Acetylcholinesterase is regulated by exposure of ultraviolet B in skin keratinocytes: A potential inducer of cholinergic urticaria.

Cholinergic urticaria is a dermatological disease characterized by the presence of large patches of red skin and transient hives triggered by factors, such as exercise, sweating, and psychological tension. This skin problem is hypothesized to be attributed to a reduced expression of acetylcholinesterase (AChE), an enzyme responsible for hydrolyzing acetylcholine (ACh). Consequently, ACh is thought to the leak from sympathetic nerves to skin epidermis. The redundant ACh stimulates the mast cells to release histamine, triggering immune responses in skin. Here, the exposure of ultraviolet B in skin suppressed the expression of AChE in keratinocytes, both in in vivo and in vitro models. The decrease of the enzyme was resulted from a declined transcription of ACHE gene mediated by micro-RNAs, that is, miR-132 and miR-212. The levels of miR-132 and miR-212 were markedly induced by exposure to ultraviolet B, which subsequently suppressed the transcriptional rate of ACHE. In the presence of low level of AChE, the overflow ACh caused the pro-inflammatory responses in skin epidermis, including increased secretion of cytokines and COX-2. These findings suggest that ultraviolet B exposure is one of the factors contributing to cholinergic urticaria in skin.

The flavonoids induce the transcription of mRNA encoding erythropoietin in cultured embryonic stem cells via the accumulation of hypoxia-inducible factor-1α.

Flavonoids are the most common phytochemicals in vegetables and herbal products. The beneficial functions of flavonoids in the brain and erythropoietic system have been proposed. Erythropoietin (EPO) is a potent protective agent in the brain; but which has difficulty to cross the blood brain barrier (BBB). Here, about 60 flavonoids were screened for their potential activation on the transcription of EPO mRNA in the neuronal embryonic stem cell lines, NT2/D1 and PC12. Amongst the screened flavonoids, formononetin, calycosin, ononin, chrysin, baicalein and apigenin showed robust up regulation of EPO production via enhancement of hypoxia response element (HRE) activity in cultured embryonic stem cells. In addition, the flavonoids showed activation of HRE activity by having increased accumulation of HIF-1α, but not on level of HIF-1ß, in the cultures. The accumulation of HIF-1α was attributed to up regulation of HIF-1α mRNA and blockade of HIF-1α degradation upon treatment of the flavonoids. These results suggested a promising trend of developing commercial products of flavonoids as food supplements tailored for brain health.

Capsaicin, a Phytochemical From Chili Pepper, Alleviates the Ultraviolet Irradiation-Induced Decline of Collagen in Dermal Fibroblast via Blocking the Generation of Reactive Oxygen Species.

Capsaicin, a major ingredient in chili pepper, has broad pharmaceutical applications, including relieving pain, anti-inflammation, and treating psoriasis. In dermatological biology, capsaicin has been shown to prevent the ultraviolet (UV)-induced melanogenesis via TRPV1 receptor. To strengthen the roles of capsaicin in skin function, the damaged skin, triggered by exposure to UV, was reversed by capsaicin in both in vitro and in vivo models. In cultured dermal fibroblasts, the exposure to UV induced a decrease of collagen synthesis and increases expression of matrix metalloproteinases (MMPs), generation of reactive oxygen species (ROS), and phosphorylation of Erk and c-Jun, and these events subsequently led to skin damage. However, the UV-mediated damages could be reversed by pre-treatment with capsaicin in a dose-dependent manner. The effect of capsaicin in blocking the UV-mediated collagen synthesis was mediated by reducing generation of ROS in dermal fibroblasts, instead of the receptor for capsaicin. Hence, capsaicin has high potential value in applying as an agent for anti-skin aging in dermatology.

Regulation of acetylcholinesterase during the lipopolysaccharide-induced inflammatory responses in microglial cells.

The non-classical function of acetylcholine (ACh) has been reported in neuroinflammation that represents the modulating factor in immune responses via activation of α7 nicotinic acetylcholine receptor (α7 nAChR), i.e., a cholinergic anti-inflammatory pathway (CAP). Acetylcholinesterase (AChE), an enzyme for ACh hydrolysis, has been proposed to have a non-classical function in immune cells. However, the involvement of AChE in neuroinflammation is unclear. Here, cultured BV2 cell, a microglial cell line, and primary microglia from rats were treated with lipopolysaccharide (LPS) to induce inflammation and to explore the regulation of AChE during this process. The expression profiles of AChE, α7 nAChR, and choline acetyltransferase (ChAT) were revealed in BV2 cells. The expression of AChE (G4 form) was induced significantly in LPS-treated BV2 cells: the induction was triggered by NF-κB and cAMP signaling. Moreover, ACh or α7 nAChR agonist suppressed the LPS-induced production of pro-inflammatory cytokines, as well as the phagocytosis of microglia, by activating α7 nAChR and followed by the regulation of NF-κB and CREB signaling. The ACh-induced suppression of inflammation was abolished in AChE overexpressed cells, but did not show a significant change in AChE mutant (enzymatic activity knockout) transfected cells. These results indicate that the neuroinflammation-regulated function of AChE may be mediated by controlling the ACh level in the brain system.

Integrated Omics Reveals the Orchestrating Role of Calycosin in Danggui Buxue Tang, a Herbal Formula Containing Angelicae Sinensis Radix and Astragali Radix, in Inducing Osteoblastic Differentiation and Proliferation.

Systems biology unravels the black box of signaling pathway of cells; but which has not been extensively applied to reveal the mechanistic synergy of a herbal formula. The therapeutic efficacies of a herbal formula having multi-target, multi-function and multi-pathway are the niches of traditional Chinese medicine (TCM). Here, we reported an integrated omics approach, coupled with the knockout of an active compound, to measure the regulation of cellular signaling, as to reveal the landscape in cultured rat osteoblasts having synergistic pharmacological efficacy of Danggui Buxue Tang (DBT), a Chinese herbal formula containing Angelicae Sinensis Radix and Astragali Radix. The changes in signaling pathways responsible for energy metabolism, RNA metabolism and protein metabolism showed distinct features between DBT and calycosin-depleted DBT. Here, our results show that calycosin within DBT can orchestrate the osteoblastic functions and signaling pathways of the entire herbal formula. This finding reveals the harmony of herbal medicine in pharmacological functions, as well as the design of drug/herbal medicine formulation. The integration of systems biology can provide novel and essential insights into the synergistic property of a herbal formula, which is a key in modernizing TCM.

Rapid Geographical Origin Identification and Quality Assessment of Angelicae Sinensis Radix by FT-NIR Spectroscopy.

Angelicae Sinensis Radix is a widely used traditional Chinese medicine and spice in China. The purpose of this study was to develop a methodology for geographical classification of Angelicae Sinensis Radix and determine the contents of ferulic acid and Z-ligustilide in the samples using near-infrared spectroscopy. A qualitative model was established to identify the geographical origin of Angelicae Sinensis Radix using Fourier transform near-infrared (FT-NIR) spectroscopy. Support vector machine (SVM) algorithms were used for the establishment of a qualitative model. The optimum SVM model had a recognition rate of 100% for the calibration set and 83.72% for the prediction set. In addition, a quantitative model was established to predict the content of ferulic acid and Z-ligustilide using FT-NIR. Partial least squares regression (PLSR) algorithms were used for the establishment of a quantitative model. Synergy interval-PLS (Si-PLS) was used to screen the characteristic spectral interval to obtain the best PLSR model. The coefficient of determination for calibration (R2C) for the best PLSR models established with the optimal spectral preprocessing method and selected important spectral regions for the quantitative determination of ferulic acid and Z-ligustilide was 0.9659 and 0.9611, respectively, while the coefficient of determination for prediction (R2P) was 0.9118 and 0.9206, respectively. The values of the ratio of prediction to deviation (RPD) of the two final optimized PLSR models were greater than 2. The results suggested that NIR spectroscopy combined with SVM and PLSR algorithms could be exploited in the discrimination of Angelicae Sinensis Radix from different geographical locations for quality assurance and monitoring. This study might serve as a reference for quality evaluation of agricultural, pharmaceutical, and food products.

The Cholinesterase Inhibitory Properties of Stephaniae Tetrandrae Radix.

Stephaniae tetrandrae radix (STR) is a commonly used traditional Chinese medicine in alleviating edema by inducing diuresis. In the clinic, STR extracts or its components are widely used in the treatment of edema, dysuria, and rheumatism for the regulation of water metabolism. Furthermore, STR has been used in treating emotional problems for years by combining with other Chinese herbs. However, the material basis and mechanism of STR on the nervous system have not been revealed. Here, the main components of STR extracts with different extracting solvents were identified, including three major alkaloids, i.e., cyclanoline, fangchinoline, and tetrandrine. The cholinesterase inhibitory activity of STR extracts and its alkaloids was determined using the Ellman assay. Both cyclanoline and fangchinoline showed acetylcholinesterase (AChE) inhibitory activity, demonstrating noncompetitive enzyme inhibition. In contrast, tetrandrine did not show enzymatic inhibition. The synergism of STR alkaloids with huperzine A or donepezil was calculated by the median-effect principle. The drug combination of fangchinoline-huperzine A or donepezil synergistically inhibited AChE, having a combination index (CI) < 1 at Fa = 0.5. Furthermore, the molecular docking results showed that fangchinoline bound with AChE residues in the peripheral anionic site, and cyclanoline bound with AChE residues in the peripheral anionic site, anionic site, and catalytic site. In parallel, cyclanoline bound with butyrylcholinesterase (BChE) residues in the anionic site, catalytic site, and aromatic site. The results support that fangchinoline and cyclanoline, alkaloids derived from STR, could account for the anti-AChE function of STR. Thus, STR extract or its alkaloids may potentially be developed as a therapeutic strategy for Alzheimer's patients.

Dehydrocorydaline Accounts the Majority of Anti-Inflammatory Property of Corydalis Rhizoma in Cultured Macrophage.

Corydalis Rhizoma (CR) is a commonly used traditional Chinese medicine for its potency in activating blood circulation and analgesia. In clinic, CR extracts or components are commonly used in the treatment of myocardial ischemia, rheumatism, and dysmenorrhea with different types of inflammation. However, due to different mechanism of pain and inflammation, the anti-inflammatory property of CR has not been fully revealed. Here, the major chromatographic peaks of CR extracts in different extracting solvents were identified, and the anti-inflammatory activities of CR extracts and its major alkaloids were evaluated in LPS-treated macrophages by determining expressions of proinflammatory cytokines, IκBα and NF-κB. The most abundant alkaloid in CR extract was dehydrocorydaline, having >50% of total alkaloids. Besides, the anti-inflammatory activities of dehydrocorydaline and its related analogues were demonstrated. The anti-inflammatory roles were revealed in LPS-treated cultured macrophages, including (i) inhibiting proinflammatory cytokines release, for example, TNF-α, IL-6; (ii) suppressing mRNA expressions of proinflammatory cytokines; (iii) promoting IκBα expression and suppressing activation of NF-κB transcriptional element; and (iv) reducing the nuclear translocation of NF-κB. The results supported that dehydrocorydaline was the major alkaloid in CR extract, which, together with its analogous, accounted the anti-inflammatory property of CR.

Capsaicin Inhibits the Expression of Melanogenic Proteins in Melanocyte via Activation of TRPV1 Channel: Identifying an Inhibitor of Skin Melanogenesis.

Chili pepper belongs to the genus Capsicum of Solanaceae family. Capsaicin is the primary capsaicinoid in placenta and flesh of chili pepper fruit, which has been shown to have various pharmacological functions, including gastric protection, anti-inflammation, and obesity treatment. Here, we revealed that capsaicin as well as chilli extract was able to inhibit synthesis of melanin in melanocytes. In cultured melanocytes, the melanin content was reduced to 54 ± 6.55% and 42 ± 7.41% with p < 0.001 under treatment of 50 µM capsaicin for 24 and 72 h, respectively. In parallel, the protein levels of tyrosinase and tyrosinase-related protein-1 were reduced to 62 ± 8.35% and 48 ± 8.92% with p < 0.001. Such an inhibitory effect of capsaicin was mediated by activation of transient receptor potential vanilloid 1-induced phosphorylation of extracellular signal-regulated kinase. This resulted in a degradation of microphthalmia-associated transcription factor, leading to reduction of melanogenic enzymes and melanin. These results revealed that capsaicin could be an effective inhibitor for skin melanogenesis. Hence, chili pepper, as our daily food, has potential in dermatological application, and capsaicin should be considered as a safe agent in treating hyperpigmentation problems.

Solar light induces expression of acetylcholinesterase in skin keratinocytes: Signalling mediated by activator protein 1 transcription factor.

Acetylcholinesterase (AChE) hydrolyses acetylcholine to choline and acetate, playing an important role in terminating the neurotransmission in brain and muscle. Recently, the non-neuronal functions of AChE have been proposed in different tissues, in which there are various factors to regulate the expression of AChE. In mammalian skin, AChE was identified in melanocytes and keratinocytes. Our previous study has indicated that AChE in keratinocyte affects the process of solar light-induced skin pigmentation; however, the expression of AChE in keratinocytes in responding to sunlight remains unknown. Here, we provided several lines of evidence to support a notion that AChE could be upregulated at transcriptional and translational levels in keratinocytes when exposed to solar light. The light-mediated AChE expression was triggered by Ca2+, supported by an induction of Ca2+ ionophore A23187 and a blockage by Ca2+ chelator BAPTA-AM. In addition, this increase on AChE transcriptional expression was eliminated by mutagenesis on the activating protein 1 (AP1) site in ACHE gene. Hence, the solar light-induced AChE expression is mediated by Ca2+ signalling through AP1 site. This finding supports the role of solar light in affecting the cholinergic system in skin cells, and which may further influence the dermatological function.

Fermentation of Danggui Buxue Tang, an ancient Chinese herbal mixture, together with Lactobacillus plantarum enhances the anti-diabetic functions of herbal product.

BACKGROUND: Danggui Buxue Tang (DBT), an ancient Chinese herbal decoction containing Astragali Radix and Angelicae Sinensis Radix at a ratio of 5: 1, is prescribed for menopausal women. Flavonoids and its flavonoid glycosides are considered as the major active ingredients within the herbal decoction; however, their amount is not controllable during the preparation. Besides, the aglycons within DBT are believed to have better gut absorption and pharmacological efficacy. METHODS: The herbal extract of DBT was fermented with Lactobacillus plantarum. The amounts of flavonoid glucosides and its aglycones in the fermented product were analyzed by using UPLC-MS/MS. In addition, in vitro assays were employed to evaluate the efficacy of the fermented DBT in regulating the activities of α-glucosidase, α-amylase and lipase, as well as their antioxidant capacity (DPPH and T-AOC assays) and anti-glycation property (BSA-methylglyoxal, BSA-fructose, and arginine-methylglyoxal models). RESULTS: The fermentation of DBT with L. plantarum drove a completed conversion of calycosin-7-O-ß-D-glucoside and ononin to calycosin and formononetin, respectively. The chemical transformation could be probably mediated by ß-glycosidase within the fermented product. Several in vitro assays corresponding to anti-diabetic functions were compared between parental DBT against its fermented product, which included the activities against α-glucosidase, α-amylase and lipase, as well as anti-oxidation and anti-glycation. The fermented DBT showed increased activities in inhibiting α-glycosidase, suppressing DPPH radical-scavenging and anti-glycation, as compared to the original herbal product. CONCLUSION: These results suggested that DBT being fermented with the probiotic L. plantarum could pave a new direction for fermentation of herbal extract, as to strengthen its pharmacological properties in providing health benefits.

Solar light induces the release of acetylcholine from skin keratinocytes affecting melanogenesis.

Cholinergic system conducts signal transmission in brain and muscle. Besides nervous system, the nonneuronal functions of cholinergic system have been proposed in various tissues. The expression of cholinergic proteins and release of acetylcholine in human skin have been reported, but its mechanism and influence on dermatological functions is not elucidated. Here, the expression profile of cholinergic markers was further investigated in skin and keratinocyte. The expression levels of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular acetylcholine transporter (VAChT), and synaptophysin, were upregulated during differentiation of keratinocytes. In cultured keratinocytes, a transient exposure of solar light induced the release of acetylcholine, which was mediated by intracellular Ca2+ mobilization. The light-induced acetylcholine release was mediated by the present of opsin. The light-induced melanogenesis was inhibited by acetylcholine or AChE inhibitor in melanocyte in vitro and mouse skin ex vivo. These results indicated that the potential role of cholinergic system could be a negative regulator in skin pigmentation.

Synergistic Inhibition of Acetylcholinesterase by Alkaloids Derived from Stephaniae Tetrandrae Radix, Coptidis Rhizoma and Phellodendri Chinensis Cortex.

Alkaloids having acetylcholinesterase (AChE) inhibitory activity are commonly found in traditional Chinese medicine (TCM); for example, berberine from Coptis chinensis, galantamine from Lycoris radiata, and huperzine A from Huperzia serrata. In practice of TCM, Stephaniae Tetrandrae Radix (STR) is often combined with Coptidis Rhizoma (CR) or Phellodendri Chinensis Cortex (PCC) as paired herbs during clinical application. Fangchinoline from STR and coptisine and/or berberine from CR and/or PCC are active alkaloids in inhibiting AChE. The traditional usage of paired herbs suggests the synergistic effect of fangchinoline-coptisine or fangchinoline-berberine pairing in AChE inhibition. HPLC was applied to identify the main components in herbal extracts of STR, CR, and PCC, and the AChE inhibition of their main components was determined by Ellman assay. The synergism of herb combination and active component combination was calculated by median-effect principle. Molecular docking was applied to investigate the underlying binding mechanisms of the active components with the AChE protein. It was found that fangchinoline showed AChE inhibitory potency; furthermore, fangchinoline-coptisine/berberine pairs (at ratios of 1:5, 1:2, 1:1, and 2:1) synergistically inhibited AChE; the combination index (CI) at different ratios was less than one when Fa = 0.5, suggesting synergistic inhibition of AChE. Furthermore, the molecular docking simulation supported this enzymatic inhibition. Therefore, fangchinoline-coptisine/berberine pairs, or their parental herbal mixtures, may potentially be developed as a possible therapeutic strategy for Alzheimer's patients.

Isorhynchophylline ameliorates cognitive impairment via modulating amyloid pathology, tau hyperphosphorylation and neuroinflammation: Studies in a transgenic mouse model of Alzheimer's disease.

Isorhynchophylline (IRN) has been demonstrated to have distinct anti-Alzheimer's disease (AD) activity in several animal models of AD. In this study, we aimed at evaluating the preventive effect of IRN on the cognitive deficits and amyloid pathology in TgCRND8 mice. Male TgCRND8 mice were administered with IRN (20 or 40 mg/kg) by oral gavage daily for 4 months, followed by assessing the spatial learning and memory functions with the Radial Arm Maze (RAM) test. Brain tissues were determined immunohistochemically or biochemically for changes in amyloid pathology, tau hyperphosphorylation and neuroinflammation. Our results revealed that IRN (40 mg/kg) significantly ameliorated cognitive deficits in TgCRND8 mice. In addition, IRN (40 mg/kg) markedly reduced the levels of Aß40, Aß42 and tumor necrosis factor (TNF-α), interleukin 6 (IL-6) and IL-1ß, and modulated the amyloid precursor protein (APP) processing and phosphorylation by altering the protein expressions of ß-site APP cleaving enzyme-1 (BACE-1), phosphorylated APP (Thr668), presenilin-1 (PS-1) and anterior pharynx-defective-1 (APH-1), as well as insulin degrading enzyme (IDE), a major Aß-degrading enzyme. IRN was also found to inhibit the phosphorylation of tau at the sites of Thr205 and Ser396. Immunofluorescence showed that IRN reduced the Aß deposition, and suppressed the activation of microglia (Iba-1) and astrocytes (GFAP) in the cerebral cortex and hippocampus of TgCRND8 mice. Furthermore, IRN was able to attenuate the ratios of p-c-Jun/c-Jun and p-JNK/JNK in the brains of TgCRND8 mice. IRN also showed marked inhibitory effect on JNK signaling pathway in the Aß-treated rat primary hippocampus neurons. We conclude that IRN improves cognitive impairment in TgCRND8 transgenic mice via reducing Aß generation and deposition, tau hyperphosphorylation and neuroinflammation through inhibiting the activation of JNK signaling pathway, and has good potential for further development into pharmacological treatment for AD.

Ophiopogon Polysaccharide Promotes the In Vitro Metabolism of Ophiopogonins by Human Gut Microbiota.

Gut microbiota play an important role in metabolism of intake saponins, and parallelly, the polysaccharides deriving from herbal products possess effects on gut microbiota. Ophiopogonis Radix is a common Chinese herb that is popularly used as functional food in China. Polysaccharide and steroidal saponin, e.g., ophiopogonin, mainly ophiopogonin D (Oph-D) and ophiopogonin D' (Oph-D'), are the major constituents in this herb. In order to reveal the role of gut microbiota in metabolizing ophiopogonin, an in vitro metabolism of Oph-D and Oph-D' by human gut microbiota, in combination with or without Ophiopogon polysaccharide, was conducted. A sensitive and reliable UPLC-MS/MS method was developed to simultaneously quantify Oph-D, Oph-D' and their final metabolites, i.e., ruscogenin and diosgenin in the broth of microbiota. An elimination of Oph-D and Oph-D' was revealed in a time-dependent manner, as well as the recognition of a parallel increase of ruscogenin and diosgenin. Ophiopogon polysaccharide was shown to stimulate the gut microbiota-induced metabolism of ophiopogonins. This promoting effect was further verified by increased activities of ß-D-glucosidase, ß-D-xylosidase, α-L-rhamnosidase and ß-D-fucosidase in the broth. This study can be extended to investigate the metabolism of steroidal saponins by gut microbiota when combined with other herbal products, especially those herbs enriched with polysaccharides.

Danggui Buxue Tang, a Chinese Herbal Decoction Containing Astragali Radix and Angelicae Sinensis Radix, Modulates Mitochondrial Bioenergetics in Cultured Cardiomyoblasts.

Danggui Buxue Tang (DBT) is an ancient herbal mixture containing Astragali Radix and Angelicae Sinensis Radix, and which are commonly consumed for "qi-invigorating" (i.e., stimulating vital energy/energy metabolism) as traditional Chinese medicine (TCM). The pharmacological activities of DBT in anti-oxidation, estrogenic, hematopoietic, and immunogenic have been reported; however, the role of DBT in cellular energy metabolism has not been determined. Here, we employed an extracellular flux analyzer to evaluate the mitochondrial respiration of cultured H9C2 cardiomyoblasts in present of DBT. The herbal extract of DBT was qualified chemically for the major ingredients, i.e. astragaloside, calycosin, formononetin, Z-ligustilide, and ferulic acid. The anti-oxidant activities of DBT, as well as its major ingredients, were determined by Folin-Ciocalteu assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and protective effect in tert-butyl hydroperoxide (tBHP)-treated cultured cardiomyoblasts. In addition, a real-time oxygen consumption rate (OCR) in herbal extract-treated cultured cardiomyoblasts was revealed by using a Seahorse extracellular flux analyzer. In addition, the transcript expressions of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PCG-1α) and other genes relating to mitochondria biogenesis were determined in cardiomyoblasts under different herbal treatments. DBT possessed the strongest anti-oxidant activity and protective effects on the oxidatively stressed cardiomyoblasts. By revealing the OCR in mitochondria, the health state of cultured cardiomyoblasts under DBT was improved via increase of basal respiration, proton leak, non-mitochondria, and adenosine triphosphate (ATP) production. Furthermore, the transcriptional activities of genes responsible for mitochondrial biogenesis and DNA replication were stimulated by application of DBT in cultures.

An Ancient Chinese Herbal Decoction Containing Angelicae Sinensis Radix, Astragali Radix, Jujuba Fructus, and Zingiberis Rhizoma Recens Stimulates the Browning Conversion of White Adipocyte in Cultured 3T3-L1 Cells.

BACKGROUND: Abnormal storage of white adipocyte tissue (WAT) is the major factor causing obesity. The promising strategies for obesity treatment are building up the brown adipocyte tissue (BAT) and/or expedite fatty acid catabolism. Traditional Chinese Medicine (TCM) sheds light on preventing obesity. Ginger is one of the most effective herbs for antiobesity by accelerating browning WAT. To fortify the antiobesity effect of ginger, an ancient Chinese herbal decoction composed of four herbs, Angelicae Sinensis Radix (ASR), Astragali Radix (AR), Jujuba Fructus (JF), and Zingiberis Rhizoma Recens (ZRR; ginger), was tested here: this herbal formula was written in AD 1155, named as Danggui Buxue Tang (DBT1155). Therefore, the antiobesity function of this ancient herbal decoction was revealed in vitro by cultured 3T3-L1 cells. MATERIALS AND METHOD: The lipid accumulation was detected by Oil Red O staining. Furthermore, the underlying working mechanisms of antiobesity functions of DBT1155 were confirmed in 3T3-L1 cells by confocal microscopy, western blot, and RT-PCR. RESULTS: DBT1155 was able to actuate brown fat-specific gene activations, which included (i) expression of PPARγ, UCP1, and PCG1α and (ii) fatty acid oxidation genes, i.e., CPT1A and HSL. The increase of browning WAT, triggered by DBT1155, was possibly mediated by a Ca2+-AMPK signaling pathway, because the application of Ca2+ chelator, BAMPTA-AM, reversed the effect. CONCLUSION: These findings suggested that the herbal mixture DBT1155 could potentiate the antiobesity functions of ginger, which might have potential therapeutic implications.

Polysaccharide deriving from Ophiopogonis Radix promotes metabolism of ginsenosides in the present of human gut microbiota based on UPLC-MS/MS assay.

The combined usage of Ginseng Radix et Rhizoma (ginseng) and Ophiopogonis Radix is common in oriental countries for thousands of years. The major active constituents of ginseng are ginsenosides, and the conversion of ginsenosides to different metabolites by gut microbiota has been reported. However, the effect of Ophiopogonis Radix, especially its polysaccharides, on the metabolism of ginsenosides by gut microbiota is not known. Here, an in vitro metabolism of ginseng extract, or ginsenosides, in combination with or without Ophiopogon polysaccharide was conducted. A sensitive and reliable UPLC-MS/MS approach using multiple reaction monitoring (MRM) in positive ion mode was developed simultaneously to quantify 22 ginsenosides in the broth of gut microbiota. After fermentation with the microbiota, 15 ginsenosides were detected and quantified, including 6 primary ginsenosides, i.e. Rb1, Rc, Rb2, Rb3, Rd and Re, and 9 metabolites, i.e. F2, Rg3, compound K, Rh2, PPD, Rg1, Rh1, Rg2 and PPT. The quantitative results therefore revealed the elimination of primary ginsenosides and the formation of their metabolites in time-dependent manners. Furthermore, Ophiopogon polysaccharide was shown to stimulate the metabolism of ginsenosides, triggered by gut microbiota. Our study can be extended to investigate the metabolism of different Panax species by gut microbiota when combining with other herbs.

Differentiation of erythroblast requires the dimeric form of acetylcholinesterase: Interference with erythropoietin receptor.

Acetylcholinesterase (AChE) hydrolyzes acetylcholine at cholinergic synapses, and which has various isoforms of AChE, i.e. AChER, AChEH and AChET, deriving from single gene. AChEH exists as a glycophosphatidylinositol (GPI)-linked dimer (G2), presents mainly in plasma membrane of mammalian erythrocyte. Transgenic mice with ACHE gene depletion were employed here to investigate the possible role of AChE in blood cell formation. ACHE knock-out mice were found to suffer normocytic anemia. In erythrocyte of ACHE-/- mice, the amount of hemoglobin, especially α-globin, was found to be markedly reduced. In addition, the number of erythrocyte and hematocrit of ACHE-/- mice were significantly lowered. To probe the role of AChE isoforms in erythroid differentiation, erythroblast-like cells (TF-1) over-expressed with different AChE isoforms were induced to differentiate by erythropoietin (EPO): this differentiation induced the expression of each AChE isoform. Only in the TF-1 cells over-expressed with AChEH, the EPO-induced transcriptions and protein expressions of α- and ß-globins could be significantly enhanced, which therefore suggested that AChEH might regulate the responsiveness of TF-1 cells to EPO. The alternation of EPO-induced downstream signaling might be accounted by association of AChE with EPO receptor in cell surface. The findings indicated the significance of AChE in erythroblast maturation, which provided an insight in elucidating possible mechanisms in regulating erythropoiesis.

Asarones from Acori Tatarinowii Rhizoma stimulate expression and secretion of neurotrophic factors in cultured astrocytes.

Acori Tatarinowii Rhizoma (ATR, the dried rhizome of Acorus tatarinowii Schott.) is a traditional Chinese medicine widely used to treat brain diseases, e.g. depression, forgetfulness, anxiety and epilepsy. Several lines of evidence support that ATR has neuronal beneficial functions in animal models, but its action mechanism in cellular level is unknown. Here, we identified α-asarone and ß-asarone could be the major active ingredients of ATR, which, when applied onto cultured rat astrocytes, significantly stimulated the expression and secretion of neurotrophic factors, i.e. nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF), in dose-dependent manners. These results suggested that the neuronal action of ATR, triggered by asarone, might be mediated by an increase of expression of neurotrophic factors in astrocytes, which therefore could support the clinical usage of ATR. In addition, application of PKA inhibitor, H89, in cultured astrocytes partially blocked the asarone-induced neurotrophic factor expression, suggesting the involvement of PKA signaling. The results proposed that α-asarone and ß-asarone from ATR could serve as potential candidates for drug development in neurodegenerative diseases.