Judy Ting Ting Zhu

Danggui Buxue Tang – A Chinese herbal decoction activates the phosphorylations of extracellular signal‐regulated kinase and estrogen receptor α in cultured MCF‐7 cells

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, DBTs mechanism of action has never been described. Here, the estrogenic effects of DBT were investigated by determining the phosphorylations of estrogen receptor α (ERα) and extracellular signal‐regulated kinase 1/2 (Erk1/2) in cultured MCF‐7 cells. The application of DBT triggered the phosphorylation of ERα and Erk1/2 in a time‐dependent manner. In contrast to the effect of estrogen, DBT triggered ERα 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.

A Chinese Herbal Decoction, Danggui Buxue Tang, Stimulates Proliferation, Differentiation and Gene Expression of Cultured Osteosarcoma Cells: Genomic Approach to Reveal Specific Gene Activation

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). When DBT was applied onto cultured MG-63 cells, an increase of cell proliferation and differentiation of MG-63 cell were revealed: both of these effects were significantly higher in DBT than RA or RAS extract. To search for the biological markers that are specifically regulated by DBT, DNA microarray was used to reveal the gene expression profiling of DBT in MG-63 cells as compared to that of RA- or RAS-treated cells. Amongst 883 DBT-regulated genes, 403 of them are specifically regulated by DBT treatment, including CCL-2, CCL-7, CCL-8, and galectin-9. The signaling cascade of this DBT-regulated gene expression was also elucidated in cultured MG-63 cells. The current results reveal the potential usage of this herbal decoction in treating osteoporosis and suggest the uniqueness of Chinese herbal decoction that requires a well-defined formulation. The DBT-regulated genes in the culture could serve as biological responsive markers for quality assurance of the herbal preparation.

A flavonol glycoside, isolated from roots of Panax notoginseng, reduces amyloid-beta-induced neurotoxicity in cultured neurons: signaling transduction and drug development for Alzheimer's disease.

A Radix Notoginseng flavonol glycoside (RNFG), quercetin 3-O-beta-D-xylopyranosyl-beta-D-galactopyranoside, was isolated from roots of Panax notoginseng. Among different biological properties tested, RNFG possessed a strong activity in preventing amyloid-beta (Abeta)-induced cell death. In an in vitro assay, RNFG inhibited the aggregation of Abeta in a dose-dependent manner. Moreover, application of RNFG in cultured cortical neurons, or PC12 cells, reduced the Abeta-induced cell death in time- and dose-dependent manners, with the suppression of Abeta-induced DNA fragmentation and caspase-3 activation. In cultured neurons, the pre-treatment of RNFG abolished the increase of Ca(2+) mobilization triggered by Abeta. The neuroprotective properties of RNFG required a specific sugar attachment within the main chemical backbone because the flavonol backbone by itself did not show any protective effect. In memory impairment experiments using the passive avoidance task, the administration of RNFG reduced brain damage in scopolamine-treated rats. These results therefore reveal a novel function of Radix Notoginseng and its flavonol glycoside that could be very useful in developing food supplements for the prevention, or potential treatment, of Alzheimers disease.

Estrogenic and Neuroprotective Properties of Scutellarin from Erigeron breviscapus: A Drug against Postmenopausal Symptoms and Alzheimer's Disease

Besides the classical hormonal effect, estrogen possesses neuroprotective effects in the brain, which leads to the searching of novel treatments for neurodegenerative diseases such as Alzheimers disease. Scutellarin is a major flavone derived from Herba Erigerontis, a Chinese medicine derived from Erigeron breviscapus, which has been shown here to possess both estrogenic and neuroprotective properties. Scutellarin showed the estrogenic effects by activating the estrogen responsive elements and phosphorylation of estrogen receptor alpha in cultured MCF-7 cells: the activation was in a dose-dependent manner. On the other hand, scutellarin inhibited the aggregation of beta-amyloid in vitro, and prevented the cell death mediated by beta-amyloid when applied to cultured neuronal PC12 cells. These results therefore suggested that Herba Erigerontis and its component scutellarin might have therapeutic effects against postmenopausal symptoms and Alzheimers disease.

Synergistic Action of Flavonoids, Baicalein, and Daidzein in Estrogenic and Neuroprotective Effects: A Development of Potential Health Products and Therapeutic Drugs against Alzheimer’s Disease

Despite the classical hormonal effect, estrogen has been reported to mediate neuroprotection in the brain, which leads to the searching of estrogen-like substances for treating neurodegenerative diseases. Flavonoids, a group of natural compounds, are well known to possess estrogenic effects and used to substitute estrogen, that is, phytoestrogen. Flavonoid serves as one of the potential targets for the development of natural supplements and therapeutic drugs against different diseases. The neuroprotection activity of flavonoids was chosen for a possible development of anti-Alzheimers drugs or food supplements. The estrogenic activity of two flavonoids, baicalein and daidzein, were demonstrated by their strong abilities in stimulating estrogen receptor phosphorylation and transcriptional activation of estrogen responsive element in MCF-7 breast cells. The neuroprotection effects of flavonoids against β-amyloid (Aβ) were revealed by their inhibition effects on in vitro Aβ aggregation and Aβ-induced cytotoxicity in PC12 neuronal cells. More importantly, the estrogenic and neuroprotective activities of individual flavonoid could be further enhanced by the cotreatment in the cultures. Taken together, this synergistic effect of baicalein and daidzein might serve as a method to improve the therapeutic efficacy of different flavonoids against Aβ, which might be crucial in developing those flavonoidsin treating Alzheimers disease in the future.

Ligustilide suppresses the biological properties of Danggui Buxue Tang: a Chinese herbal decoction composed of radix astragali and radix angelica sinensis.

Danggui Buxue Tang (DBT), a herbal decoction composed of Radix Astragali (RA) and Radix Angelica sinensis (RAS), has been used for treating menopausal irregularity in women for more than 800 years in China. According to the old tradition, RAS had to be processed with yellow wine before DBT preparation, which markedly reduced the amount of ligustilide in RAS and DBT, as well as enhanced the bioactivities of DBT. Here, we hypothesized that ligustilide would be an ingredient that possessed suppressive effects on DBTs functions. In the presence of ligustilide, the amount of astragaloside IV, calycosin, formononetin, and total polysaccharides extracted from RA were decreased. An increase of ligustilide caused a decrease of DBTs osteogenic activity in stimulating proliferation and differentiation of cultured bone cells. In addition, in the presence of a high level of ligustilide, DBT caused a side effect inducing the proliferation of breast MCF-7 cells. The current results strongly suggest that ligustilide is a negative regulator that hinders DBT to achieve its biological efficacy, which supports the traditional practice of preparing DBT using the ethanol-treated RAS.

Hibifolin, a flavonol glycoside, prevents β-amyloid-induced neurotoxicity in cultured cortical neurons

The toxicity of aggregated beta-amyloid (A beta) has been implicated as a critical cause in the development of Alzheimers disease (AD). Hibifolin, a flavonol glycoside derived from herbal plants, possessed a strong protective activity against cell death induced by aggregated A beta. Application of hibifolin in primary cortical neurons prevented the A beta-induced cell death in a dose-dependent manner. In cultured cortical neurons, the pre-treatment of hibifolin abolished A beta-induced Ca(2+) mobilization, and also reduced A beta-induced caspase-3 and caspase-7 activation. Moreover, DNA fragmentation induced by A beta could be suppressed by hibifolin. In addition to such protection mechanisms, hibifolin was able to induce Akt phosphorylation in cortical neurons, which could be another explanation for the neuroprotection activity. These results therefore provided the first evidence that hibifolin protected neurons against A beta-induced apoptosis and stimulated Akt activation, which would be useful in developing potential drugs or food supplements for treating AD.

Fo shou san, an ancient herbal decoction prepared from rhizoma chuanxiong and radix angelicae sinensis, stimulates the production of hemoglobin and erythropoietin in cultured cells.

Fo Shou San (FSS) is an ancient herbal decoction comprised of Rhizoma Chuanxiong (RC; Chuanxiong) and Radix Angelicae Sinensis (RAS; Danggui) in a ratio of 2 : 3. It is mainly prescribed for patients having a blood deficiency. This combination is considered the most popular herb pair among Chinese medicines; however, the rationale of having these two chemically similar herbs within the decoction has historically not been made clear. Here, we attempted to reveal the chemical and biological properties of this decoction as a means to deduce its mechanism of action. The effects of FSS were determined in different cell culture models. With respect to stimulation of blood circulation, FSS inhibited ADP-mediated platelet aggregation in a dose-dependent manner. In order to reveal the hematopoietic effect of this decoction, FSS was applied onto cultured K562 human leukemia cells and Hep3B human hepatocellular carcinoma cells. Application of FSS in cultured K562 cells inhibited cell proliferation and subsequently induced the production of hemoglobin. Additionally, the mRNA expression of erythropoietin (EPO) was induced in a dose-dependent manner when FSS was applied to Hep3B cells. The current results reveal the effects of FSS in different cell models, paving a direction for mechanistic studies.

Expression and Localization of PRiMA-Linked Globular Form Acetylcholinesterase in Vertebrate Neuromuscular Junctions

Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.

A new variant of proline-rich membrane anchor (PRiMA) of acetylcholinesterase in chicken: Expression in different muscle fiber types

Proline-rich membrane anchor (PRiMA) is a molecule to organize acetylcholinesterase (AChE) into tetrameric globular form (G(4)) that anchors onto the plasma membrane in brain and muscle. In mammal, PRiMA is encoded by a single gene with two splicing variants, PRiMA I and PRiMA II: PRiMA II is different to PRiMA I by its absence of a C-terminal cytoplasmic domain. The existence of these isoforms has not been revealed in avian specie. By using RT-PCR and bioinformatic analyses, two splicing variants of PRiMA were identified in chicken cerebrum. One variant contains very similar domains as compared to mammalian PRiMA I. The other variant, named as PRiMA II, has a very distinct cytoplasmic C-terminus of having 26 amino acids. Both forms of chicken PRiMA were able to organize the formation of G(4) AChE when that was over expressed together with AChE(T) subunit in cultured cells. The level of PRiMA mRNA, mainly PRiMA I, was higher in slow-twitch muscle than that of in fast-twitch muscle of chicken. This finding suggests that the muscle fiber type-specific expression of G(4) AChE in chicken could be a result of the different expression pattern of PRiMA in fast- and slow-twitch muscles.