Re-epithelialization is a crucial step towards wound healing. The traditional Chinese medicine, Astragalus membranaceus (Fisch) Bge, has been used for hundreds of years for many kinds of ulcerated wounds. Recent research has identified the active compound in this drug as astragaloside IV (AS-IV), but the underlying molecular mechanisms of its therapeutic action on keratinocytes remain poorly understood. In this study, we used an in vitro model of ulcer-like wound processes, lithium chloride (LiCl)-induced cultured mouse keratinocytes, to investigate the effects of AS-IV treatment. The effects on cell proliferation were evaluated by the MTS/PMS colorimetric assay, effects on cell migration were determined by a wound-healing scratch experiment, effects on the cell cycle were analyzed by flow cytometry, and effects on protein expression were analyzed by immunoblotting and immunofluorescence. LiCl strongly inhibited cell proliferation and migration, up-regulated β-catenin expression, and down-regulated proliferating cell nuclear antigen (PCNA) expression. AS-IV treatment attenuat the inhibition of proliferation and migration, significantly reducing the enhanced β-catenin expression, and recovering PCNA and β-tubulin expression. Thus, AS-IV mediates mouse keratinocyte proliferation and migration via regulation of the Wnt signaling pathway. Down-regulating β-catenin to increase keratinocyte migration and proliferation is one mechanism by which AS-IV can promote ulcerated wound healing.

A traditional Chinese medicine (TCM) preparation was formulated from orange peel (Pericarpium Citri Reticulatae), hawthorn (Crataegus pinnatifida), astragalus (Astragalus membranaceus (Fisch.) Bunge), pilose asiabell root (Radix codonopsis), indigowoad root (Radix isatidis), taraxacum (Herba taraxaci) and malt (Fructus Hordei Germinatus) at a weight ratio of 1:1:1.5:1.5:1.5:1.5:2. A feeding experiment was conducted to determine the effects of TCM on innate immunity of abalone, Haliotis discus hannai Ino. Artificial diets containing 1%, 3%, 5% TCM preparation, 1% hawthorn or 1% astragalus, respectively, were fed to juvenile abalone (initial weight 10.38+/-2.51 g; initial shell length 44.15+/-4.15 mm) for 80 days. A TCM-free diet was used as a control. Each diet was fed to three replicate groups of abalone using a randomized design. The results indicated that phagocytic activity was significantly higher in abalone fed 3%, 5% TCM preparation, 1% astragalus or 1% hawthorn (P<0.05). Respiratory burst activity was significantly higher in abalone fed 1%, 3%, 5% TCM preparation, 1% astragalus or 1% hawthorn (P<0.05). Agglutination titre was significantly higher in abalone fed 5% TCM preparation (P<0.05). Weight gain ratio (WGR), daily increment in shell length (DISL), total haemocyte count (THC), plasma protein concentration, and the activity of acid phosphatase (ACP) were not significantly affected by the TCM preparation (P>0.05). These results indicate that TCM preparation can modulate the immunity of H. discus hannai, and it is very possible that TCM might be used as immunostimulants in abalone farming.

4-Hydroxy-5-hydroxymethyl-[1,3]dioxolan-2,6'-spirane-5',6',7',8'-tetrahydro-indolizine-3'-carbaldehyde (HDTIC)-1 and HDTIC-2 are two isomers extracted from Astragalus membranaceus (Fisch) Bunge Var. mongholicus (Bge) Hsiao. Our previous study had demonstrated that they could extend the lifespan of human fetal lung diploid fibroblasts (2BS). To investigate the mechanisms of the HDTIC-induced delay of replicative senescence, in this study, we assessed the effects of these two compounds on telomere shortening rate and DNA repair ability in 2BS cells. The telomere shortening rates of the cells cultured with HDTIC-1 or HDTIC-2 were 31.5 and 41.1 bp with each division, respectively, which were much less than that of the control cells (71.1 bp/PD). We also found that 2BS cells pretreated with HDTIC-1 or HDTIC-2 had a significant reduction in DNA damage after exposure to 200 microM H(2)O(2) for 5 min. Moreover, the 100 microM H(2)O(2)-induced DNA damage was significantly repaired after the damaged cells were continually cultured with HDTIC for 1 h. These results suggest that HDTIC compounds slow down the telomere shortening rate of 2BS cells, which is mainly due to the biological properties of the compounds including the reduction of DNA damage and the improvement of DNA repair ability. In addition, the slow down of telomere shortening rate, the reduction of DNA damage, and the improvement of DNA repair ability induced by HDTIC may be responsible for their delay of replicative senescence.

The potential immunostimulatory effects of Astralagus membranaceus polysaccharides (APS) on sea cucumber, Apostichopus japonicus (Selenka), were investigated in vitro. Phagocytosis and superoxide anion (O(2)(-)) production by phagocytic amoebocytes (PA) from A. japonicus coelomic fluid were measured during incubation at 18 degrees C, 22 degrees C, or 25 degrees C with APS at 0, 10, 20, or 40 microg mL(-1) (n=3). Phagocytic activity against yeast cells was quantified by direct visualization, and O(2)(-) production by nitroblue tetrazolium (NBT) reduction assay. Compared with controls, including APS at 20 microg mL(-1) significantly increased (P<0.05) the percentage of phagocytic capacity (PC) and phagocytic index (PI) at 18 degrees C and 22 degrees C, but no significant enhancement was observed at 25 degrees C. In contrast, the coelmocytes of A. japonicus can have an obvious generation of O(2)(-) after the stimulation. The concentration of 20 microg mL(-1) APS resulted in a significant increase in nitroblue tetrazolium (NBT) positive cells (P<0.05) at different temperature and even 10 microg mL(-1) APS could increase O(2)(-) generation significantly at 18 degrees C and 22 degrees C. Both phagocytosing and O(2)(-) production increased with the increase of APS concentration from 0 to 20 microg mL(-1) at different temperature, and when APS at 40 microg mL(-1), they were decreased. It suggested that immunocytes activity in A. japonicus decreased with the temperature increasing from 18 degrees C to 25 degrees C, and APS could be an effective immunostimulant to enhance phagocytic activity and O(2)(-) production.

BACKGROUND

Qian-Yu decoction (QYD) is a traditional Chinese medicinal recipe composed of Radix astragali (Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao, Fabaceae ), Herba epimedii (Epimedium brevicornum Maxim., Berberidaceae), Herba leonuri (Leonurus japonicus Houtt., Lamiaceae), Cortex phellodendri (Phellodendron chinense Schneid., Rutaceae) and Radix achyranthis bidentatae (Achyranthes bidentata Bl., Amaranthaceae). This study aimed to evaluate the therapeutic activity of QYD against carrageenan-induced chronic prostatic/chronic pelvic pain syndrome (CP/CPPS) in rats and further elucidate its effective components.

METHODS

Three types of components, total polysaccharides, total flavonoids and total saponins were separately extracted from QYD. Carrageenan-induced CP/CPPS rats were intragastrically administered with lyophilized product of QYD, individual extracts and all the combined forms of extracts for three weeks. Prostatic index (PI) was determined and histopathological analysis was performed. The levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2) and prostaglandin E2 (PEG2) in rat prostate tissues were measured using ELISA. The production of inducible nitric oxide synthase (iNOS) was evaluated by an enzymatic activity assay, and the release of nitric oxide (NO) was determined by a nitrate/nitrite assay.

RESULTS

Treatment with QYD significantly ameliorated the histological changes of CP/CPPS rats and reduced the PI by 44.3%, with a marked downregulation of TNF-α (42.8% reduction), IL-1β (45.3%), COX-2 (36.6%), PGE2 (44.2%), iNOS (54.1%) and NO (46.0%). Each of three extracts attenuated the symptom of CP/CPPS, but much more weakly than QYD. The combined administration of three extracts showed efficacy comparable to that of QYD while better than that of any combination of two extracts. A principal component analysis of the six inflammatory mediators as variables indicated that the effects of TS on CP/CPPS were rather different from those of TF and TP, which were similar.

CONCLUSIONS

QYD can be beneficial in prevention and treatment of CP/CPPS. Polysaccharides, flavonoids and saponins, as the major effective components of QYD, exert a cooperative effect on CP/CPPS.

Dangguiliuhuang decoction (DGLHD) is a traditional Chinese medicine (TCM) formula, which mainly consists of angelica, radix rehmanniae, radix rehmanniae praeparata, scutellaria baicalensis, coptis chinensis, astragalus membranaceus, and golden cypress, and used for the treatment of diabetes and some autoimmune diseases. In this study, we explored the potential mechanism of DGLHD against insulin resistance and fatty liver in vivo and in vitro. Our data revealed that DGLHD normalized glucose and insulin level, increased the expression of adiponectin, diminished fat accumulation and lipogenesis, and promoted glucose uptake. Metabolomic analysis also demonstrated that DGLHD decreased isoleucine, adenosine, and cholesterol, increased glutamine levels in liver and visceral adipose tissue (VAT) of ob/ob mice. Importantly, DGLHD promoted the shift of pro-inflammatory to anti-inflammatory cytokines, suppressed T lymphocytes proliferation, and enhanced regulatory T cells (Tregs) differentiation. DGLHD also inhibited dendritic cells (DCs) maturation, attenuated DCs-stimulated T cells proliferation and secretion of IL-12p70 cytokine from DCs, and promoted the interaction of DCs with Tregs. Further studies indicated that the changed PI3K/Akt signaling pathway and elevated PPAR-γ expression were not only observed with the ameliorated glucose and lipid metabolism in adipocytes and hepatocytes, but also exhibited in DCs and T cells by DGLHD. Collectively, our results suggest that DGLHD exerts anti-insulin resistant and antisteatotic effects by improving abnormal immune and metabolic homeostasis. And DGLHD may be a novel approach to the treatment of obesity-related insulin resistance and hepatic steatosis.

Astragalus membranaceus is one of the important medicinal plant in China and Korea. It is used to increase metabolism and digestion, enhance the immune system, and promote the healing of wounds and injuries. In the present study, we used quantitative real-time PCR to investigate the expression of genes related to the biosynthesis of flavonoids, in addition to high-performance liquid chromatography to assess calycosin and calycosin-7-O-β-D-glucoside accumulation, in the different plant organs of A. membranaceus. The transcript levels of all genes (AmPAL, AmC4H, Am4CL, AmCHS, AmCHR, AmCHI, AmIFS, AmI3'H, and AmUCGT) involved in calycosin and calycosin-7-O-β-D-glucoside biosynthesis were the highest in the flower. Calycosin content was ordered as follows: leaf (145.56 μg/g dry weight [DW])>> stem (18.3 μg/g DW)>> root (1.64 μg/g DW)>> flower (0.09 μg/g DW), whereas calycosin-7-O-β-D-glucoside content was ordered as follows: root (4.88 μg/g DW)>> stem (3.86 μg/g DW)>> leaf (2.0 μg/g DW)>> flower (not detected). All genes exhibited the highest transcription levels in the flower, whereas calycosin and its glycoside content were the highest in the leaf and root, respectively. Our results indicate that the enhancement of calycosin-7-O-β-D-glucoside in the roots may originate from high calycosin accumulation in the stem and leaf. Thus, the mechanisms regulating calycosin and calycosin-7-O-β-D-glucoside content differ in the different organs of A. membranaceus. The results are expected to provide baseline information from which the mechanism of flavonoid biosynthesis in the different organs of A. membranaceus may be elucidated.

BACKGROUND

Jia-Wei-Jiao-Tai-Wan (JWJTW), composed of Jiao-Tai-Wan (Cinnamomum cassia and Rhizoma coptidis) and other antidiabetic herbs, including Astragalus membranaceus, Herba Gynostemmatis, Radix Puerariae Lobatae, Folium Mori and Semen Trigonellae, is widely used to treat diabetes and has demonstrated a curative effect in the clinic, but the potential mechanism is unknown. This study aimed to explore the effects of JWJTW on diabetic rats and to clarify the underlying mechanism.

METHODS

JWJTW was prepared, and the main components contained in the formula were identified by high-performance liquid chromatography (HPLC) fingerprint analysis. Diabetic rats induced by streptozotocin (STZ) and a high-sucrose-high-fat diet were treated with two concentrations of JWJTW (1.025 and 2.05 g/kg/d) for 100 days. The oral glucose tolerance test (OGTT), insulin release test (IRT) and insulin tolerance test (ITT) were performed to measure the glycometabolism of the diabetic rats at the end of the treatment period. Blood was collected to determine the serum lipid levels of the diabetic rats. Nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were detected in pancreas homogenates to analyze the oxidative stress in the pancreata of diabetic rats, and the expression levels of pancreatic and duodenal homeobox 1 (PDX-1) and insulin in the pancreas were tested by Western blot to measure pancreatic islet function. In addition, Western blots were used to measure the expression of proteins related to the insulin signaling pathway in skeletal muscle of the diabetic rats.

RESULTS

The results showed that the administration of JWJTW could ameliorate impairments in glucose tolerance, insulin release function and insulin tolerance in diabetic rats. JWJTW could also dose-dependently reduce serum lipid levels in diabetic rats. JWJTW restrained oxidative stress by decreasing the expression of NO and MDA and increasing the expression of SOD and GSH-px. JWJTW improved the function of pancreatic β cells by increasing PDX-1 and insulin expression. In addition, JWJTW restored the impaired insulin signaling; upregulated phospho-insulin receptor (pInsR) expression, insulin receptor substrate (IRS) tyrosine phosphorylation, phosphatidylinositol 3-kinase (PI3K) (p85), and glucose transporter 4 (GLUT4) expression; and downregulated the serine phosphorylation of IRS.

CONCLUSIONS

This study suggests that JWJTW can ameliorate type 2 diabetes by improving β cell function and reducing insulin resistance in diabetic rats.

Astragalus membranaceus (AM) is a traditional Chinese medicinal herb, whose cytoprotective effects remain largely unknown. Here, the bacterial endotoxin lipopolysaccharide (LPS) was applied to a human pulmonary type II‑like epithelial lung adenocarcinoma cell line, a human umbilical vein endothelial cell line, and a human bladder carcinoma cell line to construct in vitro models of intracellular oxidative stress. The authors assayed the cellular and mitochondrial cytoprotective effects of varying doses of AM root extract upon these cell lines. The cell lines were cultured as follows: LPS‑only group, four LPS+AM groups treated with various AM concentrations plus LPS, and an untreated control group. Flow cytometry was used to assess apoptosis and cell cycle progression. A 2',7'‑dichlorofluorescein‑diacetate assay was used to quantitate reactive oxygen species (ROS) generation. Mitochondrial membrane potential (Δψmit) was analyzed by Rhodamine 123 assay. Western blotting was performed to detect cleaved caspase‑3, p53, and B cell lymphoma (Bcl)‑2 levels. Across all cell lines, LPS significantly elevated apoptosis rates, shifted cells to S/G2 phase, increased ROS production, reduced Δψmit, upregulated cleaved caspase‑3, upregulated p53, and downregulated Bcl‑2 relative to controls (all P<0.05). As a general trend, increasing AM concentrations produced progressively greater reductions in the apoptosis rate, greater reductions in S/G2 phase %, greater reductions in ROS production, greater increases in Δψmit, greater reductions in cleaved caspase‑3 and p53 expression, and greater increases in Bcl‑2 expression. AM treatment protects human pulmonary and bladder epithelial cells, in addition to human endothelial cells, from LPS‑induced apoptosis, in a dose‑dependent manner.

The main objective of this research is to investigate the effects of astragaloside IV, calycosin separately glucoside, formononetin on oxidative stress in Chang Liver cells induced by H2O2. In the experiments, Chang Liver cells (a kind of normal human hepatocytes) were used as the research object, bifendate which has a clear hepatoprotective effect was used as the positive control drug, then the oxidative damage model of Chang Liver cells were established by H2O2. Cells were divided into six groups: blank control group, oxidative stress group, astragaloside IV group, calycosin separately glucoside group, formononetin group and positive control group. Then endogenous antioxidant system related indexes were detected by micro plate and colorimetric method; intracellular reactive oxygen species (ROS) were detected by DCFH-DA fluorescent probe; and the expressions of CYP2E1 were evaluated by liver microsomes, mRNA, and protein, respectively with spectrophotometry, Real-time PCR method, and Western blot technique. Results showed that H2O2 decreased antioxidant activity, and increased ROS level and expression of CYP2E1. The above oxidative stress status had been changed with protections of the three components of Astragalus membranaceus (compared with oxidative stress group, P < 0.05, P < 0.01), which taken as a whole had equivalent effects as the drug of positive control group( bifendate). Taken together, three Astragalus membranaceus ingredients all had significant or extremely significant inhibiting effects on oxidative damaged Chang Liver cells which were induced by H2O2, and the oxidative damage of Chang Liver cells had been relieved.

The effects of six kinds of aqueous extracts of Chinese herbal medicine (Astragalus membranaceus, Acanthopanacis senticosi, Panax genseng and Ophiopogon japonicus, P. genseng and Aconitum carmichaeli, Salviae miltiorrhiae, Polyporus umbellatus polysaccharide) on sperm motility characteristics of 30 infertile male volunteers were studied in vitro with a computer-assisted sperm analysis at 15, 60 and 180 min after incubated with the drugs. The results showed that per cent viability, number of progressive motile spermatozoa, curvilinear velocity, average path velocity and amplitude of lateral head displacement were significantly enhanced by A. membranaceus (P < 0.05 or < 0.01), per cent viability, average path velocity and amplitude of lateral head displacement were significantly enhanced by A. senticosi (P < 0.05), but all the above were not affected by P. genseng and O. japonicus, P. genseng and A. carmichaeli, S. miltiorrhiae and P. umbellatus polysaccharide. It is suggested that A. membranaceus and A. senticosi can enhance the motility of human spermatozoa in vitro.

Astragalus polysaccharide (APS), a natural anti-oxidant found in Astragalus membranaceus emerging as a novel anticancer agent, exerts antiproliferative and pro-apoptotic activity in various cancer cell types, but its effect on ovarian cancer (OC) remains unknown. In this study, we tried to elucidate the role and mechanism of APS in ovarian cancer cells. Our results showed that APS treatment suppressed the proliferation and induced apoptosis in OC cells. Afterwards, the miRNA profiles in APS-treated cells were determined by a microarray assay, and whether APS affected OV-90 cells through regulation of miRNA was determined. Among these aberrant miRNAs, miR-27a was selected for further study as its oncogenic roles in various human cancers. Moreover, we found overexpression of miR-27a reversed the anti-proliferation and pro-apoptotic effects of APS on ovarian cancer cells. F-box and WD-40 domain protein 7 (FBXW7), a classical tumor suppressor, was found directly targeted by miR-27a and its translation was suppressed by miR-27a in ovarian cancer cells. Finally, it was also observed that knockdown of FBXW7 by si-FBXW7 reversed the tumor suppressive activity of APS in ovarian cancer cells, which is similar with the effects of miR-27a overexpression. Our findings demonstrate that APS can suppress ovarian cancer cell growth in vitro via miR-27a/FBXW7 axis, and this observation reveals the therapeutic potential of APS for treatment of OC.

Isoflavonoids are a group of phenolic secondary metabolites found almost exclusively in leguminous plants. Formononetin, calycosin and calycosin-7-O-β-d-glucoside (CG) are isoflavonoid products in the CG pathway. Accumulation of the three isoflavonoids plus daidzein and expression of six genes of enzymes involved in the CG pathway were studied in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao with ultraviolet (UV) irradiation. Our results showed that (1) main isoflavonoids in roots, stems and leaves were CG, daidzein and calycosin, respectively; they accumulated significantly under the induction of UV irradiation during 8 days but their content declined later; (2) expression of six genes of enzymes involved in the CG pathway was inhibited slightly at early stage but the expression was increased greatly afterward; (3) chalcone synthase, chalcone reductase and chalcone isomerase were expressed to their individual maximum level within shorter hours than were cinnamate 4-hydroxylase, isoflavone synthase (IFS) and isoflavone 3'-hydroxylase and (4) more calycosin but less daidzein accumulated in leaves. IFS was highly expressed in leaves, which might lead to high accumulation of the common precursor of daidzein and 2,7-dihydroxy-4'-O-methoxy-isoflavanone, the latter of which would be converted to formononetin, calycosin and CG via a series of reactions. Little daidzein accumulated in leaves, which suggested that rather than be converted to daidzein, the 2,7,4'-trihydroxyisoflavanone was probably more easily caught by 2-hydroxyisoflavanone 4'-O-methyltransferase and hence provided more precursors for formononetin. The findings were discussed in terms of the influence of UV irradiation in the accumulation of isoflavonoids.

Compound Astragalus and Salvia miltiorrhiza extract (CASE) is a Chinese herbal formula consisting of astragalosides, astragalus polysaccharide and salvianolic acids extracted from Astragalus membranaceus and Salvia miltiorhiza. Previous studies by our group have demonstrated that CASE effectively suppresses diethylinitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats via modulating transforming growth factor β/Mothers against decapentaplegic (TGFβ/Smad) signaling. To further elucidate the mechanism of CASE, the effects of CASE on TGF-β1, the serine/threonine kinase receptors of TGF-β [TGF-β receptor type-I (TβRI) and TβRII] and karyopherins [Importin 7 (Imp7) and Imp8], which are crucial for TGF-β/Smad signaling in fibro-hepatocarcinogenesis, were assessed in the present study using in vivo (DEN-induced HCC in rats) and in vitro [TGF-β1-stimulated rat myofibroblasts (MFBs) and HepG2 cells] models of fibro-hepatocarcinogenesis. Hematoxylin and eosin staining revealed that CASE may suppress inflammatory reactions and fibrosis in HCC as well as increasing the differentiation of HCC cells. Positive TGF-β1 staining was increased in HCC nodule areas and in adjacent normal liver tissues in DEN-treated rats, while TβRI staining was increased only in normal adjacent liver tissues. The elevated expression of TGF-β1, TβRI and TβRII was suppressed by CASE. CASE treatment also reduced glutathione S-transferase P 1 and Imp7/8 protein expression in fibro-hepatocarcinogenesis. In vitro experiments confirmed that CASE was able to decrease the expression of TβRI and TβRII in TGF-β1-stimulated MFBs and HepG2 cells. These results indicate that the anti-HCC effect of CASE may be achieved by mediating TGF-β/TβR and Imp7/8 protein expression, suggesting that CASE has multiple targets in HCC treatment.

At high concentrations selenium (Se) exerts phytotoxic effects in non-tolerant plant species partly due to the induction of secondary nitro-oxidative stress; however, these processes are not fully understood. In order to get a more accurate view about the involvement of nitro-oxidative processes in plant Se sensitivity, this study aims to characterize and compare Se-triggered changes in reactive oxygen (ROS) and nitrogen species (RNS) metabolism and the consequent protein tyrosine nitration as a marker of nitrosative stress in non-accumulator Astragalus membranaceus and in Se hyperaccumulator Astragalus bisulcatus.The observed parameters (Se accumulation, microelement homeostasis, tissue-level changes in the roots, germination, biomass production, root growth, cell viability) supported that A. membranaceus is Se sensitive while the hyperaccumulator A. bisulcatus tolerates high Se doses. We first revealed that in A. membranaceus, Se sensitivity coincides with the Se-induced disturbance of superoxide metabolism leading to its accumulation. Furthermore, Se increased the production or disturbed the metabolism of RNS (nitric oxide, peroxynitrite, S-nitrosoglutathione) consequently resulting in intensified protein tyrosine nitration in sensitive A. membranaceus. In the (hyper)tolerant and hyperaccumulator A. bisulcatus, Se-induced ROS/RNS accumulation and tyrosine nitration proved to be negligible suggesting that this species is able to prevent Se-induced nitro-oxidative stress which can contribute to the Se tolerance of this species.

Astragalus membranaceus has been shown to possess anti-inflammation and antitumor properties. Several studies have indicated that extracts of Astragalus membranaceus (PG2) have growth inhibitory effects on tumor. However, the effect of PG2 on enhancing the chemotherapy, modulating tumor immune escape and their mechanism of action is unknown and need further investigation. Connexin (Cx) 43 is ubiquitous in cells and involved in facilitating the passage of chemotherapeutic drugs to bystander tumor cells. The indoleamine 2, 3-dioxygenase (IDO) depletes tryptophan, reduces the active T cell number and destroys immune surveillance. Herein, we provide evidence that the treatment of PG2 induced Cx43 expression, decreases IDO expression and enhances the distribution of chemotherapeutic drug. However, the effects of combination therapy (PG2 plus cisplatin) in animal models significantly retarded tumor growth and prolonged the survival. We believe that the information provided in this study may aid in the design of future therapy of PG2, suggest suitable combinations with chemotherapies.

ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), often reduce drug efficacy and are the major cause of drug resistance. Astragaloside IV (ASIV), one of the bioactive saponins isolated from Astragalus membranaceus, has been demonstrated to alleviate the progression of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model for multiple sclerosis (MS). In the present study, we found for the first time that ASIV induced the upregulation of P-gp and BCRP in the central nervous system (CNS) microvascular endothelial cells of EAE mice. Further study disclosed that tariquidar, a P-gp inhibitor, could facilitate the penetration of ASIV into CNS. On bEnd.3 cells, a mouse brain microvascular endothelial cell line, tariquidar benefited the net uptake and transport of ASIV. Additional molecular docking experiment suggested that ASIV might be a potential substrate of P-gp. In EAE mice, tariquidar was demonstrated to enhance the efficacy of ASIV, as shown by attenuated clinical symptom and reduced incidence rate as well as mitigated inflammatory infiltration and decreased demyelination in the CNS. Collectively, our findings implicate that P-gp inhibitor can promote the therapeutic efficacy of ASIV on EAE mice, which may boost its clinical usage together with ASIV in the therapy of MS.

Excessive nitric oxide (NO) and pro-inflammatory cytokines are produced during the pathogenesis of inflammatory diseases and cancer. It has been demonstrated that anti-inflammation contributes Astragalus membranaceus saponins (AST)'s beneficial effects in combination of conventional anticancer drugs. However, the immunomodulating property of AST has not been well characterized. In this study, we found that AST suppressed lipopolysaccharide (LPS)-induced generation of NO without causing cytotoxicity in the mouse macrophage RAW264.7. The gene and protein overexpression of inducible NO synthase (iNOS) as well as the production of tumor necrosis factor-[Formula: see text], evoked by LPS, was consistently down-regulated by AST. AST also inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and suppressed nuclear factor (NF)-[Formula: see text]B activation and the associated I[Formula: see text]B[Formula: see text] degradation during LPS insult. Furthermore, AST induced growth inhibition in promyelocytic leukemic HL-60 cells and T-lymphocyte leukemic Jurkat cells, but exerted no cytotoxic effects in normal human peripheral blood mononuclear cells (PBMC). It is known that the chemotherapeutic drug 5-FU can suppress the immune system, which can be identified by a reduced white blood cell count and decreased hematocrit, while the combination of AST and 5-FU can reverse the above hematologic toxicities. To summarize, non-cytotoxic concentrations of AST suppress LPS-induced inflammatory responses via the modulation of p38 MAPK signaling and the inhibition of NO and cytokine release. Importantly, AST can alleviate the hematologic side effects of current chemotherapeutic agents. These findings can facilitate the establishment of AST in the treatment of inflammatory diseases and inflammation-mediated tumor development.

BACKGROUND

The content of medicinal bioactive constituents in huangqi is affected by plant age. In this study, we devised a quick and convenient method for determining the age of huangqi, which was cultivated in Hunyuan County (Shanxi Province).

METHODS

1, 2, 3, 4, 5, 8, 10 growth years huangqi had 38 samples, all samples were collected separately. The growth rings in these samples were observed after making paraffin section and freehand-section. The relationship between growth rings and its growth years was analyzed by SPSS 19.0 software. Histochemical localization of total flavones and saponins in huangqi was determined by color reactions. The concentration of four flavonoids and two saponins in the roots of huangqi of different ages and different organizational structure (normal roots and rotten heart roots) were determined by HPLC-DAD and HPLC-ELSD. The results were analyzed by SPSS 19.0 software.

RESULTS

All huangqi samples had clear growth rings, and the statistical result about growth rings (X) and growth years (Y) showed significant correlation (r = 1, P = 0.000). The calibration curves of these six ingredients showed good linearity respectively, with significant correlation. All relative standard deviations (RSDs) of precision, recovery, repeatability, and stability experiments were less than 2%. Roots of 5-year-old plants contained the highest concentrations of total flavonoids and saponins. Saponin concentrations increased toward the center of the roots, whereas the four flavonoids showed an opposite trend in tissue distribution.

CONCLUSIONS

The growth year of huangqi (Hunyuan County, Shanxi Province) could be determined soon and conveniently by naked eyes after staining phloroglucinol-HCl solution on freehand section. The content of saponins and flavonoids in rotten heart root and the surrounding normal tissues were affected by the formation and the extent of rotten heart.

Hepatic fibrosis (HF) represents the excessive wound healing where an excess amount of connective tissues is formed within the liver, finally resulting in cirrhosis or even hepatocellular carcinoma (HCC). Therefore, it is significant to discover the efficient agents and components to treat HF, thus restraining the further progression of hepatopathy. Astragalus membranaceus (Fisch.) Bunge [also called Astragali Radix (AR)] is a famous herb in traditional Chinese medicine (TCM), which possesses a variety of biological activities and exerts good therapeutic effects in the treatment of HF. Flavonoids account for the major active ingredients related to the AR pharmacological effects. Total AR flavonoids have been proved to exert inhibitory effects on hepatic fibrosis. This study aimed to further undertake network pharmacology analysis coupled with experimental validation and molecular docking to investigate the effects and mechanism of multiple flavonoid components from AR against liver fibrosis. The results of the network pharmacology analysis showed that the flavonoids from AR exerted their pharmacological effects against liver fibrosis by modulating multiple targets and pathways. The experimental validation data showed that the flavonoids from AR were able to suppress transforming growth factor beta 1 (TGF-β1)-mediated activation of hepatic stellate cells (HSCs) and reduce extracellular matrix deposition in HSC-T6 cells via regulating the nuclear factor kappa B (NF-κB) signal transduction pathway. The results of the molecular docking study further showed that the flavonoids had a strong binding affinity for IκB kinase (IKKβ) after docking into the crystal structure. The above results indicated that, flavonoids possibly exerted the anti-inflammatory effect on treating HF by mediating inflammatory signaling pathways. The potential mechanism of these flavonoids against liver fibrosis may be related to suppression of the NF-κB pathway through effective inhibition of IKKβ. This study not only provides a scientific basis for clarifying the effects and mechanism of AR flavonoids against liver fibrosis but also suggests a novel promising therapeutic strategy for the treatment of liver fibrosis.

Keywords: Astragalus membranaceus (fisch) bunge; anti-inflammation; flavonoids; liver fibrosis; network pharmacology.

Two cyclolanostane-type saponins, astragalosides I and II, were first identified by TLC-MS/MS in the ethyl acetate extract of the roots of Astragalus membranaceus Bge var. mongholicus (Bge.) Hsiao without chemical reference substances. They were then isolated by high-speed counter-current chromatography with a two-step two-phase solvent system of ethyl acetate-2-propanol-water (5:1:5, 50:1:50, v/v/v). The quantities of astragalosides I and II isolated from 1 g of the crude extract were 30.2 mg and 16.5 mg, respectively. Their purities were found to be over 95% by HPLC-ELSD analysis. Their chemical structures were confirmed by 1H and 13C nuclear magnetic resonance analysis.

Astragaloside IV is the main active compound of Astragalus membranaceus. Astragaloside IV has strong anti-oxidative activities and protective effects against progression of peripheral neuropathy. In this study, we determined whether astragaloside IV protects retinal ganglion cells (RGC) from oxidative stress injury using the rat RGC-5 cell line. Hydrogen peroxide (H2O2) was used to induce oxidative stress injury, with the protective effect of astragaloside IV examined. Cell Counting Kit-8 and 4',6-diamidino-2-phenylindole staining showed that astragaloside IV increased cell survival rate and decreased apoptotic cell number. Flow cytometry showed that astragaloside IV decreased H2O2-induced reactive oxygen species levels. While laser confocal microscopy showed that astragaloside IV inhibited the H2O2-induced decrease of mitochondrial membrane potential. Western blot assay showed that astragaloside IV reduced cytochrome c release induced by H2O2, inhibited Bax and caspase-3 expression, and increased Bcl-2 expression. Altogether, these results indicate that astragaloside IV has potential protective effects against H2O2-induced oxidative stress in retinal ganglion cells.

Radix Astragali is commonly used in traditional Chinese medicine, and its quality is closely related to ecological factors, such as climate and soil, in the production area. To provide high-quality Radix Astragali to Chinese and foreign markets, we used maximum entropy model and statistical analysis method, combined with data on ecological factors, Astragalus membranaceus var. mongholicus geographical distribution, and index component content to predict the ecological suitability distribution of A. membranaceus var. mongholicus and establish the relationship between astragaloside IV and calycosin-7-glucoside in this species and ecological factors. Subsequently, we could determine the suitability regionalization of high-quality A. membranaceus var. mongholicus in Inner Mongolia, China. The results showed that the standard deviation of seasonal changes in temperature (40.6%), precipitation in October (15.7%), vegetation type (14.3%), soil type (9.2%), and mean sunshine duration in the growing season (9.1%) were the top five most influential factors out of the 17 main ecological factors affecting the distribution of A. membranaceus var. mongholicus. The standard deviation of seasonal changes in temperature, precipitation in October, precipitation in April, soil pH, and mean sunshine duration in the growing season were found to be the key ecological factors affecting the accumulation of astragaloside IV and calycosin-7-glucoside in A. membranaceus var. mongholicus. The regions with the highest-quality A. membranaceus var. mongholicus were distributed in Baotou (Guyang County), Hohhot (Wuchuan County), and central Wulanchabu (Chahar Right Middle Banner, Chahar Right Back Banner, and Shangdu County) and its surroundings in Inner Mongolia. Baotou, Hohhot, and their surrounding areas were the main traditional production areas of A. membranaceus var. mongholicus, and central Wulanchabu was a potentially suitable distribution area of this species. The main production areas were consistent with the actual production base of A. membranaceus var. mongholicus. This study therefore provides a scientific basis to guide the cultivation of A. membranaceus var. mongholicus.