Despite the classical hormonal effect, estrogen possesses a neuroprotective effect in the brain, which has led many to search for novel treatments for neurodegenerative diseases. Flavonoids, a group of compounds mainly derived from vegetables, share a resemblance, chemically, to estrogen, and indeed, some have been used as estrogen substitutes. To search for potential therapeutic agents against neurodegenerative diseases, different subclasses of flavonoids were analyzed and compared with estrogen. First, the estrogenic activities of these flavonoids were determined by activating the estrogen-responsive elements in cultured MCF-7 breast cancer cells. Second, the neuroprotective effects of flavonoids were revealed by measuring its inhibition effects on the formation of reactive oxygen species, the aggregation of beta-amyloid, and the induction of cell death by beta-amyloid in cultured neuronal PC12 cells. Among these flavonoids, baicalein, scutellarin, hibifolin, and quercetin-3'-glucoside possessed the strongest effect in neuroprotection; however, the neuroprotective activity did not directly correlate with the estrogenic activity of the flavonoids. Identification of these flavonoids could be very useful in finding potential drugs, or food supplements, for treating Alzheimer's disease.

In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y(1) receptor. The activation of the P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca(2+) mobilization in cultured chick myotubes. P2Y(1) receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y(1) receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus, and rat) but not in the chick embryo. In chicks after hatching, this P2Y(1) localization develops over approximately 3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y(1) transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR alpha-subunit were induced when the P2Y(1) receptors were activated by specific agonists or by overexpression of P2Y(1) receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter-reporter gene constructs for those subunits, actions that were blocked by a P2Y(1)-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.

Radix Astragali (RA) is commonly used as a health food supplement to reinforce the body vital energy. Flavonoids, including formononetin, ononin, calycosin, and calycosin-7-O-β-d-glucoside, are considered to be the major active ingredients within RA. Here, we provided different lines of evidence that the RA flavonoids stimulated the expression of erythropoietin (EPO), the central regulator of red blood cell mass, in cultured human embryonic kidney fibroblasts (HEK293T). A plasmid containing hypoxia response element (HRE), a critical regulator for EPO transcription, was tagged upstream of a firefly luciferase gene, namely, pHRE-Luc, which was being transfected into fibroblasts. The application of RA flavonoids onto the transfected cells induced the transcriptional activity of HRE. To account for the transcriptional activation after the treatment of flavonoids, the expression of hypoxia-inducible factor-1α (HIF-1α) was markedly increased: The increase was in both mRNA and protein levels. In addition, the degradation of HIF-1α was reduced under the effect of flavonoids. The regulation of HIF-1α therefore could account for the activation of EPO expression mediated by the RA flavonoids. The current results therefore reveal the function of this herb in enhancing hematopoietic functions.

Yu Ping Feng San (YPFS), a Chinese herbal decoction, is composed of Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu) and Saposhnikoviae Radix (SR; Fangfeng) in a weight ratio of 1∶2∶1. Clinically, YPFS has been widely used to regulate immune functions; however, the action mechanism of it is not known. Here, we addressed this issue by providing detail analyses of chemical and biological properties of YPFS. By using rapid resolution liquid chromatography coupled with mass spectrometry, fifteen chemicals deriving from different herbs of YPFS were determined, and which served as a control for the standardization of the herbal extract of YPFS. In general, the amounts of chosen chemical markers were higher in a preparation of YPFS as compared to that of single herb or two-herb compositions. In order to reveal the immune functions of YPFS, the standardized extract was applied onto cultured murine macrophages. The treatment of YPFS stimulated the mRNA and protein expressions of pro-inflammatory cytokines via activation of NF-κB by enhancing IκBα degradation. In contrast, the application of YPFS suppressed the expressions of pro-inflammatory cytokines significantly in the lipopolysaccharide (LPS)-induced chronic inflammation model. In addition, YPFS could up regulate the phagocytic activity in cultured macrophages. These results therefore supported the bi-directional immune-modulatory roles of YPFS in regulating the releases of cytokines from macrophages.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter-reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

Chinese date, the fruit of Ziziphus jujuba Mill., has thousands of years cultivation history, and about 700 cultivars of dates in China. Two types of dates are commonly found in the market: (i) fresh immature dates consumed as fruits, and (ii) dried mature dates used as Chinese medicines. Here, chemical and biological properties of these dates were revealed. Different sources of dates showed similar chemical profiles; however, the amounts of identified chemicals showed a great variation. The amount of nucleotides, flavonoids and polysaccharides in dates could be affected by its maturity and drying process. In parallel, the antioxidative functions of their extracts were compared. The date extracts protected PC12 cells against tBHP-induced cytotoxicity, and which also stimulated the transcriptional activity of antioxidant response element. The antioxidative effects were varied among different dates. The current results suggested the optimization of sources and specific usage of different maturity dates.

Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-)induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.

Danggui Buxue Tang (DBT), a Chinese herbal decoction used to treat ailments in women, contains Radix Astragali (Huangqi; RA) and Radix Angelicae Sinensis (Danggui; RAS). The weight ratio of RA to RAS used in DBT must be 5:1 as stipulated as early as AD 1247; however, DBT's mechanism of action has never been described. Here, the estrogenic effects of DBT were investigated by determining the phosphorylations of estrogen receptor a (ERa) and extracellular signal-regulated kinase 1/2 (Erk1/2) in cultured MCF-7 cells. The application of DBT triggered the phosphorylation of ERa and Erk1/2 in a time-dependent manner. In contrast to the effect of estrogen, DBT triggered ERa phosphorylation at both S118 and S167. This DBT-specific phosphorylation was not triggered by an extract of one of the individual herbs, or by mixing the extracts of RA and RAS. DBT-induced downstream signals are described here. These signals suggest the uniqueness of this Chinese herbal decoction that requires a well-defined formulation.