黄芪叶中黄芪甲苷的含量测定

目的 :测定黄芪叶中黄芪甲苷的含量 ,寻找提取黄芪甲苷的新药源。方法 :本实验以黄芪甲苷为标准品 ,采用薄层色谱—分光光度法测定黄芪叶中黄芪甲苷的含量。结果 :叶中的黄芪甲苷含量是根中的 2 .8倍。结论 :黄芪叶有潜在的开发价值     

黄芪甲苷联合骨髓间充质干细胞对糖尿病肾病大鼠的肾脏保护作用研究

目的:研究黄芪甲苷(Astragaloside Ⅳ, AS-Ⅳ)联合骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)对链脲佐菌素(Streptozocin, STZ)诱导的糖尿病肾病(Diabetic nephrology, DN)大鼠肾脏的保护作用。方法:8周龄正常雄性SD(Sprague Dawley, SD)随机选取8只作为正常对照组,其余大鼠给予腹腔注射60 mg/kg剂量STZ建立糖尿病模型。造模成功的大鼠随机分为模型组、BMSCs组和BMSCs+AS-Ⅳ组,每组8只。BMSCs+AS-Ⅳ组给予AS-Ⅳ口服灌胃治疗。BMSCs组与BMSCs+AS-Ⅳ组给予尾静脉注射BMSCs,正常对照组和模型组尾静脉注射相同剂量的无血清DMEM培养基。于第10周末收集大鼠24小时尿液,并留取肾脏组织,测定24 h尿白蛋白排泄,观察肾脏病理改变,并采用免疫组织化学方法检测肾组织中α-SMA、Desmin、Nephrin、NOX4的表达。结果:与模型组相比,BMSCs组与BMSCs+AS-Ⅳ组大鼠的肾脏病理损伤明显改善,肾小球系膜细胞增生,系膜基质增多;肾组织中NOX4、Desmin和α-SMA蛋白表达降低,Nephrin蛋白表达增加,24 h尿白蛋白排泄均明显减轻,且BMSCs联合AS-Ⅳ治疗组效果更显著(P0.05)。结论:AS-Ⅳ联合BMSCs治疗可缓解糖尿病肾病大鼠肾脏病理损伤,改善DN大鼠肾组织的氧化应激,改善肾脏足细胞转分化,从而减轻蛋白尿排泄,具有肾脏保护作用。     

黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响

目的：探讨黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响。方法：采用Percoll密度离心法和贴壁法分离纯化hBMSC;流式细胞术检测hBMSC表面标志;MTT法检测不同浓度黄芪甲苷干预72h后hBMSC的增殖情况;Real-timePCR法检测经黄芪甲苷干预后hBMSC对SCF、VEGF、SDF-1、GM-CSFmRNA的表达水平。结果：成功分离培养出laBMSC;不同浓度（20、40、80、160、320mg／mL）的黄芪甲苷可促进hBMSC增殖（P〈0．05）,其中160mg／mL组促增殖最明显;黄芪甲苷可促进hBMSC对SCF、VEGF、SDF-1mRNA的表达,而GM—CSFmRNA表达无明显变化。结论：黄芪甲苷可促进hBMSC体外增殖,可能与其促进hBMSC对SCF、VEGF、SDF-1mRNA表达有关。     

黄芪甲苷对人骨髓间充质干细胞体外增殖 及细胞因子表达的影响*

摘要目的：探讨黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响。方法：采用Percoll 密度离心法和贴壁法分离 纯化hBMSC;流式细胞术检测hBMSC 表面标志;MTT 法检测不同浓度黄芪甲苷干预72 h后hBMSC 的增殖情况;Real-time PCR 法检测经黄芪甲苷干预后hBMSC对SCF、VEGF、SDF-1、GM-CSF mRNA 的表达水平。结果：成功分离培养出hBMSC;不同 浓度（20、40、80、160、320 mg / mL）的黄芪甲苷可促进hBMSC增殖(P<0.05),其中160 mg/mL 组促增殖最明显;黄芪甲苷可促进 hBMSC对SCF、VEGF、SDF-1 mRNA的表达,而GM-CSF mRNA 表达无明显变化。结论：黄芪甲苷可促进hBMSC 体外增殖,可 能与其促进hBMSC 对SCF、VEGF、SDF-1 mRNA表达有关。     

黄芪甲苷后处理对乳鼠心肌细胞缺氧复氧损伤的作用研究

目的:观察黄芪甲苷(AstragalosideⅣ,AsⅣ)后处理对缺氧复氧损伤(simulated ischemia reperfusion injury,SI/RI)的SD乳鼠心肌细胞是否具有保护作用。方法:将乳鼠原代心肌细胞平均分为五组,即空白对照组(Control)、缺氧复氧处理组(SI/RI)、黄芪甲苷预处理(5,10、20μM)+SI/RI组(AsIV+SI/RI)。各组细胞经处理后,四氮唑溴盐比色法(MTT)检测各组细胞存活率;TUNEL染色法测定各组细胞凋亡率;SOD测试盒检测培养液中超氧化物歧化酶(SOD)含量,总嘌呤氧化酶(XOD)测试盒检测丙二醛(MDA)含量。Western blot法检测各组细胞抗凋亡蛋白Bcl-2和促凋亡蛋白Caspase-3的表达。结果:与空白组相比,缺氧复氧损伤组细胞活力显著下降(P0.05),凋亡率显著上升(P0.05),其培养液中SOD水平显著降低(P0.05),MDA水平显著升高。而不同浓度AsⅣ后处理组的心肌细胞存活率显著上升,凋亡率显著下降,培养液中SOD水平显著上升,MDA水平显著下降(P0.05),且呈浓度呈依赖性。Western blot结果显示AsⅣ后处理组细胞中的Bcl-2表达明显上升,Caspase-3明显下降。结论:黄芪甲苷后处理对缺氧复氧诱导的乳鼠心肌细胞损伤具有显著的保护作用,能够显著上调抗凋亡蛋白Bcl-2的表达,下调促凋亡蛋白Caspase-3的表达。     

黄芪甲苷对阿霉素诱导的扩张型心肌病大鼠心肌纤维化和Th17细胞分化的影响

摘要 目的：探究黄芪甲苷（AS-Ⅳ）对阿霉素诱导的扩张型心肌病（DCM）大鼠心肌纤维化和Th17细胞分化的影响。方法：采用剂量为2.5 mg/kg的阿霉素腹腔注射诱导构建DCM大鼠模型,将DCM大鼠分为模型组、黄芪甲苷低剂量组（低AS-Ⅳ组）、黄芪甲苷中剂量组（中AS-Ⅳ组）和黄芪甲苷高剂量组（高AS-Ⅳ组）,每组10只;另设置10只健康SD大鼠为对照组。低AS-Ⅳ组、中AS-Ⅳ组和高AS-Ⅳ组大鼠每日灌胃给予剂量为20 mg/kg、40 mg/kg和80 mg/kg的AS-Ⅳ,每日1次,共给药6周;对照组和模型组大鼠同时给予等体积生理盐水灌胃。给药结束后,超声检测各组大鼠心脏功能指数：左室收缩末期内径（LVESD）、左室舒张末期内径（LVEDD）和左室射血分数（LVEF）。采用苏木精-伊红（HE）染色和Masson染色检测大鼠心肌组织病理变化,流式细胞术检测大鼠脾脏组织Th17细胞水平,ELISA法检测大鼠血清中IL-17、IL-21和TNF-α水平,qRT-PCR检测大鼠心肌组织RORγt和FoxP3基因表达水平,Western blot检测大鼠心肌组织α-SMA、collagenⅠ、TGF-β1、RORγt和FoxP3蛋白表达水平。结果：HE和Masson染色结果显示,模型组大鼠心肌组织出现炎性细胞浸润,细胞肥大,间质组织中蓝色染色胶原沉积量增加。与模型组比较,低AS-Ⅳ组、中AS-Ⅳ组和高AS-Ⅳ组大鼠心肌组织中炎性细胞浸润和细胞肥大情况逐渐缓解,胶原沉积量减少。与对照组比较,模型组大鼠LVESD和LVEDD指标均升高（P<0.05）,LVEF指标降低（P<0.05）;心肌组织α-SMA、collagen Ⅰ、TGF-β1和RORγt水平均升高（P<0.05）,FoxP3水平降低（P<0.05）;脾脏组织Th17细胞比例升高（P<0.05）;血清中IL-17、IL-21和TNF-α水平均升高（P<0.05）。与模型组比较,低AS-Ⅳ组、中AS-Ⅳ组和高AS-Ⅳ组大鼠LVESD和LVEDD指标均降低（P<0.05）,LVEF指标升高（P<0.05）;心肌组织α-SMA、collagen Ⅰ、TGF-β1和RORγt水平均降低（P<0.05）,FoxP3水平升高（P<0.05）;脾脏组织Th17细胞比例均降低（P<0.05）;血清中IL-17、IL-21和TNF-α水平均降低（P<0.05）。结论：AS-Ⅳ对阿霉素诱导的DCM大鼠具有良好的治疗效果,其机制可能与抗心肌组织纤维化和抑制Th17细胞分化相关。     

外泌体介导的辐射旁效应对心肌成纤维细胞的干预及黄芪甲苷的防护作用

为探讨放射性外泌体介导的辐射旁效应对心肌成纤维细胞(cardiac fibroblasts,CFs)的影响并观察黄芪甲苷(astragaloside IV,AST)的防护效应,该研究以2 Gy X线辐照大鼠CFs,48 h后超速离心提取放射性外泌体(X-exo),进行外泌体形态、浓度和表面标志蛋白鉴定。将CFs分为对照组(Control)、照射组(X-CFs)、CFs与放射性外泌体共培养组(X-exo+CFs)和黄芪甲苷干预组(X-exo+AST-CFs),采用流式细胞术检测各组细胞周期;划痕实验检测细胞迁移能力;Western blot检测纤维化相关分子TGF-β1、Col-I的表达情况。结果显示,X-CFs组和X-exo+CFs组在干预后24 h、48 h处于G₀/G₁期的细胞比例均低于Control组(P<0.01),S期和G₂/M期的细胞比例均高于Control组(P<0.01);X-CFs和X-exo+CFs组细胞迁移率在12 h、24 h、48 h时均显著高于Control组(P<0.05)...     

柱前衍生化HPLC法测定黄芪中黄芪甲苷的含量

目的：建立黄芪中黄芪甲苷的柱前衍化高效液相色谱测定法,并用该法对不同产地黄芪中黄芪甲苷的含量进行比较,方法：以吡啶－苯甲酰氯（２．５：１）为衍生化试剂,对黄芪甲苷分子中的羟基进行苯甲酰化,以甲醇－四氢呋喃－水（９０：４：６,０．２％三乙胺）为流动相,ＶＤ３为内标物,在２３０ｎｍ波长处检测。结果黄芪甲苷在０．００４－０．０８０ｍｇ．ｍＬ＾－１范围内呈线性,相关系数为０．９９９９,平均回收率为９４．７     

黄芪甲苷对辐射诱发肾损伤的干预作用及其机制

目的:探讨黄芪甲苷(AS-IV)对辐射诱导的小鼠肾脏损伤的防护作用及其硫氧还蛋白相互作用蛋白(TXNIP)/NOD样受体蛋白3(NLRP3)通路机制.方法:将小鼠分为正常对照组(Control)、二甲基亚砜(DMSO)溶剂组、辐射组(IR)、20 mg/kg AS-IV预防组(IR+AS-20 mg/kg)和40 mg...     

HPLC-ELSD法测定消栓通颗粒中黄芪甲苷的含量

目的:建立测定消栓通颗粒中黄芪甲苷含量的新方法.方法:采用高效液相色谱-蒸发光散射检测法测定,色谱柱为kromasilC18柱,以乙腈-水(32:68)为流动相,流速为1.0ml/min,ELSD条件为飘逸管温度为105℃,载气流速为2.5L/min;测定消栓通颗粒中黄芪甲苷的含量.结果:黄芪甲苷在1.08μg-6.48μg范围内与峰面积线性关系良好(r=0.9999),平均加样回收率为100.2%,RSD为0.93%.结论:方法灵敏、可靠、准确、重复性好,可作为消栓通颗粒的质量控制方法.     

Activation of cardiac progenitor cells through paracrine effects of mesenchymal stem cells

Mesenchymal stem cells (MSC) transplantation has been proved to be promising strategy to treat the failing heart. The effect of MSC transplantation is thought to be mediated mainly in a paracrine manner. Recent reports have suggested that cardiac progenitor cells (CPC) reside in the heart. In this study, we investigated whether MSC had paracrine effects on CPC in vitro. CPC were isolated from the neonatal rat heart using an explant method. MSC were isolated from the adult rat bone marrow. MSC-derived conditioned medium promoted proliferation of CPC and inhibited apoptosis of CPC induced by hypoxia and serum starvation. Chemotaxis chamber assay demonstrated that MSC-derived conditioned medium enhanced migration of CPC. Furthermore, MSC-derived conditioned medium upregulated expression of cardiomyocyte-related genes in CPC such as β-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP). In conclusion, MSC-derived conditioned medium had protective effects on CPC and enhanced their migration and differentiation.     

Clusterin induces CXCR4 expression and migration of cardiac progenitor cells

Clusterin (CST) is a stress-responding protein with multiple biological functions, including the inhibition of apoptosis and inflammation and transport of lipids. It may also participate in cell traffic and migration. In the process of post-infarct cardiac tissue repair, stem cells migrate into the damaged myocardium under the influence of chemoattractive substances such as stromal cell-derived factor (SDF). This study aimed at testing whether CST enhances expression of stem cell homing receptor and migration of cardiac progenitor cells (CPCs). CPCs isolated from fetal canine hearts transduced by CST cDNA expressed high levels of CXCR4, a receptor for SDF-1. The transfected cells also showed an increased migratory response to SDF-1 stimulation. The SDF-1-mediated migration of the CST-expressing CPCs was attenuated by PI3 kinase inhibitor LY294002 but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Analysis of cell cycle by flow cytometry revealed no significant difference in cell cycle between the transduced and control CPCs. Thus, CST expression may increase CPCs migration via increasing CXCR4 expression and SDF-1/chemokine receptor signaling in a PI3/Akt-dependent manner.     

PIWI-interacting RNA (piRNA) signatures in human cardiac progenitor cells

Cardiac progenitors, such as cardiospheres and cardiosphere-derived cells, represent an attractive cell source for cardiac regeneration. The PIWI-interacting RNAs, piRNAs, are an intriguing class of small non-coding RNAs, implicated in the regulation of epigenetic state, maintenance of genomic integrity and stem cell functions. Although non-coding RNAs are an exploiting field in cardiovascular research, the piRNA signatures of cardiac progenitors has not been evaluated yet.We profiled, through microarrays, 15,311 piRNAs expressed in cardiospheres, cardiosphere-derived cells and cardiac fibroblasts. Results showed a set of differentially expressed piRNAs (fold change ≥2, p < 0.01): 641 piRNAs were upregulated and 1,301 downregulated in the cardiospheres compared to cardiosphere-derived cells, while 255 and 708 piRNAs resulted up- and down-regulated, respectively, if compared to cardiac fibroblasts. We also identified 181 piRNAs that are overexpressed and 129 are downregulated in cardiosphere-derived cells respect to cardiac fibroblasts.Bioinformatics analysis showed that the deregulated piRNAs were mainly distributed on few chromosomes, suggesting that piRNAs are organized in discrete genomic clusters.Furthermore, the bioinformatics search showed that the most upregulated piRNAs target transposons, especially belonged to LINE-1 class, as validated by qRT-PCR. This reduction is also associated to an activation of AKT signaling, which is beneficial for cardiac regeneration.The present study is the first to show a highly consistent piRNA expression pattern for human cardiac progenitors, likely responsible of their different regenerative power. Moreover, this piRNome analysis may provide new methods for characterize cardiac progenitors and may shed new light on the understanding the complex molecular mechanisms of cardiac regeneration.     

GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells

Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.     

Irisin promotes cardiac progenitor cell-induced myocardial repair and functional improvement in infarcted heart

Irisin, a newly identified hormone and cardiokine, is critical for modulating body metabolism. New evidence indicates that irisin protects the heart against myocardial ischemic injury. However, whether irisin enhances cardiac progenitor cell (CPC)-induced cardiac repair remains unknown. This study examines the effect of irisin on CPC-induced cardiac repair when these cells are introduced into the infarcted myocardium. Nkx2.5⁺ CPC stable cells were isolated from mouse embryonic stem cells. Nkx2.5 ⁺ CPCs (0.5 × 10 ⁶) were reintroduced into the infarcted myocardium using PEGlylated fibrin delivery. The mouse myocardial infarction model was created by permanent ligation of the left anterior descending (LAD) artery. Nkx2.5 ⁺ CPCs were pretreated with irisin at a concentration of 5 ng/ml in vitro for 24 hr before transplantation. Myocardial functions were evaluated by echocardiographic measurement. Eight weeks after engraftment, Nkx2.5 ⁺ CPCs improved ventricular function as evident by an increase in ejection fraction and fractional shortening. These findings are concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Transplantation of Nkx2.5 ⁺ CPCs promoted cardiac regeneration and neovascularization, which were increased with the pretreatment of Nkx2.5 ⁺ CPCs with irisin. Furthermore, irisin treatment promoted myocyte proliferation as indicated by proliferative markers Ki67 and phosphorylated histone 3 and decreased apoptosis. Additionally, irisin resulted in a marked reduction of histone deacetylase 4 and increased p38 acetylation in cultured CPCs. These results indicate that irisin promoted Nkx2.5 ⁺ CPC-induced cardiac regeneration and functional improvement and that irisin serves as a novel therapeutic approach for stem cells in cardiac repair.     

Moderate dose insulin promotes function of endothelial progenitor cells

EPCs (endothelial progenitor cells) regenerate the vascular endothelial cells and keep the integrity of the vascular endothelium and thus may retard the onset of atherosclerosis. Steady state levels of EPCs in the circulation were found to be correlated with cardiovascular event risks. Given the close relationship between insulin and the cardiovascular system, we tested the long-term effects of moderate-dose insulin treatment on bone marrow-derived EPCs. Rat bone marrow EPCs were exposed to various levels of insulin under normal (5 mmol/l) or high (40 mmol/l) glucose conditions for 7 days. Insulin at levels near the physiological range (0.1, 1 nmol/l) up-regulated EPCs proliferation, stimulated NO (nitric oxide) production and reduced EPC senescence and ROS (reactive oxygen species) generation under both normal- and high-glucose conditions. Glucose exerted deleterious effects on EPCs contrary to insulin. Western blot analysis suggested concomitant decrease of Akt phosphorylation and eNOS (endothelial nitric oxide synthase) expression by high-glucose treatment and increase with insulin administration. Thus, insulin promoted several activities of EPCs, which suggested a potential endothelial protective role of insulin. Akt/eNOS pathway may be involved in the modulation of EPCs function by glucose and insulin.     

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.     

Adiponectin promotes endothelial progenitor cell number and function

Obesity-linked diseases are associated with suppressed endothelial progenitor cell (EPC) function. Adiponectin is an adipose-derived protein that is downregulated in obese and diabetic subjects. Here, we investigated the effects of adiponectin on EPCs. EPC levels did not increase in adiponectin deficient (APN-KO) in response to hindlimb ischemia. Adenovirus-mediated delivery of adiponectin increased EPC levels in both WT and APN-KO mice. Incubation of human peripheral blood mononuclear cells with adiponectin led to an increase of the number of EPCs. Adiponectin induced EPC differentiation into network structures and served as a chemoattractant in EPC migration assays. These data suggest that hypoadiponectinemia may contribute to the depression of EPC levels that are observed in patients with obesity-related cardiovascular disorders.     

Foetal and adult cardiomyocyte progenitor cells have different developmental potential

In the past years, cardiovascular progenitor cells have been isolated from the human heart and characterized. Up to date, no studies have been reported in which the developmental potential of foetal and adult cardiovascular progenitors was tested simultaneously. However, intrinsic differences will likely affect interpretations regarding progenitor cell potential and application for regenerative medicine. Here we report a direct comparison between human foetal and adult heart‐derived cardiomyocyte progenitor cells (CMPCs). We show that foetal and adult CMPCs have distinct preferences to differentiate into mesodermal lineages. Under pro‐angiogenic conditions, foetal CMPCs form more endothelial but less smooth muscle cells than adult CMPCs. Foetal CMPCs can also develop towards adipocytes, whereas neither foetal nor adult CMPCs show significant osteogenic differentiation. Interestingly, although both cell types differentiate into heart muscle cells, adult CMPCs give rise to electrophysiologically more mature cardiomyocytes than foetal CMPCs. Taken together, foetal CMPCs are suitable for molecular cell biology and developmental studies. The potential of adult CMPCs to form mature cardiomyocytes and smooth muscle cells may be essential for cardiac repair after transplantation into the injured heart.     

Toll-like receptors in the functional orientation of cardiac progenitor cells

Cardiac progenitor cells (CPCs) have the potential to differentiate into several cell lineages with the ability to restore in cardiac tissue. Multipotency and self-renewal activity are the crucial characteristics of CPCs. Also, CPCs have promising therapeutic roles in cardiac diseases such as valvular disease, thrombosis, atherosclerosis, congestive heart failure, and cardiac remodeling. Toll-like receptors (TLRs), as the main part of the innate immunity, have a key role in the development and differentiation of immune cells. Some reports are found regarding the effect of TLRs in the maturation of stem cells. This article tried to find the potential role of TLRs in the dynamics of CPCs. By showing possible crosstalk between the TLR signaling pathways and CPCs dynamics, we could achieve a better conception related to TLRs in the regeneration of cardiac tissue.