人参皂苷Rg1对寡聚肽Aβ_(1-42)增加JNK活性及诱导凋亡的影响

目的在寡聚肽Aβ1-42诱导的神经元凋亡过程中,探讨人参皂苷Rg1的保护作用和可能机制;方法运用寡聚肽Aβ1-42来诱导原代培养的皮层神经元损害,把神经元分为空白对照组、Aβ组、sp600125与Aβ共同孵育组以及人参皂苷Rg1与Aβ共同孵育组,然后观察JNK、p-JNK、caspase-3活性和TUNEL细胞数的变化。结果寡聚肽Aβ1-42作用15 min,皮层神经元的JNK磷酸化水平明显增加;经过预先孵育24 h人参皂苷Rg1(2.5~10μmol·L-1)后,再与寡聚肽Aβ1-42共同孵育15 min,JNK磷酸化水平明显降低;寡聚肽Aβ1-42孵育24 h,caspase-3活性和TUNEL数明显升高;人参皂苷Rg1(10μmol·L-1)预处理24 h后,再与Aβ1-42共孵育24 h组中caspase-3和TUNEL阳性数目明显下降。结论人参皂苷Rg1可通过JNK通路减轻寡聚肽Aβ1-42诱导的神经元凋亡。     

p25/cdk5可能参与人参皂苷Rb1减轻Aβ_(25-35)诱导的tau蛋白过度磷酸化

目的探讨在Aβ25-35诱导的皮层神经元tau蛋白过度磷酸化中,人参皂苷Rb1对周期依赖性蛋白激酶(cyc lin-de-pendent k inase 5,CDK 5)的激动亚基p35/p25的影响。方法通过蛋白免疫印迹法和免疫细胞化学染色法检测胎鼠皮层神经元CDK 5的两个亚基cdk5和p35/p25的蛋白水平,以及CDK 5的磷酸化底物tau蛋白在Ser199/202、Thr205、Ser396和Ser404位点的磷酸化水平。结果凝聚态Aβ25-35(20μmol.L-1)作用于皮层神经元12 h,可使皮层神经元中p25的数量增多,以及tau蛋白在Ser199/202、Thr205、Ser396和Ser404位点的磷酸化水平增高,但对cdk 5亚基表达水平影响并不明显。Rb1和calpain特异性抑制剂calpeptin可减少皮层神经元p25的生成,同时人参皂苷Rb 1和CDK 5特异性抑制剂roscovitine可减轻凝聚态Aβ25-35诱导的皮层神经元tau蛋白的过度磷酸化水平。结论p25/cdk 5可能参与人参皂苷Rb1减轻Aβ25-35诱导的tau蛋白过度磷酸化。     

人参皂苷Rg1可能通过抗氧化作用来保护帕金森病鼠黑质神经元

研究人参皂苷Rgl对1—甲基—4—苯基—1，2，3，6—四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的小鼠黑质神经元凋亡的抗氧化保护作用。方法：采用MPTP制备帕金森病(PD)小鼠模型，经人参皂苷Rg1预处理后，用尼氏(Nissl)染色和TH组化染色观察黑质神经元的存活情况，通过活化型Caspase3的免疫组化染色和TUNEL染色了解黑质神经元的凋亡情况，并用生化技术对黑质区域谷胱甘肽（GSH)浓度及超氧化物歧化酶(SOD)活力进行检测。结果：人参皂苷Rg1预处理能提高黑质区域GSH的浓度及降低SOD活力，相应地减少PD鼠黑质致密带Nissl阳性神经元和TH阳性神经元的脱失现象，使活化型Caspase3表达阳性细胞减少，降低黑质神经元TUNEL染色的阳性率。结论：人参皂苷Rg1可能对MPTP诱导的小鼠黑质神经元凋亡有抗氧化保护作用。     

人参皂苷Rg1对Aβ_(25-35)诱导大鼠海马神经元tau蛋白异常磷酸化的影响

目的探讨人参皂苷Rg1对Aβ2535所致大鼠海马神经tau蛋白异常磷酸化的抑制作用及其可能机制。方法应用脑立体定向技术向成年大鼠海马背侧注射凝聚态Aβ25355nmol制备AD样大鼠模型,术后分别给予腹腔内注射不同浓度的人参皂苷Rg1(625、125、25μmol·kg-1)处理,14d后处死,采用镀银染色方法观察海马组织神经元病理改变;免疫组织化学染色方法和免疫蛋白印迹技术显示大鼠脑内[pS396]tau、[pSpS199/202]tau、[pT231]tau的表达水平情况,以及总tau蛋白的水平(tau5);免疫蛋白印迹技术检测海马组织中GSK3β和磷酸化GSK3β的水平变化。结果凝聚态Aβ2535注射组神经元纤维走行紊乱,增粗、肿胀密集成宽带状,轴突深染;而人参皂苷Rg1对神经元具有明显的保护作用,脑内总tau的水平下降,[pS396]tau、[pSpS199/202]tau、[pT231]tau的表达明显低于Aβ2535注射组(P<001),以25μmol·kg-1保护作用最明显;GSK3β和磷酸化GSK3β的水平亦明显低于Aβ2535注射组,与正常和假注射组差异无显著性(P>005),以25μmol·kg-1作用最明显。结论人参皂苷Rg1对Aβ2535诱导的AD样大鼠海马神经元具有保护作用,其机制可能是通过阻断GSK3β的活性而降低磷酸化tau蛋白的表达而实现的。     

人参皂苷Rg1对细胞衰老过程中p21，cyclin E和CDK2表达的影响

目的探讨p21、细胞周期蛋白E (cyclin E) 和周期蛋白依赖性蛋白激酶2 (cyclin-dependent kinase 2, CDK2) 在人参皂苷Rg1对抗三丁基过氧化氢(t-BHP)诱导WI-38细胞衰老过程中的可能作用。方法细胞超微结构、流式细胞分析和β-半乳糖苷酶细胞化学染色观察衰老细胞，蛋白印迹法检测p21,cyclin E和CDK2蛋白的表达。结果Rg1预处理可明显减弱t-BHP对WI-38细胞衰老的诱导作用，同时p21表达水平明显降低，cyclin E和CDK2表达水平增加。结论人参皂苷Rg1对抗三丁基过氧化氢对细胞衰老的诱导作用可能与其改变p21，cyclin E和CDK2的表达水平有关。     

人参皂苷Rg1对Aβ_(25-35)诱导的神经细胞核因子-κB活化的影响

目的探讨人参皂苷Rg1拮抗β淀粉样蛋白(Aβ)、保护神经细胞的作用是否与核因子κB(NF-κB)活化机制变化有关。方法分别以原代培养的大鼠海马神经元和星形胶质细胞为靶标建立Alzheimer病(AD)细胞模型。采用四唑盐显色(MTT)法检测细胞活力,进行Aβ25-35造模浓度、时间以及人参皂苷Rg1预处理最适宜浓度的选择。经Aβ25-35和Rg1干预后,用激光共聚焦显微镜检测分析FITC/PI双重标记的NF-κB在细胞内的激活程度,结合形态学观察分析人参皂苷Rg1对以上两种细胞的保护作用。结果40μmol.L-1的Aβ25-35作用24h后可激活原代培养星形胶质细胞的NF-κB(P<0.01),下调原代培养神经元的NF-κB活性(P<0.01)。2μmol.L-1的人参皂苷Rg1能明显提高神经元的NF-κB活性(P<0.01),减弱星形胶质细胞的NF-κB活性(P<0.01)。同时结合形态学观察和细胞活力检测发现,人参皂苷Rg1对两种细胞都有保护作用。结论人参皂苷Rg1通过启动神经元中NF-κB活化、下调星形胶质细胞的NF-κB活性,从两方面发挥其保护神经细胞的作用,从而有可能达到减缓AD发病进程的效果。     

人参皂苷Rc、Re、Rf和Rg1对药物代谢酶CYP1A1活性诱导作用研究

目的研究人参皂苷Rc、Re、Rf和Rg1能否激活h AhR受体信号通路诱导CYP1A1基因及蛋白表达。方法利用前期实验室构建的p GL4.17-CYP1A1报告基因质粒、pc DNA3.1-h AhR表达质粒与pRL-TK内参质粒共转染Hep G2细胞,检测人参皂苷Rc、Re、Rf和Rg1对AhR的转录激活效应;并利用Real-time PCR及Western blot技术对人参皂苷Rc、Re、Rf和Rg1的不同浓度、不同时间处理组进行mRNA及蛋白的检测。结果报告基因模型检测结果显示,人参皂苷Rc、Re、Rf和Rg1对AhR具有转录激活作用,其中人参皂苷Re与Rf对AhR转录激活效应明显;同时不同浓度人参皂苷Rc、Re、Rf和Rg1均能上调CYP1A1 mRNA与蛋白表达水平。结论人参皂苷Rc、Re、Rf和Rg1可以诱导CYP1A1mRNA与蛋白质水平的表达,这种诱导作用可能与上述皂苷成分激活AhR并提高其对CYP1A1的转录活性有关。     

人参皂苷 Rg1减轻大鼠离体心脏缺血再灌注损伤的作用研究

目的：探讨人参皂苷 Rg1对大鼠离体心脏缺血再灌注损伤的减轻作用及机制。方法制备大鼠离体心脏 I/ R 模型,将18只大鼠随机分为正常对照组、I/ R 组、人参皂苷 Rg1组。100μmol/ L 人参皂苷 Rg1进行预处理10 min,观察血流动力学指标左心室内压最大上升/下降速率和心率压力乘积的变化,检测灌流液中肌酸激酶（CK）、乳酸脱氢酶（LDH）活性。Western blotting 检测心肌组织 PI3K 下游激酶 Akt、ERK1/2及其磷酸化水平的变化,磷酸化GSK-3β的蛋白表达。结果与 I/ R 组比较,人参皂苷 Rg1预处理显著改善心脏功能,降低灌流液中 CK、LDH 活性（ P ﹤0.05）;升高 p-Akt 和 p-GSK-3β的蛋白表达（ P ﹤0.05）。结论人参皂苷 Rg1对大鼠离体心脏缺血再灌注损伤有减轻作用,其机制与激活 PI3K-Akt 通路有关。     

人参皂苷Rg1抗黑质神经元凋亡的可能机制

目的研究人参皂苷Rg1抗1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的小鼠黑质神经元凋亡的作用及其机制。方法MPTP制备的帕金森病(Parkinson′s disease,PD)小鼠模型,经人参皂苷Rg1预处理后,用尼氏(Nissl)染色和TH组化染色观察黑质神经元的损害情况,借助TUNEL染色了解黑质神经元的凋亡情况,并用免疫组织化学方法检测黑质神经元caspase-3的活化以及iNOS和nNOS的表达情况。结果人参皂苷Rg1预处理能减少PD鼠模型黑质致密带Nissl阳性神经元和TH阳性神经元的脱失现象,降低黑质神经元TUNEL染色的阳性率。结论人参皂苷Rg1预处理对MPTP诱导的小鼠黑质神经元凋亡有明显的保护作用。     

五味子乙素抑制视网膜母细胞瘤细胞生物学行为的分子机制研究

摘要：目的:研究五味子乙素对人视网膜母细胞瘤Y79细胞增殖、凋亡、迁移及侵袭的影响,及其对共济失调毛细血管扩张突变基因（ATM）/转录因子E2F1信号通路的作用。方法:体外培养Y79细胞,ATM inhibitor质粒和ATM mimics质粒转染Y79细胞进行ATM沉默或过表达,细胞计数试剂盒-8（CCK-8）法测定细胞活力和增殖情况,流式细胞仪分析Y79细胞凋亡及周期分布,划痕实验检测Y79细胞迁移,Transwell实验分析细胞侵袭,Western Blotting实验检测Y79细胞中细胞周期蛋白（cyclin D1、cyclin B1和CDK2）、抑癌基因周期蛋白依赖性激酶抑制4b（INK4b）、INK4a、肿瘤抑制蛋白ARF、人体抑癌基因p53、人视网膜母细胞抑制蛋白pRB、ATM及E2F1蛋白表达。结果:五味子乙素呈浓度依赖性抑制Y79细胞活力、克隆、迁移及侵袭,将Y79细胞阻滞在G0/G1期,诱导其凋亡;另外,五味子乙素显著诱导INK4b、INK4a、ARF、p53、pRB、ATM及E2F1蛋白表达,显著抑制cyclin D1、cyclin B1及CDK2蛋白水平（P<0.05）; ATM沉默能部分降低五味子乙素对Y79细胞生物学行为的抑制作用。结论:五味子乙素通过激活ATM/E2F1信号传导,进而抑制视网膜母细胞瘤细胞增殖,提示ATM/E2F1信号通路可能是视网膜母细胞瘤治疗的新靶标。     

CDK4，pRB和E2F1参与人参皂片Rg1对淀粉样β蛋白诱导的大鼠皮层神经元凋亡的抑制作用

AIM: To explore the possible mechanism of beta-amyloid (Abeta)-induced apoptosis in rat cortical neurons and the protective effect of ginsenoside Rg1. METHODS: AO-EB staining was used to quantify the apoptotic cells. DNA fragmentation was observed by gel electrophoresis. The levels of cyclin-dependent kinases-4 (CDK4) and phosphorylated pRB were detected by Western blot. RT-PCR was used to examine the expression of E2F1 mRNA. RESULTS: Treatment with Abeta1-40 at the concentration of 20, 40, 80 mg/L for 48 h induced rat cortical neuron apoptosis from 12.5 %+/-1.5 % (control) to 22.3 %+/-1.4 %, 38.8 %+/-1.3 %, 36.7 %+/-1.4 %, respectively. Pretreatment with Rg1 at the dose of 0.5, 1, 2, 4, 8, 16 micromol/L for 24 h, then treatment with Abeta1-40 40 mg/L for 24 h, the percentage of apoptotic neurons decreased from 38.8 %+/-1.3 % to 14.5 %+/-1.3 %, 13.3 %+/-1.0 %, 11.6 %+/-0.29 %, 11.8 %+/-1.0 %, 6.2 %+/-0.8 %, 5.8 %+/-0.8 %, respectively. After treatment with Abeta1-40 40 mg/L for 24 h, there were transient increases in CDK4 and phosphorylated pRB protein level, as well as the expression of E2F1 mRNA. However, the above levels decreased markedly after pretreatment with Rg1 8 micromol/L for 24 h. CONCLUSION: Ginsenoside Rg1 attenuated Abeta1-40-induced apoptosis in rat cortical neurons via inhibiting the activity of CDK4, decreasing the phosphorylation of pRB and downregulating the expression of E2F1 mRNA.     

Ginsenoside Rg1 protects primary cultured rat hippocampal neurons from cell apoptosis induced by β-amyloid protein

Objectives: Estrogen is known to prominently benefit neuronal syndromes and neurodegenerative diseases. Ginsenoside Rg1, an active ingredient found in a Chinese plant, ginseng root, was previously demonstrated to exert estrogen-like activity. This study was performed to assess the neuroprotective effect of ginsenoside Rg1 against apoptosis induced by β-amyloid protein 25–35 (Aβ_25–35) in primary cultured rat hippocampal neuronal cells as well as in the underlying mechanisms.

Methods: We first measured cell viability and lactate dehydrogenase (LDH) release from primary cultured rat hippocampal neurons. After that, the inhibition effects of ginsenoside Rg1 on neuronal cell apoptosis were evaluated with flow cytometric analysis. Furthermore, western blot analysis was used for detecting the expression of apoptosis-related proteins Bcl-2, Bax, and active caspase 3.

Results: The results show that ginsenoside Rg1 could increase neuronal viability and reduce LDH release; rescue cell apoptosis induced by Aβ_25–35; decrease the expression of caspase 3, increase the ratio of Bcl-2/Bax at the protein levels compared with the cells only treated with Aβ_25–35.

Conclusions: Taken together, our results indicate that the apoptosis induced by Aβ_25–35 could be reversed by ginsenoside Rg1. Furthermore, this neuroprotective effect is probably mediated by up-regulating the ratio of Bcl-2/Bax that activates caspase 3.     

人参皂甙 Rg1 抗神经细胞凋亡作用机制的研究

研究人参皂甙Rg1对神经细胞凋亡的抑制作用。用原代培养的大鼠脑皮层神经细胞为实验模型。结果表明,Rg1能增强细胞活性,降低LDH的释放,减轻细胞核形态的改变,减少DNA断裂,增加细胞膜流动性,抑制细胞凋亡。提示细胞凋亡与细胞膜流动性关系密切;Rg1的抗衰老作用与抗凋亡作用有关。     

Ginsenoside Rg5 attenuates hypoxia-induced cardiomyocyte apoptosis via regulating the Akt pathway

Ginsenoside Rg5 has been implicated in a variety of diseases. However, it is unknown whether Ginsenoside Rg5 can protect against hypoxia-induced neonatal rat cardiomyocytes (NRMs). The purpose of this study was to look into the effect of Ginsenoside Rg5 on hypoxia-induced NRMs apoptosis as well as the underlying molecular mechanism. In this study, following isolation and culture of ventricular myocardial cells from neonatal rats, the appropriate concentration of Rg5 was determined using the MTT assay, the effect of Rg5 on apoptosis was assessed employing TUNEL staining and flow cytometry assays. Levels of apoptosis-related proteins and phosphorylated level of Akt (ser 473 and ser 308) were analyzed using the western blot analysis. Finally, the experimental results shown that Ginsenoside Rg5 significantly inhibited hypoxia-induced NRMs apoptosis, decreased the expression pro-apoptotic protein Bax, increased the expression of anti-apoptotic protein Bcl-2 ratio and the level of cleaved caspase 3. Akt signaling activation was found to be the mechanism of Ginsenoside Rg5s protective effect on hypoxia-induced NRMs apoptosis, as an Akt inhibitor eliminated the anti-apoptotic effects of Ginsenoside Rg5. Various analyses were performed and verified, ginsenoside Rg5 suppressed hypoxia-induced apoptosis in NRMs via activation of the Akt signaling.     

Impairment of preimplantation and postimplantation embryonic development through intrinsic apoptotic processes by ginsenoside Rg1 in vitro and in vivo

Ginsenoside Rg1, which is the most abundant compound found in Asian ginseng (Panax ginseng), has demonstrated various pharmacological actions, including neuroprotective, immune‐stimulatory, and antidiabetic effects. Pregnant women, especially in the Asian community, consume ginseng as a nutritive supplement. Thus, the effects of ginsenoside‐Rg1 on embryonic development need to be investigated, such as in a mouse model. As previous investigations have found that ginsenoside Rg1 appears to either trigger or prevent apoptosis in different cell lines, the effects of this agent on apoptosis remain to be clarified. In this study, we investigated whether ginsenoside Rg1 exerts a hazardous effect on mouse blastocysts and/or affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25–100 μM ginsenoside Rg1 exhibited significant induction of apoptosis and a corresponding decrease in the inner cell mass (ICM) cell number. Importantly, the implantation rate was lower among ginsenoside Rg1‐treated blastocysts compared to untreated controls. Moreover, embryo transfer assays revealed that blastocysts treated with 100 μM ginsenoside Rg1 exhibited increased resorption of postimplantation embryos and decreased weight among surviving fetuses. In vivo, intravenous injection of mice with ginsenoside Rg1 (2, 4, or 6 mg/kg body weight/day) for 4 days was associated with increased apoptosis of blastocyst‐stage embryos and negatively impacted early embryonic development. Further experiments revealed that these effects may reflect the ability of ginsenoside Rg1 to trigger oxidative stress‐mediated intrinsic apoptotic signaling. Our in vitro results indicate that ginsenoside Rg1 treatment increases intracellular oxidative stress, decreases mitochondrial membrane potential, increases the Bax/Bcl‐2 ratio, and activates caspase‐9 and caspase‐3, but not caspase‐8. Taken together, our study results strongly suggest that ginsenoside Rg1 induces apoptosis and impairs the early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.     

RP-HPLC法同时测定丹七片中三七皂苷R_1和人参皂苷Rg_1的含量

目的:建立以反相高效液相色谱法同时测定丹七片中三七皂苷R1和人参皂苷Rg1含量的方法。方法:色谱柱为KromasilC18(250mm×4.6mm,5μm),流动相为乙腈-水-磷酸(20.5∶79.5∶0.02),流速为1.0mL.min-1,柱温为室温,检测波长为203nm。结果:三七皂苷R1、人参皂苷Rg1的检测浓度分别在4.67~46.68(r=0.999 0)、4.62~115.50μg.mL-1(r=0.999 9)范围内与各自峰面积积分值呈良好线性关系;平均回收率分别为97.93%和97.98%,RSD分别为1.31%(n=6)和1.38%(n=6)。结论:本方法简便、快速、准确,可用于丹七片的质量控制。     

HPLC法测定益心复脉颗粒中人参皂苷Rg_1、Re、Rb_1的含量

目的:建立以高效液相色谱法测定益心复脉颗粒中人参皂苷Rg1、Re、Rb1含量的方法。方法:色谱柱为Diamond C18(150mm×4.6mm,5μm),流动相为乙腈-水(梯度洗脱),流速为1mL·min-1,检测波长为203nm,柱温为25℃,进样量为10μL。结果:人参皂苷Rg1、Re和Rb1分别在0.645～6.450μg(r=0.999 8)、0.54～5.40μg(r=0.999 7)和0.605～6.050μg(r=0.999 9)范围内与各自峰面积积分值呈良好线性关系;三者平均加样回收率分别为100.59%(RSD=2.03%)、98.70%(RSD=1.46%)和98.99%(RSD=1.19%)。结论:本方法灵敏度高、简便、准确,可用于益心复脉颗粒的质量控制。     

人参皂苷Rg3抑制人膀胱癌细胞增殖作用的研究

目的：探讨人参皂苷R静对人膀胱癌细胞增殖作用的影响及作用机制。方法：采用人膀胱癌T24细胞株进行细胞培养,将人参皂苷Rg3分别以0,5,10,20和40p,mol·L^-1的剂量处理细胞24h后,采用四甲基偶氮唑盐（MTT）方法检测人参皂苷R邸对人膀胱癌他4细胞增殖的抑制作用,倒置显微镜和流式细胞术观察人参皂苷Rg3对T24细胞凋亡的诱导作用,应用RT-PCR和Westernblot方法检测不同浓度人参皂苷Rg3处理后124细胞中EphB4mRNA及其蛋白的表达情况。结果：人参皂苷Rg3对膀胱癌T24细胞具有较强的抑制作用,且这种抑制作用随浓度和时间增加而增大,呈浓度依赖关系（P〈0．05）。不同浓度人参皂苷Rg3作用下的膀胱癌T24细胞中,EphB4mRNA及其蛋白的表达明显减弱,且呈剂量梯度下降（P〈0．05）。结论：人参皂苷Rg3的抗癌活性与其抑制EphB4表达有关。