Astragaloside suppresses tumor necrosis factor receptor-associated factor 5 signaling pathway and alleviates neurodegenerative changes in retinal pigment epithelial cells induced by isoflurane

Epidemiological studies showed that isoflurane, a general anesthetic widely used in surgery including those for the children, is associated with impairment of neurodevelopment and neurodegenerative diseases, such as Alzheimer’s disease (AD) and age-related macular degeneration (AMD), which are related to the accumulation of reactive oxygen species (ROS). Astragaloside (AS) is an antioxidant derivative from a traditional Chinese herbal medicine Astragalus membraneaceus Bunge. In this study, we used retinal pigment epithelial cells, which share plenty of features with neurodegenerative diseases such as AD and AMD to investigate the effect of AS. Cell cycle re-entry and proapoptosis were seen in retinal pigment epithelium (RPE) cells treated with isoflurane, which was alleviated by pretreatment of AS. Further, tumor necrosis factor receptor-associated factor 5 (TRAF5) and downstream nuclear factor-κB (NF-κB) were investigated to elucidate the molecular mechanism underlying protective effect of AS. RPE cells exposed to isoflurane expressed higher TRAF5 and NF-κB than those pretreated with AS, suggesting a critical role of TRAF5 therein. In Morris water maze (MWM) assay, Sprague-Dawley rats pretreated with AS and then exposed to isoflurane spent less time in swimming to the platform, and their TRAF5 expression was significantly lower than those received anesthesia alone. Further studies on the consequence of forced downregulation or upregulation are warranted that may employ cutting-edge technologies such as optogenetics to overcome the difficulties in manipulating expression of TRAF5. Although the link between TRAF5 and neurodegeneration requires more in-depth investigations, our study provide a novel hint on the pathological mechanism of isoflurane and suggest a potential target for eliminating persistent side effect of anesthesia.     

黄芪甲甙对急性心肌梗死大鼠心室重构和NOX/ROS/TNF⁃α信号通路的影响

研究旨在探究黄芪甲甙对急性心肌梗死大鼠心室重构的作用及其机制。选取36只Wistar大鼠随机分为假手术组、模型组、黄芪甲甙低剂量组、黄芪甲甙中剂量组、黄芪甲甙高剂量组和阳性对照组,每组6只。除假手术组外,其余大鼠手术建立急性心肌梗死模型,假手术组大鼠开胸后仅分离冠状动脉左前降支,不做结扎处理便逐层缝合,造模后第2天开始药物干预：黄芪甲甙低、中、高剂量组分别给予20、40、60 mg·kg^-1黄芪甲甙灌胃处理,阳性对照组给予100 mg·kg^-1阿司匹林灌胃处理。比较各组治疗第0、2、4周心功能指标[左心室射血分数（left ventricular ejection fractions,LVEF）、左心室短轴缩短率（left ventricular fractional shortening,LVFS）、左心室质量指数（left ventricular mass index,LVMI）]的变化情况。采用心脏血流动力学监测系统监测心脏血流动力学指标[左心室舒张末压（left ventricular end diastolic pressure,LVEDP）、左心室收缩压（left ventricular systolic pressure,LVSP）和左心室等容收缩/舒张压上升最大速率（maximal rate of left ventricular pressure increase/decrease,±dp/dt_max）]的变化情况,采用酶联免疫吸附法测定血清血管紧张素Ⅱ（angiotensin Ⅱ,AngⅡ）、内皮素1（endothelin-1,ET-1）、脑钠肽（brain natriuretic peptide,BNP）含量,试剂盒检测心肌组织活性氧（reactive oxygen species,ROS）含量,实时荧光定量PCR和Western blot检测大鼠心肌组织NADPH氧化酶（reduced nicotinamide adenine dinucleotide phosphate oxidase,NOX）、肿瘤坏死因子α（tumor necrosis factor α,TNF-α）mRNA相对表达量和蛋白表达水平。结果显示,治疗4周后,黄芪甲苷高剂量组和阳性对照组LVEF、LVFS随着治疗时间延长不断升高（P<0.05）;与相同治疗时间假手术组比较,模型组LVEF、LVFS均显著降低（P<0.05）;与治疗第0周模型组比较,各治疗组LVEF、LVFS差异无统计学意义（P>0.05）;治疗第2、4周与模型组比较,黄芪甲苷中剂量组、黄芪甲苷高剂量组和阳性对照组LVEF、LVFS显著升高（P<0.05）;与模型组比较,黄芪甲苷中剂量组、黄芪甲苷高剂量组和阳性对照组LVMI显著降低（P<0.05）,LVSP、LVEDP、±dp/dt显著升高（P<0.05）,血清AngⅡ、ET-1、BNP显著降低（P<0.05）,心肌组织ROS含量显著降低（P<0.05）,NOX、TNF-α基因和蛋白表达量显著降低。研究结果表明,黄芪甲甙可改善AMI大鼠心功能和血流动力学紊乱,抑制心室重构,可能与下调NOX/ROS/TNF-α信号通路表达有关。     

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g^–1 astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l^–1, astragaloside IV 0.9 mg g^–1). Polysaccharide yields were similar amongst the different bioreactors (range 25–32 mg g^–1). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.     

Astragaloside IV synergizes with ferulic acid to suppress hepatic stellate cells activation in vitro

Because hepatic fibrosis usually involves more than one pathological process, combination therapy with modalities that target aberrant signaling cascade in activated hepatic stellate cells (HSCs) represents an alternative strategy. This study evaluates the hypothesis that astragaloside IV (AS-IV) and ferulic acid (FA) synergize to inhibit HSCs activation via simultaneous activating nuclear factor erythroid-2-related factor-2 (Nrf2) and blocking transforming growth factor-β (TGF-β) pathways. The combination of FA and AS-IV, hereafter referred to as the AS-IV/FA, at suboptimal concentrations synergistically inhibited HSCs activation, as measured by expressions of α-smooth muscle actin (α-SMA), collagen α type I (Col I) and fibronectin. Nrf2 nuclear accumulation, glutathione (GSH) increase, and reactive oxygen species (ROS) reduction by AS-IV were not potentiated by co-treatment with FA. Similarly, inhibition of TGF-β1 secretion and Smad activity by FA also was not enhanced by combined treatment with AS-IV. AS-IV/FA synergistically suppresses the p38 mitogen-activated protein kinase (MAPK) activity. Inhibition of HSCs activation by AS-IV/FA could be completely blocked by TGF-βs-neutralizing antibody plus shRNA-mediated knockdown of Nrf2. Dual blockade of the TGF-β1/Smad pathway by FA and activation of Nrf2/ARE pathway by AS-IV contributed to the synergistic effects of this combination treatment. These results suggest that combinatorial treatments that target different pathway may afford a more effective strategy to inhibit HSC activation.     

干旱胁迫对蒙古黄芪生长和生理生化指标及其黄芪甲苷积累的影响

以二年生蒙古黄芪实生苗为试验材料,采用盆栽模拟自然界干旱的方法,连续控水14d,分析持续性干旱胁迫对蒙古黄芪实生苗生长、生理生化指标及其黄芪甲苷的影响。结果显示:(1)随着土壤相对含水量的降低,黄芪叶片相对含水量逐渐降低,而其中丙二醛含量升高,其根茎干重也降低。(2)随着胁迫时间的延长,黄芪根叶中抗氧化酶类SOD、POD、CAT、APX和GR活性呈先增加后降低的趋势,根中最高值较对照分别增加了72.1%、108.6%、178.0%、299.4%和303.4%,且根叶中渗透调节物质脯氨酸和可溶性糖含量也逐渐升高。(3)黄芪甲苷积累量在干旱胁迫第12天达到最大,比对照增加了53.0%,随后降低。研究表明,随着干旱胁迫程度的加深,黄芪苗生长受到一定程度抑制,但能通过调节自身保护酶活性和渗透调节物质含量来减轻干旱的损伤,最大程度地维持植株的正常代谢;适度水分胁迫有利于黄芪甲苷的积累,而重度胁迫不利于其积累。     

Astragaloside IV modulates TGF‐β1‐dependent epithelial‐mesenchymal transition in bleomycin‐induced pulmonary fibrosis

Epithelial‐mesenchymal transition (EMT) plays an important role in idiopathic pulmonary fibrosis (IPF). Astragaloside IV (ASV), a natural saponin from astragalus membranaceus, has shown anti‐fibrotic property in bleomycin (BLM)‐induced pulmonary fibrosis. The current study was undertaken to determine whether EMT was involved in the beneficial of ASV against BLM‐induced pulmonary fibrosis and to elucidate its potential mechanism. As expected, in BLM‐induced IPF, ASV exerted protective effects on pulmonary fibrosis and ASV significantly reversed BLM‐induced EMT. Intriguing, transforming growth factor‐β1 (TGF‐β1) was found to be up‐regulated, whereas Forkhead box O3a (FOXO3a) was hyperphosphorylated and less expressed. However, ASV treatment inhibited increased TGF‐β1 and activated FOXO3a in lung tissues. TGF‐β1 was administered to alveolar epithelial cells A549 to induce EMT in vitro. Meanwhile, stimulation with TGF‐β1‐activated phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway and induced FOXO3a hyperphosphorylated and down‐regulated. It was found that overexpression of FOXO3a leading to the suppression of TGF‐β1‐induced EMT. Moreover, ASV treatment, similar with the TGF‐β1 or PI3K/Akt inhibitor, reverted these cellular changes and inhibited EMT in A549 cells. Collectively, the results suggested that ASV significantly inhibited TGF‐β1/PI3K/Akt‐induced FOXO3a hyperphosphorylation and down‐regulation to reverse EMT during the progression of fibrosis.     

ERK1／2信号通路对黄芪苷Ⅳ抗H2O2诱导H9c2细胞氧化损伤的作用

目的：研究黄芪苷Ⅳ（AST）是否通过细胞外信号调节激酶1／2（ERK1／2）通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。方法：用200μmoL／L的H2O2处理细胞6h,采用MTT法检测细胞存活率,建立H2O2诱导的H9c2细胞氧化损伤模型;比色法测定细胞培养液中乳酸脱氢酶（LDH）活性、总超氧化物歧化酶（T—SOD）和锰超氧化物歧化酶（Mn—SOD）活力以及丙二醛（MDA）含量;Western blot检测H9c2细胞ERK1／2蛋白的磷酸化水平。结果：在H2O2浓度为200μmol／L作用6h条件下,细胞存活率降低程度适中,实验结果重复性好,确定后续实验采用200μmol／L H2O2作用6h建立模型。与H2O2组比较,10mg／L及20mg／L AST均显著提高细胞存活率（P〈0．01）,使细胞培养液中LDH活性显著降低（P〈0．01）,T—SOD及Mn—SOD活力显著提高（P〈0．01）,MDA含量显著降低（P〈0．01）。10mg/L及20mg/L AST均显著增加H2O2损伤的H9c2细胞p—ERK1／2蛋白的表达（P〈0．01）,当用PD98059（ERK1／2的抑制剂）预处理后,AST的作用则被取消。结论：黄芪苷Ⅳ可以通过ERK1／2通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。     

Astragaloside IV enhances taxol chemosensitivity of breast cancer via caveolin-1-targeting oxidant damage

Accumulating evidence suggests that caveolin-1 (CAV-1) is a stress-related oncotarget and closely correlated to chemoresistance. Targeting CAV-1 might be a promising strategy to improve chemosensitivity for breast cancer treatment. Astragaloside IV (AS-IV), a bioactive compound purified from Astragalus membranaceus, has been shown to exhibit multiple bioactivities, including anticancer. However, the involved molecular targets are still ambiguous. In this study, we investigated the critical role of CAV-1 in mediating the chemosensitizing effects of AS-IV to Taxol on breast cancer. We found that AS-IV could enhance the chemosensitivity of Taxol with minimal direct cytotoxicity on breast cancer cell lines MCF-7 and MDA-MB-231, as well as the nontumor mammary epithelial cell line MCF-10A. AS-IV was further demonstrated to aggravate Taxol-induced apoptosis and G2/M checkpoint arrest. The phosphorylation of mitogen-activated protein kinase (MAPK) signaling extracellular signal-regulated kinase (ERK) and c-Jun N-terminal Kinase (JNK), except p38, was also abrogated by a synergistic interaction between AS-IV and Taxol. Moreover, AS-IV inhibited CAV-1 expression in a dose-dependent manner and reversed CAV-1 upregulation induced by Taxol administration. Mechanism study further demonstrated that AS-IV treatment triggered the eNOS/NO/ONOO⁻ pathway via inhibiting CAV-1, which led to intense oxidant damage. CAV-1 overexpression abolished the chemosensitizing effects of AS-IV to Taxol by inhibiting oxidative stress. In vivo experiments further validated that AS-IV increased Taxol chemosensitivity on breast cancer via inhibiting CAV-1 expression, followed by activation of the eNOS/NO/ONOO⁻ pathway. Taken together, our findings not only suggested the potential of AS-IV as a promising candidate to enhance chemosensitivity, but also highlighted the significance of CAV-1 as the target to reverse cancer drug resistance.     

Metastasis-associated protein 1 promotes epithelial-mesenchymal transition in idiopathic pulmonary fibrosis by up-regulating Snail expression

Idiopathic pulmonary fibrosis (IPF) is a progressive and usually fatal lung disease that lacking effective interventions. It is well known that aberrant activation of transforming growth factor-beta1 (TGF-β1) frequently promotes epithelial-mesenchymal transition (EMT) in IPF. Metastasis-associated gene 1 (MTA1) has identified as an oncogene in several human tumours, and aberrant MTA1 expression has been related to the EMT regulation. However, its expression and function in IPF remain largely unexplored. Using a combination of in vitro and in vivo studies, we found that MTA1 was significantly up-regulated in bleomycin-induced fibrosis rats and TGF-β1-treated alveolar type Ⅱ epithelial (RLE-6TN) cells. Overexpression of MTA1 induced EMT of RLE-6TN cells, as well as facilitates cell proliferation and migration. In contrast, knockdown of MTA1 reversed TGF-β1-induced EMT of RLE-6TN cells. The pro-fibrotic action of MTA1 was mediated by increasing Snail expression through up-regulating Snail promoter activity. Moreover, inhibition of MTA1 effectively attenuated bleomycin-induced fibrosis in rats. Additionally, we preliminarily found astragaloside IV (ASV), which was previously validated having inhibitory effects on TGF-β1-induced EMT, could inhibit MTA1 expression in TGF-β1-treated RLE-6TN cells. These findings highlight the role of MTA1 in TGF-β1-mediated EMT that offer novel strategies for the prevention and treatment of IPF.