Noggin or the NOS inhibitor L-NAME diminishes puerarin induced cell proliferation, Alkaline phosphatase (ALP) activity, NO production, as well as the BMP-2, SMAD4, Cbfa1/Runx2, OPG, and RANKL gene expression. CONCLUSIONS: In this in vitro study, we demonstrate that puerarin is a bone anabolic agent that exerts its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expression. This effect may contribute to its induction of osteoblast proliferation and differentiation, resulting in bone formation.

Synthesis and antibacterial activities of novel 4-hydroxy-7-hydroxy- and 3-carboxycoumarin derivatives.

Coumarin derivatives are used as fluorescent dyes and medicines. They also have some notable physiological effects, including the acute hepatoxicity and carcinogenicity of certain aflatoxins, the anticoagulant action of dicoumarol, and the antibiotic activity of novobicin and coumerymycin A1. Because the number of drug resistant strains is increasing at present, the synthesis of new antibacterial compounds is one of the critical methods for treating infectious diseases. Therefore, a series of coumarinsubstituted derivatives, namely 4-hydroxy- and 7-hydroxycoumarins, and 3-carboxycoumarins were synthesized. 4-Hydroxycoumarin derivatives 4a-c underwent rearrangement reactions. Both 4- and 7-hydroxycoumarins were treated with activated aziridines which produced series of ring-opened products 7, 8, 10, and 11. 3-Carboxy-coumarin amide dimer derivatives 14-21 were prepared by reacting aliphatic alkylamines and alkyldiamines with PyBOP and DIEA. In this study, we use a new technique called modified micro-plate antibiotic susceptibility test method (MMAST), which is more convenient, more efficient, and more accurate than previous methods and only a small amount of the sample is required for the test. Some of the compounds were produced by reactions with acid anhydrides and demonstrated the ability to inhibit Gram-positive microorganisms. The dimer derivatives displayed lower antibacterial activities.

The protective effects of coumestrol against amyloid-ß peptide- and lipopolysaccharide-induced toxicity on mice astrocytes.

OBJECTIVES: Estrogen replacement therapy can decrease the risk of developing Alzheimer's disease. Phytoestrogens have been proposed as potential alternatives to estrogen replacement therapy. The purpose of this study was to evaluate the in vitro protective effects of coumestrol on mice astrocytes. METHODS: Different concentrations of coumestrol were tested for their protective efficacy against two toxic insults, lipopolysaccharide (LPS) and amyloid-beta peptide, on astrocytes. The mitochondrial activity of astrocytes was determined, and the protective efficacy and pathway were examined by their specific gene expression and protein change. RESULTS: The results showed that coumestrol induced a modest but significant increase in viability of astrocytes, while the viability of astrocytes was reduced following exposure to LPS and amyloid-beta peptide. The addition of coumestrol could reverse the toxic effect induced by LPS and amyloid-beta peptide. Both the LPS and amyloid-beta peptide enhanced interleukin 1, interleukin 6, and tumor necrosis factor-alpha synthesis and these effects were inhibited by 10(-9)M coumestrol. This effect was more obvious on the LPS-induced inflammation. The estrogen receptor expression was upregulated by coumestrol, while the effect was more obvious on estrogen receptor-beta (ER-beta). These effects can be inhibited by extracellular signal-regulated kinase and c-Jun N-terminal kinase inhibitors but not p38 inhibitor. DISCUSSION: The current data support a possible role for astrocytes in the mediation of neuroprotection by coumestrol. An indirect extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling pathway to downregulate the expression of interleukin 1, interleukin 6, and the tumor necrosis factor-alpha cytotoxic effect may act in concert with the proposed direct ER-beta biosynethsis pathway to achieve a widespread, global protection of ER-beta positive neurons.

Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis.

Icariin has been reported to enhance bone healing and treat osteoporosis. In this study, we examined the detail molecular mechanisms of icariin on lipopolysaccharide (LPS)-induced osteolysis. Our hypothesis is that icariin can inhibit osteoclast differentiation and bone resorption by suppressing MAPKs/NF-κB regulated HIF-1α and PGE(2) synthesis. After treatment with icariin, the activity of osteoclasts differentiation maker, tatrate resistances acid phosphatease (TRAP), significantly decreased at the concentration of 10(-8)M. Icariin (10(-8)M) reduced the size of LPS-induced osteoclasts formation, and diminished their TRAP and acid phosphatease (ACP) activity without inhibition of cell viability. Icariin also inhibited LPS-induced bone resorption and interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) expression. The gene expression of osteoprotegerin (OPG) was up-regulated, while receptor activator of NF-κB ligand (RANKL) was down-regulated. Icariin also inhibited the synthesis of cyclo-oxygenase type-2 (COX-2) and prostaglandin E(2) (PGE(2)). In addition, icariin had a dominant repression effect on LPS-induced hypoxia inducible factor-1α (HIF-1α) expression of osteoclasts. On osteoclasts, icariin suppresses LPS-mediated activation of the p38 and JNK; while on the osteoblasts, icariin reduced the LPS-induced activation of ERK1/2 and I-kappa-B-alpha (IκBα), but increased the activation of p38. In conclusion, we demonstrated that icariin has an in vitro inhibitory effects on osteoclasts differentiation that can prevent inflammatory bone loss. Icariin inhibited LPS-induced osteoclastogenesis program by suppressing activation of the p38 and JNK pathway.

Icariin protects murine chondrocytes from lipopolysaccharide-induced inflammatory responses and extracellular matrix degradation.

Septic arthritis is an inflammatory arthropathy characterized by degeneration of articular cartilage. Icariin, the main active flavonoid glucoside isolated from Epimedium pubescens, is used as antirheumatics (or antiinflammatory), tonics, and aphrodisiacs in traditional Chinese medicine. In this study, we used lipopolysaccharide (LPS) to simulate the in vitro inflammatory response of chondrocytes during septic arthritis. Our hypothesis is that the icariin can protect chondrocytes from LPS-induced inflammation and extracellular matrix degradation. The inflammation of neonatal mice chondrocytes was induced by LPS and the antiinflammatory effects were examined. The synthesis of nitric oxide was analyzed, whereas the titer of glycosaminoglycan and total collagen were measured and the gene expressions (including inducible nitric oxide synthase [iNOS], matrix metalloproteinase [MMP]-1, MMP-3, and MMP-13) were evaluated. The results showed that the viability of chondrocytes, extracellular matrix synthesis, was significantly decreased, whereas nitric oxide synthesis was significantly increased in the presence of 10(-5) g/mL LPS. Icariin pretreatment can partially reverse these effects. The up-regulated expressions of MMP-1, 3, 13, cyclooxygenase-2 (COX-2), and iNOS genes by LPS treatment were also significantly down-regulated by the pretreatment of icariin to 1.8%, 0.056%, 7.7%, 3.1%, and 5.3% of the LPS-positive control sample, respectively. Our results demonstrate that icariin is a safe anabolic agent of chondrocytes. Icariin may exert its protective effects through inhibition of nitric oxide and MMP synthesis, and may then reduce the extracellular matrix destruction.

Icariin isolated from Epimedium pubescens regulates osteoblasts anabolism through BMP-2, SMAD4, and Cbfa1 expression.

Epimedii herba is one of the most frequently used herbs in formulas prescribed for the treatment of osteoporosis in China. The main active flavonoid glucoside extracted from Epimedium pubescens is Icariin, which has been reported to enhance bone healing and reduce osteoporosis occurrence. However, the detailed molecular mechanisms remain unclear. In this present study, we examine the molecular mechanisms of icariin by using primary osteoblast cell cultures obtained from adult mice. The osteoblast cells were harvested from 8-month old female Imprinting Control Region (ICR) mice. The effects of icariin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The production of nitric oxide (NO) and caspase-3 were analyzed, along with the gene expressions of bone morphogenetic protein-2 (BMP-2), SMAD4, Cbfa1/Runx2, OPG, and RANKL. The viability of the osteoblasts reached its maximum at 10(-8)M icariin. At this concentration, icariin increased the proliferation and matrix mineralization of osteoblasts and promoted NO synthesis. With icariin treatment, the BMP-2, SMAD4, Cbfa1/Runx2, and OPG gene expressions were up-regulated; the RANKL gene expression was however down-regulated. Concurrent treatment involving the BMP antagonist (Noggin) or the NOS inhibitor (L-NAME) diminished the icariin-induced cell proliferation, ALP activity, NO production, as well as the BMP-2, SMAD4, Cbfa1/Runx2, OPG, RANKL gene expressions. In this study, we demonstrate that in vitro icariin is a bone anabolic agent that may exert its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expressions. This effect may contribute to its action on the induction of osteoblasts proliferation and differentiation, resulting in bone formation.

Ghrelin improves LPS-induced gastrointestinal motility disturbances: roles of NO and prostaglandin E2.

Ghrelin, an important orexigenic peptide, exerts gastroprokinetic and anti-inflammatory effects. We investigated the role of ghrelin in LPS-induced gastrointestinal (GI) motility disturbances through NO and prostaglandin E2 pathways in mice. Ghrelin-containing cells and its receptor, growth hormone secretagogue receptor 1 (GHSR-1), were localized in the stomach and duodenum using an immunohistochemical method. The distribution of ghrelin-containing cells or GHSR-1 immunoreactivity in both the mucosal and the muscle layers was heterogeneous within both tissues. The i.p. administration of ghrelin (1-20 microg/kg) had no effect on gastric emptying but markedly increased the GI transit (GIT) in normal mice. LPS (20 mg/kg i.p.)-treated mice showed significant decreases in the gastric emptying and GIT. Ghrelin attenuated the LPS-induced delay in gastric emptying and GIT. We also performed immunohistochemical experiments on both tissues. Immunohistochemistry showed the presence of iNOS and cyclooxygenase 2 in both tissues of LPS-treated mice. Treatment of LPS-exposed mice with ghrelin (20 microg/kg) diminished the presence of iNOS but not cyclooxygenase 2 in both tissues. The effect of ghrelin on regulating LPS-induced GI motility disturbance was further found to be associated with a reduction in iNOS expression in the GI tract and plasma NO overproduction rather than regulation of neural or endothelial NO synthase expression in the GI tissue. In addition, ghrelin was found to elevate prostaglandin E2 levels in the GI tissue but showed no significant change in LPS-treated mice. These findings indicate that the action of ghrelin binding to GHSR-1 improves endotoxemia-induced GI motility disturbances mainly through down-regulating the NO pathway in the GI tract.

Anti-inflammatory effects of daidzein on primary astroglial cell culture.

INTRODUCTION: Alzheimer's disease is the common cause of dementia in old people. The pathological hallmarks of Alzheimer's disease include neuronal loss, deposition of amyloid-beta, and presence of neurofibrillary tangles. The endogenous steroid estrogen has been shown to affect neuronal growth, differentiation and survival, while isoflavones also have a neuroprotective effect on human cortical neurons. Daidzein, however, has a superior neuron-protective effect to other isoflavones. The present study is to determine whether daidzein is able to inhibit the production of pro-inflammatory mediators under amyloid-beta and lipopolysaccharide stimulation. MATERIALS AND METHODS: Astrocyte cells were stimulated with amyloid-beta or lipopolysaccharide in the absence and presence of diadzein. Nitric oxide released into the culture media was determined using the Griess reaction, and concentrations of IL-1, IL-6, TNF-alpha and estrogen receptor gene expression were measured by semi-quantitative real-time polymerase chain reaction assay. RESULTS: Diadzein-treatment increases astrocyte cell counts and attains its maximal effect at the 10(-12)M concentration. The addition of 20 microM amyloid-beta or 10(-6) g/ml LPS can significantly decrease the viability of astrocytes, up-regulated IL-1, IL-6, TNF-alpha mRNA and estrogen receptor expression; in addition, 1-h daidzein pre-treatment can restore the decreased viability of astrocytes induced by amyloid-beta or lipopolysaccharide as well as down-regulate their mRNA expression. CONCLUSIONS: It seems that this response is estrogen receptor-mediated. These results further increase the possibility that daidzein may have potential to ameliorate the inflammatory process and also alleviate the risk of Alzheimer's disease progression.

Effects of coumestrol on neonatal and adult mice osteoblasts activities.

Estrogen replacement therapy has been shown to reduce postmenopausal osteoporosis. In the present study, we examined the effects of the phytoestrogen coumestrol on neonatal and adult osteoblasts metabolism. Two different sources of osteoblast cells (neonatal mice calvaria and adult mice long bone) cultures were used in this study. The effects of coumestrol on the cellular activities were analyzed by the mitochondrial tetrazolium (MTT) assay, secretion of alkaline phosphatase (ALP), intracellular calcium content (Ca), and the gene expression of bone matrix protein, estrogen receptors (ER-alpha, ER-beta), and osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL). The results showed that the proliferation of neonatal mice osteoblast cells was enhanced by treatment of coumestrol. In the presence of 10(-9)M coumestrol, the osteoblast proliferation attained 139.5% of the control and that the coumestrol can increase the intracellular calcium contents. Type I collagen gene expression was upregulated 167% at the 1st day's culture; ALP gene expression was upregulated 360% at the 7th day's culture; while the osteocalcin gene expression was upregulated 222% at the 14th day's culture. When adult mice osteoblasts were cultured in the presence of 10(-9)M coumestrol, the osteoblasts population increased significantly earlier and attained its maximal effect at the 21st day's culture with 207.4% of control group. The content of ER-beta and osteoprotegerin secretion by neonatal mice control cells gradually increased during osteoblasts differentiation, whereas the ER-alpha and OPGL content were decreased in this study. The cellular responses to the estradiol and counmestrol were quite different in the osteoblasts derived from different age.

Study on the stevioside analogues of steviolbioside, steviol, and isosteviol 19-alkyl amide dimers: synthesis and cytotoxic and antibacterial activity.

A new group of steviolbioside amide dimers 2a-g, derivatives 2h-i and their related steviol and isosteviol amide dimers 3a and 4a were prepared by reacting aliphatic alkylamine and alkyldiamines with PyBOP and DIEA. The synthesized compounds had cytotoxic effects on cancer and human embryonic lung cells. Compounds 3a, 4a, 2b and 2h were cytotoxic to cancer cells and to a lesser extent to human embryo lung cells. Compounds 2f, 2g and 4 of this series had favorable antibacterial effects, and were superior to penicillin G at inhibiting growth of Bacillus subtilis (BCRC 10029). The cytotoxicity and antibacterial effects may depend on the dimerization and derivative moieties in relation to the respective aglycons.