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.
The regulatory mechanism of DBT-induced EPO gene expression via HIF-1α in HEK293T cell was revealed. This regulation involves two distinct mechanisms: (i) transcriptional level by increasing the ...HIF-1α mRNA and (ii) translational level by enhancing the HIF-1α protein.
Danggui Buxue Tang (DBT), a Chinese medicinal decoction that is being commonly used as hematopoietic medicine to treating woman menopausal irregularity, contains two herbs: Radix Astragali and Radix Angelicae Sinensis. Pharmacological results indicate that DBT can stimulate the production of erythropoietin (EPO), a specific hematopoietic growth factor, in cultured cells.
In order to revealing the mechanism of DBT's hematopoietic function, this study investigated the activity of the DBT-induced EPO expression and the upstream regulatory cascade of EPO via hypoxia-induced signaling in cultured kidney fibroblasts (HEK293T).
DBT-induced mRNA expressions were revealed by real-time PCR, while the change of protein expressions were analyzed by Western blotting. For the analysis of hypoxia-dependent signaling, a luciferase reporter was used to report the transcriptional activity of hypoxia response element (HRE).
The plasmid containing HRE, being transfected into HEK293T, was highly responsive to the challenge of DBT application. To account for the transcriptional activation of HRE, DBT treatment was shown to increase the mRNA and protein expressions of
hypoxia-
inducible
factor-1α (HIF-1α). In addition, the activation of Raf/MEK/ERK signaling pathway by DBT could also enhance the translation of HIF-1α, suggesting the dual actions of DBT in stimulating the EPO expression in kidney cells.
Our study indicates that HIF pathway plays an essential role in directing DBT-induced EPO expression in kidney. These results provide one of the molecular mechanisms of this ancient herbal decoction for its hematopoietic function.
Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on regarding their estrogen-like activities and particularly their ...ability to affect bone metabolism. Here, different subclasses of flavonoids were screened for their osteogenic properties by measuring alkaline phosphatase activity in cultured rat osteoblasts. The flavone baicalin derived mainly from the roots of Scutellaria baicalensis showed the strongest induction of alkaline phosphatase activity. In cultured osteoblasts, application of baicalin increased significantly the osteoblastic mineralization and the levels of mRNAs encoding the bone differentiation markers, including osteonectin, osteocalcin, and collagen type 1α1. Interestingly, the osteogenic effect of baicalin was not mediated by its estrogenic activity. In contrast, baicalin promoted osteoblastic differentiation via the activation of the Wnt/β-catenin signaling pathway; the activation resulted in the phosphorylation of glycogen synthase kinase 3β and, subsequently, induced the nuclear accumulation of the β-catenin, leading to the transcription activation of Wnt-targeted genes for osteogenesis. The baicalin-induced osteogenic effects were fully abolished by DKK-1, a blocker of Wnt/β-catenin receptor. Moreover, baicalin also enhanced the mRNA expression of osteoprotegerin, which could regulate indirectly the activation of osteoclasts. Taken together, our results suggested that baicalin could act via Wnt/β-catenin signaling to promote osteoblastic differentiation. The osteogenic flavonoids could be very useful in finding potential drugs, or food supplements, for treating post-menopausal osteoporosis.
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-Alzheimer's 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 Alzheimer's disease in the future.
Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body ...resistance against hypoxia in mountain sickness. The mechanism of this adaptogenic property deriving from Rhodiola, however, has not been revealed. Erythropoietin (EPO) is an erythrocyte-specific hematopoietic hormone that increases the production of red blood cells: this hormone is a crucial factor in regulating the body balance in responding to hypoxia. In cultured kidney fibroblasts (HEK293T), application of water extract deriving from Rhodiola induced the expression of EPO both in mRNA and protein levels. The activation of the hypoxia response element (HRE) located on the promoter region of the EPO gene is one of the mechanisms accounting for transcriptional activation. In addition, the Rhodiola-induced EPO expression was triggered by an increase of hypoxia-inducible factor-1α (HIF-1α) protein, via the reduction of HIF-1α degradation but not the induction of HIF-1α mRNA. Moreover, the same EPO induction effect by Rhodiola was also observed in cultured liver cells since liver is another vital organ to provide EPO regulation apart from the kidney. These results therefore elucidate one of the molecular mechanisms of this herb in mediating the anti-hypoxia function.
The discovery of nitrite contamination aroused public concern on the safety of cubilose consumption. Here, forty-eight batches of cubilose were randomly purchased from Hong Kong market. The median ...nitrite content of different types of cubilose was as follows: Red cubilose (600 ppm) > Yellow cubilose (510 ppm) ;> White cubilose (100 ppm). Under a developed standardized processing method, up to 98% of nitrite was removed from cubilose; nitrite was not detected in the stewed cubilose. To search for the source of nitrite, droppings from swiftlets and water samples were collected from production sites of cubilose in Malaysia and Indonesia, and which contained a high amount of nitrate. On the other hand, the protein extract of cubilose was subjected to proteomics analysis. A microbial nitrate reductase was identified by mass spectroscopy, which converted nitrate to nitrite in cubilose. A specific inhibitor of nitrate reductase successfully abolished the nitrate reduction activity found in cubilose, which subsequently reduced the nitrite in cubilose. This phenomenon was successfully proven by the field study. Thus, the nitrite on cubilose could be a result of the contaminating nitrate reductase.
•All cubilose in this surveillance had different levels of nitrite contamination.•The medians of nitrite content of cubiloses were as follows: Red > Yellow > White.•No nitrite was detected from cubilose after the standard processing.•A nitrate reductase was identified which converted nitrate to nitrite in cubilose.•Sodium tungstate abolished the nitrite formation in cubilose.
Acetylcholinesterase (AChE) inhibitors are widely used for the treatment of Alzheimer's disease (AD). Several AChE inhibitors, e.g. rivastigmine, galantamine and huperzine are originating from ...plants, suggesting that herbs could potentially serve as sources for novel AChE inhibitors. Here, we searched potential AChE inhibitors from flavonoids, a group of naturally occurring compounds in plants or traditional Chinese medicines (TCM). Twenty-one flavonoids, covered different subclasses, were tested for their potential function in inhibiting AChE activity from the brain
in vitro. Among all the tested flavonoids, galangin, a flavonol isolated from Rhizoma Alpiniae Officinarum, the rhizomes of
Alpiniae officinarum (Hance.) showed an inhibitory effect on AChE activity with the highest inhibition by over 55% and an IC
50 of 120
μM and an enzyme-flavonoid inhibition constant (
K
i
) of 74
μM. The results suggest that flavonoids could be potential candidates for further development of new drugs against AD.
The toxicity of aggregated β-amyloid (Aβ) has been implicated as a critical cause in the development of Alzheimer's disease (AD). Hibifolin, a flavonol glycoside derived from herbal plants, possessed ...a strong protective activity against cell death induced by aggregated Aβ. Application of hibifolin in primary cortical neurons prevented the Aβ-induced cell death in a dose-dependent manner. In cultured cortical neurons, the pre-treatment of hibifolin abolished Aβ-induced Ca
2+ mobilization, and also reduced Aβ-induced caspase-3 and caspase-7 activation. Moreover, DNA fragmentation induced by Aβ 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 Aβ-induced apoptosis and stimulated Akt activation, which would be useful in developing potential drugs or food supplements for treating AD.
A high-performance liquid chromatography coupled with diode array detector and mass spectrometry (HPLC–DAD-MS) method was developed to evaluate the quality of Rhizoma Belamcandae (
Belamcanda ...chinensis (L.) DC.) through establishing chromatographic fingerprint and simultaneous determination of seven phenolic compounds. The analysis was achieved on an Alltima C
18 analytical column (250
mm
×
4.6
mm i.d. 5
μm) using linear gradient elution of acetonitrile–0.1% trifluoroacetic acid. The correlation coefficients of similarity were determined from the HPLC fingerprints, and they shared a close similarity. By using an online APCI–MS/MS, twenty phenols were identified. In addition, seven of these phenols including mangiferin, 7-
O-methylmangiferin, tectoridin, resveratrol, tectorigenin, irigenin and irisflorentin were quantified by the validated HPLC–DAD method. These phenols are considered to be major constituents in Rhizoma Belamcandae, and are generally regarded as the index for quality assessment of this herb. This developed method by having a combination of chromatographic fingerprint and quantification analysis could be applied to the quality control of Rhizoma Belamcandae.
Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with ...PRiMA requires the C-terminal "t-peptide" in AChE catalytic subunit (AChE(T)). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE(T) plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE(T) mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K(m) value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.
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