Involvement of IGF-I receptor and estrogen receptor pathways in the protective effects of ginsenoside Rg1 against Aβ25–35-induced toxicity in PC12 cells

Ginsenoside Rg1 is the main pharmacologically active compound of ginsenosides and has demonstrated pharmacological effects in the cardiovascular system, central nervous system and immune system. The involvement of insulin-like growth factor-I receptor (IGF-IR)-dependent pathway and estrogen receptor (ER)-dependent pathway in the biological effect of ginsenoside Rg1 have been demonstrated in our previous study. The present study tested the hypothesis that the protective effects of Rg1 against Aβ_25–35-induced toxicity involved activation of the IGF-IR and ER signaling pathways in PC12 cells. Treatment with Aβ_25–35 decreased the cell viability in a dose-dependent manner in PC12 cells. Rg1 pretreatment resulted in an enhancement of survival and the maximum protection occurred at the concentration of 1 μM. Co-treatment with IGF-IR antagonist JB-1 or ER antagonist ICI182,780 could completely block the protective effect of Rg1. The decreased Bcl-2 mRNA expression induced by Aβ_25–35 could be restored by Rg1 pretreatment. Rg1 pretreatment could also restore the decreased mitochondrial membrane potential induced by Aβ_25–35 and these effects could be completely blocked by JB-1 or ICI182,780. In addition, Rg1 treatment alone could significantly increase the phosphorylation level of MEK and ERK in a time-dependent manner and the functional transactivation of ERα in PC12 cells. The functional transactivation of ERα by Rg1 could be completely blocked by JB-1 or ICI182,780. Taken together, our results suggest that IGF-IR and ER signaling pathways might be involved in the protective effect of Rg1 against Aβ_25–35-induced toxicity in PC12 cells.     

Ginsenoside Rg1 protects H9c2 cells against nutritional stress-induced injury via aldolase /AMPK/PINK1 signalling

Insufficient nutrients supply will greatly affect the function of cardiac myocytes. The adaptive responses of cardiac myocytes to nutritional stress are not fully known. Ginsenoside Rg1 is one of the most pharmacologically active components in Panax Ginseng and possesses protective effects on cardiomyocyte. Here, we investigate the effects of ginsenoside Rg1 on H9c2 cells which were subjected to nutritional stress. Nutritional stress-induced by glucose deprivation strongly induced cell death and this response was inhibited by ginsenoside Rg1. Importantly, glucose deprivation decreased intracellular ATP levels and mitochondrial membrane potential. Ginsenoside Rg1 rescued ATP levels and mitochondrial membrane potential in nutrient-starved cells. For molecular mechanisms, ginsenoside Rg1 increased the expressions of PTEN-induced kinase 1 (PINK1) and p-AMPK in glucose deprivation treated H9c2 cells. Reducing the expression of aldolase in H9c2 cells inhibited ginsenoside Rg1′s actions on PINK1 and p-AMPK. Further, the nutritional stress mice were used to verify the mechanisms obtained in vitro. Ginsenoside Rg1 increased the expressions of aldolase, p-AMPK, and PINK1 in starved mice heart. Taken together, our results reveal that ginsenoside Rg1 limits nutritional stress-induced H9c2 cells injury by regulating the aldolase /AMP-activated protein kinase/PINK1 pathway.     

Ginsenoside Rg1 Protects against Cardiac Remodeling in Heart Failure via SIRT1/PINK1/Parkin-Mediated Mitophagy

Adverse cardiac remodeling may lead to the development and progression of heart failure, which is lack of effective clinical treatment. Ginsenoside Rg1 (GRg1), a primary ingredient of Panax ginseng, protects against diverse cardiovascular disease, but its effects on cardiac remodeling remain unclear. Thus, we investigated the protective effect and mechanism of GRg1 on cardiac remodeling after myocardial infarction. GRg1 significantly ameliorated cardiac remodeling in mice with left anterior descending coronary artery ligation, reflected by reduced left ventricular dilation and decreased cardiac fibrosis, accompanied by improved cardiac function. Mechanistically, GRg1 considerably increased mitophagosomes formation, ameliorated cardiac mitochondria damage, and enhanced SIRT1/PINK1/Parkin-mediated mitophagy during cardiac remodeling. Consistently, GRg1 increased cell viability and attenuated apoptosis and fibrotic responses in H₂O₂-treated H9c2 cells by promoting the SIRT1/PINK1/Parkin axis. Furthermore, SIRT1-specific inhibitor (EX527) or the use of small interfering RNA against Parkin abolished the protective effect of GRg1 in vitro. These findings reveal a novel mechanism of GRg1 alleviating cardiac remodeling via enhancing SIRT1/PINK1/Parkin-mediated mitophagy.     

Ginsenoside Rg1 protects rat cardiomyocyte from hypoxia/reoxygenation oxidative injury via antioxidant and intracellular calcium homeostasis

Ginsenoside Rg1 is a major active ingredient of Panax notoginseng radix which has demonstrated a number of pharmacological actions including a cardioprotective effect in vivo. This study investigated the protective effect and mechanism of ginsenoside Rg1 in cardiomyocytes hypoxia/reoxygenation (H/R) model. Pretreatment with ginsenoside Rg1 (60–120 µM) reduced lactate dehydrogenase release and increased cell viability in a dose‐dependent manner. Fluorescence analysis demonstrated ginsenoside Rg1 reduced intracellular ROS and suppressed the intracellular [Ca²⁺] level. Cell lysate detected an increase of T‐SOD, CAT, and GSH levels. The myocardial protection of ginsenoside Rg1 during H/R is partially due to its antioxidative effect and intracellular calcium homeostasis. J. Cell. Biochem. 108: 117–124, 2009. © 2009 Wiley‐Liss, Inc.     

人参皂甙Rg1与1,6-二磷酸果糖配伍抗疲劳作用的研究

观察人参皂甙Rg1与1,6-二磷酸果糖配伍的抗疲劳效果,寻找最佳配伍剂量。根据雄性清洁级昆明种小鼠按体重随机分为安静对照组、运动对照组、人参皂甙Rg1对照组及4个配伍组。通过析因实验设计分析人参皂甙Rg1与1,6-二磷酸果糖提高运动耐力的效果及两者的交互作用。结果不同剂量人参皂甙Rg1对小鼠力竭游泳时间影响有显著差异(P<0.05)。各配伍组力竭游泳时间均有显著延长,C组延长最显著;与人参皂甙Rg1对照组相比,各配伍组力竭游泳时间均显著降低(P<0.01)。MDA含量A组显著低于运动对照组和D组,与安静对照组相比,各组均升高。SOD/MDA比值A、C组显著高于运动对照组,C组显著高于人参皂甙Rg1对照组,与安静对照组相比,各组均有下降趋势,D组下降最显著(P<0.01)。乳酸脱氢酶C、D组显著低于运动对照组和人参皂甙Rg1对照组(P<0.05)。与运动对照组及人参皂甙Rg1对照组相比,配伍在一定程度上减少心肌、骨骼肌细胞线粒体和其他细胞超微结构损伤。结论:与FDP配伍未能延长小鼠力竭游泳时间,配伍可减轻耐力运动对小鼠心肌和骨骼肌的细胞损害,一定程度上保护线粒体的呼吸功能,缓解细胞缺氧损伤。     

人参皂苷Rg1促进人牙周膜干细胞增殖及骨向分化

人参皂苷Rg1 (GS Rg1) 是人参的主要药理活性成分. GS Rg1有刺激造血干细胞的形成和促进骨髓间充质干细胞增殖和分化的作用.而人牙周膜干细胞（human periodontal ligament stem cells, hPDLSCs）具有自我更新和多向分化的干细胞特性.但目前关于GS Rg1能否促进牙周膜干细胞增殖和分化的研究尚不多见.本研究证明,1×10-5 mol/L GS Rg1作用于hPDLSCs后,能明显促进牙周膜干细胞的增殖与分化.MTT检测细胞增殖显示,培养液中加入了GS Rg1实验组在第2,3,4,5 d增殖情况明显高于对照组,提示GS Rg1成分促进了牙周膜干细胞的增殖. 检测ALP表达量,RUNX2,Collagen I,OPN,OCN表达水平,加药实验组表达水平均高于未加药对照组,它们的表达量的增高提示成骨分化的增强. 牙周膜干细胞与纳米羟基磷灰石支架结合的电镜图片及其支架植入小鼠体内后的免疫组化检测显示,含有GS Rg1成分的细胞支架能促进形成更多的骨样组织.因此,GS Rg1能促进hPDLSCs体内体外的增殖及骨向分化,并在替代传统生长因子应用于牙周组织工程方面有较好前景.     

Rg1 protects iron‐induced neurotoxicity through antioxidant and iron regulatory proteins in 6‐OHDA‐treated MES23.5 cells

Ginsenoside‐Rg1 is one of the pharmacologically active components isolated from ginseng. It was reported that Rg1 protected dopamine (DA) neurons in 6‐hydroxydopamine (6‐OHDA)‐induced Parkinson's disease (PD) models in vivo and in vitro. Our previous study also demonstrated that iron accumulation was involved in the toxicity of 6‐OHDA. However, whether Rg1 could protect DA neurons against 6‐OHDA toxicity by modulating iron accumulation and iron‐induced oxidative stress is not clear. Therefore, the present study was carried out to elucidate this effect in 6‐OHDA‐treated MES23.5 cells and the possible mechanisms were also conducted. Findings showed Rg1 restored iron‐induced decrease in mitochondrial transmembrane potential in MES23.5 cells, and increased ferrous iron influx was found in 6‐OHDA‐treated cells. Rg1 pretreatment could decrease this iron influx by inhibiting 6‐OHDA‐induced up‐regulation of an iron importer protein divalent metal transporter 1 with iron responsive element (DMT1 + IRE). Furthermore, findings also showed that the effect of Rg1 on DMT1 + IRE expression was due to its inhibition of iron regulatory proteins (IRPs) by its antioxidant effect. These results suggested that the neuroprotective effect of Rg1 against iron toxicity in 6‐OHDA‐treated cells was to decrease the cellular iron accumulation and attenuate the improper up‐regulation of DMT1 + IRE via IRE/IRP system. This provides new insight to understand the pharmacological effects of Rg1 on iron‐induced degeneration of DA neurons. J. Cell. Biochem. 111: 1537–1545, 2010. © 2010 Wiley‐Liss, Inc.     

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor

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人参皂苷Rg1组合诱导人成骨肉瘤MG-63细胞分化过程中Prohibitin的表达与定位变化

选择性抽提经人参皂苷Rg1组合（RCT）诱导处理前后的人成骨肉瘤MG-63细胞核基质,对prohibitin在核基质中的存在、分布及其与相关基因产物在RCT处理前后MG-63细胞中的共定位关系进行观察研究.蛋白质组学分析结果显示,prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在RCT处理后细胞核基质中表达下调;蛋白质印迹杂交确证了prohibitin在MG-63细胞核基质中的存在及其在RCT处理后下调变化;免疫荧光显微镜观察进一步证实prohibitin定位在核基质上,经RCT处理后出现分布位置与表达水平变化;激光共聚焦显微镜观察可见prohibitin与c-Fos、c-Myc、p53和Rb基因产物均存在共定位关系,并在RCT处理后共定位分布区域出现变化.本研究证实了prohibitin是一种新发现的核基质蛋白,其在核基质上的定位与表达在RCT诱导分化前后发生显著变化,并与相关癌基因、抑癌基因产物存在共定位关系.实验表明RCT处理引起的prohibitin的变化与人成骨肉瘤MG-63细胞的诱导分化与调控具有密切关系,为深入揭示RCT等中药有效成分诱导肿瘤细胞分化的机理提供了重要科学依据和深入探索的新方向.     

Insulin-like Growth Factor (IGF) I Down-Regulates Type 1 IGF Receptor (IGF 1R) and Reduces the IGF I Response in A549 Non-Small-Cell Lung Cancer and Saos-2/B-10 Osteoblastic Osteosarcoma Cells

The insulin-like growth factor type 1 receptor (IGF 1R) mediates the acute metabolic effects of IGF I as well as IGF I-stimulated cell proliferation and protection from apoptosis. IGF binding proteins (IGFBPs) can modulate these responses. We, therefore, investigated whether intrinsic IGFBPs interfere with IGF I-induced regulation of IGF 1R expression and with the biological response to IGF I in two human tumor cell lines, the non-small-cell lung cancer cell line A549 and the osteoblastic osteosarcoma cell line Saos-2/B-10. We compared the growth rates, IGFBP production, IGF I binding characteristics, IGF 1R protein and mRNA levels, and the acute IGF I response (stimulation of glycogen synthesis) after pretreatment of the cells in serum-free medium with or without added IGF I or medium supplemented with 5% fetal calf serum (FCS). In contrast to A549 cells, which produce IGF I and significant amounts of IGFBPs, survival and proliferation of Saos-2/B-10 cells, which do not produce IGF I or significant amounts of IGFBPs, depended on the addition of exogenous IGF I. IGF I increased the concentration of IGFBP-2 and -3 and decreased the concentration of IGFBP-4 in the medium of A549 cells. As compared to FCS, IGF I pretreatment in both cell lines decreased the number of specific IGF I binding sites, down-regulated total and membrane IGF 1R protein, and largely reduced or abolished the acute IGF I response without affecting IGF 1R mRNA levels. The data suggest that the IGF 1R protein of the two cell lines is translationally and/or posttranslationally down-regulated by its ligand in the presence and in the absence of locally produced IGFBPs and that the cell lines have retained this negative feedback to counteract IGF I stimulation.     

TGF-beta 1 modulation of IGF-I signaling pathway in rat thyroid epithelial cells

Transforming growth factor beta 1 (TGF-beta 1) and insulin-like growth factor I (IGF-I) have contrasting effects on cell cycle regulation in thyroid cells and TGF-beta 1 induces a dramatic decrease in IGF-I-induced cell proliferation. The aim of the present study was to investigate the molecular mechanism of cross-talk between TGF-beta 1 and IGF-I in FRTL-5 cells. TGF-beta 1 affected IGF-I-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with Grb2 protein. Moreover, TGF-beta 1 decreased the IGF-I-induced tyrosine phosphorylation of the adaptor protein CrkII and its association with the IGF-I receptor. These results were accompanied by TGF-beta 1 inhibition of IGF-I-stimulated mitogen-activated protein kinase phosphorylation and activation. Conversely, TGF-beta 1 did not alter IGF-I-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, IGF-I-induced tyrosine phosphorylation of Shc, and its binding to Grb2. Taken together, these findings provide a molecular basis for the growth-inhibitory action of TGF-beta 1 on the IGF-I-induced mitogenic effect.     

Ginsenoside Rg1 facilitates neural differentiation of mouse embryonic stem cells via GR-dependent signaling pathway

Ginsenoside Rg1, a steroidal saponin of high abundance in ginseng, possesses the neuroprotective effects. In this study, we tried to explore the effect of Rg1 on promoting differentiation of mouse embryonic stem (ES) cells towards the neuronal lineage and its potential role involved in glucocorticoid receptor (GR) activation. Rg1 treatment induced a remarkable increase in the population of neuron-like cells in a time-dependent manner. More than 80% of Rg1-treated embryoid bodies (EBs) differentiated into neuron-like cells on d 8 + 10. Furthermore, the gradually increased protein expression of neurofilament (NEFM) and β-tubulin III (a neuronal specific protein) was determined. GR expression gradually increased during the differentiation course. RU486, an antagonist of GR, could efficiently block the neurogenesis-promoting activity of Rg1. On the other side, Rg1 stimulated the phosphorylation of ERK1/2 and Akt at different time points through GR activation-dependent mechanisms. Treatment of both U0126 (an inhibitor of MEK) and LY294002 (an inhibitor of PI3 K), hampered the neuronal differentiation induced by Rg1. Meantime, U0126 further decreased Rg1-induced p-Akt expression. In conclusion, Rg1 possesses the effects on inducing differentiation of mouse ES cells into neurons in vitro via the GR-MEK-ERK1/2-PI3 K-Akt signaling pathway.     

Intranigral infusion of interleukin-1beta activates astrocytes and protects from subsequent 6-hydroxydopamine neurotoxicity

Activation of glial cells is a prevalent response to neuronal damage in brain disease and ageing, with potential neuroprotective and neurotoxic consequences. We were interested in studying the role of glial activation on dopaminergic neurons of the substantia nigra in an animal model of Parkinson's disease. Thus, we evaluated the effect of a pre-existing glial activation on the dopaminergic neuronal death induced by striatal infusion of 6-hydroxydopamine. We established a model of local glial activation by stereotaxic infusion of interleukin-1beta in the substantia nigra of adult rats. Interleukin-1beta (20 ng) induced a marked activation of astrocytes at days 2, 5 and 10, revealed by heat-shock protein 27 and glial fibrillary acid protein immunohistochemistry, but did not affect the microglial markers OX-42 and heat-shock proteins 32 or 47. Intranigral infusion of interleukin-1beta 5 days before a striatal injection of 6-hydroxydopamine significantly protected nigral dopaminergic cell bodies, but not striatal terminals from the 6-hydroxydopamine lesion. Also, in the animals pre-treated with interleukin-1beta, a significant prevention of 6-hydroxydopamine-induced reduction of adjusting steps, but not of 6-hydroxydopamine-induced amphetamine rotations, were observed. These data show the characterization of a novel model of local astroglial activation in the substantia nigra and support the hypothesis of a neuroprotective role of activated astrocytes in Parkinson's disease.     

人参皂苷Rg1对游离脂肪酸诱导非酒精性脂肪肝细胞炎症的改善作用及机制研究

该研究探讨人参皂苷Rg1对非酒精性脂肪性肝细胞炎症反应的作用及其分子机制。用1 mmol/L游离脂肪酸处理HepG2和L02细胞24 h,再用20μg/mL或40μg/mL人参皂苷Rg1处理6 h;设置对照组、模型组、低剂量Rg1组、高剂量Rg1组。全自动生化仪检测各组细胞上清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)的含量;酶联免疫吸附法测定细胞上清IL-1β、IL-6、TNF-α。RT-qPCR及Western blot检测NF-κB通路相关基因及蛋白的改变。免疫荧光染色观察NF-κB核转移;Western blot检测各组胞质与胞核内的NF-κB P65蛋白的表达。与对照组相比,模型组培养上清炎症指标明显增加(P<0.05);Rg1能降低炎症指标的表达(P<0.05)。Rg1能减少游离脂肪酸诱导的NF-κB磷酸化及其下游IL-1β、IL-6、TNF-α的表达,减少NF-κB P65从胞质向胞核的转移(P<0.05)。Rg1可通过抑制NF-κB活化减少NASH细胞模型炎症反应,为非酒精性脂肪性肝炎的治疗提供了可能的靶点。     

Rb1、Rg1对肾缺血／再灌注血清诱导HK-2细胞凋亡中Bcl-2、Bax表达的影响

目的：探讨人参皂甙Rb1、Rg1在肾缺血／再灌注血清诱导HK-2细胞凋亡中对Bol-2、Bax表达的影响。方法：制备家兔肾缺血／再灌注血清（SIR）和对照组血清（SC）用于HK-2细胞培养,TUNEL法检测细胞凋亡。实验分组：对照组、缺血／再灌注组、Rb1干预组、Rg1干预组,培养24h后免疫细胞化学法检测Bcl-2、Bax的表达。结果：与缺血／再灌注组比较,Rb1干预组和Rg1干预组Bax的表达明显下降（P〈0．01）,Bcl-2／Bax比值增大。结论：人参皂甙Rb1、Rg1对肾缺血／再灌注血清诱导HK-2细胞凋亡具有保护作用。     

Lipocalin-type prostaglandin D2 synthase reduces glucagon secretion in alpha TC-1 clone 6 cells via the DP1 receptor

Diabetes is associated with disturbances in the normal levels of both insulin and glucagon, both of which play critical roles in the regulation of glycemia. Recent studies have found lipocalin-type prostaglandin D₂ synthase (l-PGDS) to be an emerging target involved in the pathogenesis of type-2 diabetes. This study focused on the effect of l-PGDS on glucagon secretion from cultured pancreatic Alpha TC-1 Clone 6 cells. When cells were treated with various concentrations of l-PGDS (0, 10, 50, and 100 ug/ml) for 2 h in 1 mM glucose; glucagon secretion decreased to 670±45, 838±38, 479±11, and 437±45 pg/ml, respectively. In addition, pancreatic islets were isolated from C57BL/6 mice and stained for prostaglandin D₂ receptors, DP1 and DP2, using immunohistochemistry. Our results showed that these islets express only the DP1 receptor. Pancreatic islets were then stained for alpha and beta cells, as well as DP1, to find the primary location of the receptor within the islets using immunofluorescence. Interestingly, DP1 receptor density was found primarily in alpha cells rather than in beta cells. Our study is the first to report a correlation between l-PGDS and glucagon secretion in alpha cells. Based on our obtained results, it can be concluded that higher concentrations of l-PGDS significantly reduced the secretion of glucagon in alpha cells, which may contribute to the pathogenesis of diabetes as well as offer a novel therapeutic site for the treatment of diabetes.