山茱萸多糖对HDF细胞β-半乳糖苷酶及p16表达的影响

目的探讨山茱萸多糖对抗HDF细胞衰老的作用及其可能的调控机制。方法体外培养人胚肺二倍体成纤维细胞(HDF),实验组从40代开始加多糖含药血清,观察细胞形态,MTT法检测细胞活力,分光光度法检测β-半乳糖苷酶活性,免疫组化法检测p16蛋白的表达。结果衰老组细胞活力下降,β-半乳糖苷酶含量升高,p16蛋白表达阳性细胞数升高;山茱萸多糖可提高细胞活力、降低β-半乳糖苷酶含量,p16蛋白表达阳性细胞数下降。结论山茱萸多糖可通过改变p16的表达而发挥其抗HDF细胞衰老作用。     

人参皂甙Rg3对血管平滑肌细胞衰老的影响及机制

目的探讨人参皂甙Rg3对D-半乳糖诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMC)衰老的影响及机制。方法取SD大鼠胸主动脉中层,分离并原代培养VSMC,选取3~6代VSMC,通过D-半乳糖共孵育的方法诱导细胞衰老,β-半乳糖苷酶染色和透射电镜鉴定衰老VSMC;VSMC随机分成对照组、D-半乳糖组、Rg3高浓度组、Rg3低浓度组,Western blot方法检测p16、p21和p53蛋白的表达水平,流式细胞术检测细胞周期。结果与对照组比较,D-半乳糖组β-半乳糖苷酶阳性细胞明显升高[(58.67±2.52)%vs(4.67±0.58)%,P<0.05]。电镜下表现为细胞核膜内折,细胞线粒体肿胀,脂褐素堆积;p16、p21和p53蛋白表达水平明显升高(P<0.05),细胞周期停滞在G0/G1期。与D-半乳糖组比较,Rg3低浓度组和Rg3高浓度组β-半乳糖苷酶阳性细胞明显减少,p16、p21和p53蛋白表达水平明显降低,G0/G1期细胞数明显减少(P<0.05)。结论人参皂甙Rg3可抑制VSMC衰老,其机制可能部分通过抑制细胞生长周期p16INK4a/Rb、p53-p21Cip1/Waf1信号通路来实现。     

双歧杆菌脂磷壁酸延缓细胞衰老的作用机制

目的 探讨双歧杆菌脂磷壁酸(Lipoteichoic acid,LTA)在延缓H2O2诱导细胞衰老中的作用.方法 H2O2诱导WI-38细胞衰老,β-半乳糖苷酶细胞化学染色计算衰老细胞百分率变化,RT-PCR和Western印迹检测衰老细胞p21、细胞周期蛋白E(cyclin E)和周期蛋白依赖性蛋白激酶2(CDK2)表达水平的变化.结果 双歧杆菌LTA处理后,β-半乳糖苷酶细胞化学染色阳性细胞百分率较衰老模型组降低(P＜0.01).与年轻对照组相比,衰老模型组细胞中p21的表达增高,cyclin E和CDK2表达降低,而双歧杆菌LTA能够逆转上述变化(P＜0.01).结论 双歧杆菌能延缓H2O2诱导的细胞衰老,机制可能与改变p21,cyclin E和CDK2表达水平有关.     

富硒麦芽粉水提取物通过细胞衰老和凋亡抑制人白血病K562细胞的增殖

目的 研究富硒麦芽粉水提取物能否抑制K562细胞增殖的作用.方法 Coulter细胞计数法检测细胞增殖的抑制作用,流式细胞仪检测细胞周期分布以及细胞内活性氧自由基的水平,DNA特异染料Hoechst 33342染色检测染色质凝集,免疫印迹法检测凋亡相关蛋白的表达,衰老相关的β-半乳糖苷酶染色检测细胞衰老.结果 富硒麦芽粉水提取物能抑制K562细胞的增殖,IC50值为0.5 mg/mL.进一步研究发现：富硒麦芽粉水提取物使细胞阻滞在G2/M期、增加细胞内活性氧自由基水平,染色质发生凝集,引起细胞凋亡标志蛋白的切割.此外,4 mg/mL的水提取物能引起60％的细胞发生衰老.结论 富硒麦芽粉水提取物具有明显的抑制K562细胞增殖的作用,其机制与诱导细胞衰老和凋亡有关.     

巴戟天对D-半乳糖致衰老大鼠心肌细胞肌球蛋白和肌动蛋白基因表达的影响

目的 探讨补肾益气中药巴戟天对衰老心肌细胞肌球蛋白和肌动蛋白基因表达的影响及其可能机制.方法 以新生24h内SD大鼠心脏组织为材料,采用胶原酶消化法分离并培养心肌细胞,用8 mg/ml的D-半乳糖诱导细胞2d并常规培养5 d制得心肌细胞衰老模型,并用巴戟天处理,观察作用.实验分为三组:对照组、模型组、巴戟天组.采用倒置显微镜观察细胞形态,生化方法检测β-半乳糖苷酶活力,MTT法测定细胞活力,RT-PCR检测肌球蛋白重链基因α-MHC和β-MHC mRNA表达,Western印迹方法检测肌动蛋白的蛋白表达.结果 ①与正常组相比,模型组细胞β-半乳糖苷酶活力显著性升高,细胞活力显著性降低,肌球蛋白重链α-MHC mRNA表达下降,肌球蛋白重链β-MHC mRNA表达升高;②巴戟天组与模型组相比较,一定程度上改变了衰老心肌细胞的形态变化,β-半乳糖苷酶的活力降低,细胞活力升高,肌球蛋白重链α-MHC mRNA表达上调,肌球蛋白重链β-MHC mRNA表达下调.α-actin蛋白表达在各用药组并未表现出显著性差异.结论 巴戟天能改善衰老心肌细胞α-MHC mRNA表达下调程度和β-MHC mRNA表达的上调程度.α-actin的表达保持恒定.     

基于自噬与凋亡平衡探讨补肾活血汤对衰老耳蜗毛细胞株HEI-OC1的保护作用

目的探讨补肾活血汤对衰老耳蜗毛细胞株HEI-OC1自噬和凋亡的影响。方法应用D-半乳糖构建衰老HEI-OC1细胞的体外模型后,将细胞分为对照组、衰老组、含药血清低、中及高剂量组(5%、10%、20%),采用噻唑蓝(MTT)比色法检测耳蜗HEI-OC1细胞的增殖能力;β-半乳糖苷酶染色检测细胞衰老状态;膜联蛋白V/碘化丙啶(Annexin V-FITC/PI)双染流式细胞术检测细胞凋亡的程度;Western印迹检测细胞中自噬和凋亡相关蛋白的表达水平。结果MTT、β-半乳糖苷酶染色、Annexin V-FITC/PI双染流式细胞术和Western印迹结果显示,与对照组比较,衰老组HEI-OC1细胞增殖能力明显下降;细胞衰老状态明显增加,细胞凋亡率明显增加,Atg5、Atg12、Beclin1和Bcl-2蛋白表达水平明显下降,p62和Bax蛋白表达水平显著升高(均P<0.01)。与衰老组比较,补肾活血汤各组HEI-OC1细胞增殖能力明显上升;细胞凋亡率明显降低;Atg5、Atg12、Beclin1和Bcl-2蛋白表达水平显著增加(均P<0.05);p62和Bax蛋白表达水平显著下降(均P<0.05),且均呈现出剂量依赖性。结论补肾活血汤可通过促进自噬及抑制凋亡来延缓耳蜗毛细胞株HEI-OC1的衰老,且与其浓度相关。     

人参皂苷Rg1对衰老大鼠胸腺结构与功能的影响

目的探讨人参皂苷Rg1对衰老大鼠胸腺结构与功能的影响及机制。方法 SD大鼠随机分为4组,每组10只。衰老模型组,皮下注射D-半乳糖120 mg/kg,1次/d,42 d;Rg1衰老模型组,注射D-半乳糖剂量与时间同衰老模型组,第15天起腹腔注射Rg1 20 mg/kg,1次/d,28 d正常对照组,皮下注射生理盐水1次/d,42 d;Rg1正常对照组,注射生理盐水1次/d,14 d,第15天起腹腔注射Rg1(同Rg1衰老模型组)。模型复制或药物注射完成后第2天,取胸腺测定胸腺指数,石蜡切片观察胸腺形态学;衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测胸腺细胞衰老,CCK-8检测胸腺细胞对刀豆蛋白A刺激的增殖能力,ELISA检测胸腺细胞分泌肿瘤坏死因子(TNF)-α、GM-CSF、白细胞介素(IL)-2与IL-6的能力,流式细胞术检测细胞活性氧(ROS)、胸腺细胞凋亡,硫代巴比妥酸法检测丙二醛(MDA),酶学检测超氧化物歧化酶(SOD),DTNB法测定谷胱甘肽(GSH)、氧化谷胱甘肽(GSSG)含量,Western印迹检测细胞衰老相关蛋白p21、p53、Rb的变化。结果 Rg1衰老模型组大鼠胸腺指数升高、胸腺皮质面积比例增加、胸腺细胞的增殖能力提高,凋亡率减少、SA-β-Gal阳性胸腺细胞百分率下降、TNF-α、GM-CSF、IL-2、IL-6的分泌能力明显提高、SOD活性明显提升;ROS和MDA含量下降;p53、p21、Rb蛋白表达有显著下调。结论 D-半乳糖复制的衰老模型大鼠胸腺结构与功能损伤明显,人参皂苷Rg1对其致衰损伤有明确的保护作用,其机制可能与抑制氧化损伤和下调p16-Rb、p53/p21信号通路有关。     

睾酮对亚急性衰老小鼠下丘脑的影响及其机制

目的观察低刘量睾酮对小鼠下丘脑衰老的影响及其可能的调控机制。方法将24只健康雄性C57小鼠随机分为3组:D-半乳糖+睾酮治疗组(治疗组)、D-半乳糖组和正常对照组,每组8只。用药5个月后分离小鼠下丘脑组织,制备石蜡切片,测定小鼠下丘脑衰老相关性β-半乳糖苷酶(SA-β-Gal)染色情况,用免疫组织化学法检测p16~(INK4a)蛋白的表达水平。结果与正常对照组比较,D-半乳糖组小鼠体重降低(P<0.05),下丘脑SA-β-Gal染色阳性细胞率和p16~(INK4a)蛋白阳性细胞率明显升高(P<0.05);与D-半乳糖组比较,治疗组小鼠体重增加(P<0.05).下丘脑SA-β-Gal染色阳性细胞率和p16~(INK4a)蛋白阳性细胞率明显降低(P<0.05)。结论低剂量睾酮可能通过改变p16~(INK4a)的表达而发挥抗下丘脑细胞衰老的作用。     

丙泊酚对高糖诱导的H9c2细胞衰老的保护作用

目的研究丙泊酚对高糖诱导的H9c2细胞衰老的保护作用及其机制。方法本实验通过使用高糖长期培养H9c2细胞,在体外建立心肌细胞高糖老化模型,给予不同浓度的丙泊酚(5,10,20,40μmol/L)作用后,分别测定细胞存活率,细胞内活性氧(ROS)释放,细胞β-半乳糖苷酶染色,细胞P16、P53、Rb蛋白表达量。结果预处理丙泊酚(5~20μmol/L)能够明显减轻高糖诱导的细胞损伤;抑制细胞内ROS释放;显著降低细胞β-半乳糖苷酶染色阳性细胞数目;降低P16、P53和Rb蛋白表达量。结论丙泊酚通过抑制ROS释放,降低H9c2细胞衰老相关基因的蛋白表达量,从而产生心脏保护作用。     

Valsartan延缓血管内皮细胞衰老及p16INK4a表达变化的研究

目的 探讨Valsartan对血管内皮细胞衰老与p16INK4a表达变化的影响,为寻求廷缓内皮细胞衰老途径提供理论和实验依据.方法 体外培养人脐静脉内皮细胞,子血管紧张素Ⅱ及Valsartan干预,实验分为空白对照组、血管紧张素Ⅱ诱导组及Valsartan组,采用β-半乳糖苷酶(β-gal)染色鉴定细胞衰老;流式细胞术分析细胞周期变化;免疫细胞化学染色法、Western blot分析各组细胞p16INK4a蛋白的表达.结果 与对照组比较,血管紧张素Ⅱ诱导组β-半乳糖苷酶阳性染色率显著增多81.24％±6.46％,细胞周期停滞于G0-G1(88.36％±6.45％),p16INK4a 蛋白表达水平上调(P＜0.05);予以Valsartan干预后,β-半乳糖苷酶阳性细胞染色率减少,G0-G1细胞减少,p16INK4a蛋白表达水平下调(P＜0.05).结论 血管内皮细胞衰老分子机制可能通过下调p16INK4a的表达,使细胞周期停滞于G1期有关,Valsartan对血管内皮细胞衰老有一定保护作用,可能通过调控p16INK4a的表达发挥其延缓3血管内皮细胞衰老的作用.     

Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts

The mitochondrial theory of aging predicts that functional alterations in mitochondria contribute to the aging process. Whereas this hypothesis implicates increased production of reactive oxygen species (ROS) as a driving force of the aging process, little is known about molecular mechanisms by which mitochondrial impairment might contribute to aging. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between mitochondrial impairment and the appearance of a senescence-like phenotype in young cells. We found that treatment by antimycin A delays proliferation and induces premature senescence in a subset of the cells, associated with increased reactive oxygen species (ROS) production. Quenching of ROS by antioxidants did however not restore proliferation capacity nor prevent premature senescence. Premature senescence is also induced upon chronic exposure to oligomycin, irrespective of ROS production, and oligomycin treatment induced the up-regulation of the cdk inhibitors p16, p21 and p27, which are also up-regulated in replicative senescence. Thus, besides the well-established influence of ROS on proliferation and senescence, a reduction in the level of oxidative phosphorylation is causally related to reduced cell proliferation and the induction of premature senescence.     

细胞周期抑制因子p16可诱导人成纤维细胞发生衰老样变化

目的 探讨细胞周期抑制因子p16在细胞衰老中的作用。方法 利用逆转录病毒载体将p16基因转染入人胚肺二倍体成纤维细胞2BS中,获得高表达,检测其对2BS细胞衰老的影响。结果 与对照组细胞相比,p16基因转染后,2BS细胞生长速度下降了约50％,细胞周期阻滞于G1期,细胞对生长因子刺激的反应性下降了79．4％,细胞形态呈衰老细胞样变化。结论 p16在人二倍体成纤维细胞的衰老过程中起促进作用。     

Dicoumarol enhances gemcitabine-induced cytotoxicity in high NQO1-expressing cholangiocarcinoma cells

AIM: To investigate whether dicoumarol, a potent inhibitor of NAD(P)H quinone oxidoreductase-1 (NQO1), potentiates gemcitabine to induce cytotoxicity in chol-angiocarcinoma cells (CCA) and the role of reactive oxygen generation in sensitizing the cells. METHODS: Four human cell lines with different NQO1 activity were used; the human CCA cell lines, KKU-100, KKU-OCA17, KKU-M214, and Chang liver cells. NQO1 activity and mRNA expression were determined. The cells were pretreated with dicoumarol at relevant con...     

Effects of antioxidants on homocysteine thiolactone-induced apoptosis in human umbilical vein endothelial cells

Background and objectives Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine thiolactone (HcyT), one of the homocysteine metabolites in vivo, is toxic both in vivo and in vitro. The aim of this study was to investigate the effect of HcyT on apoptotic damage in human umbilical vein endothelial cells (HUVECs) and the role of antioxidants in the reduction of HcyT-induced apoptosis. Methods HUVECs were cultured in DMEM supplemented with 20% heat inactivated fetal bovine serum cell cultures were maintained in a humidified 5% CO2 atmosphere at 37℃. Cytotoxicity was determined by MTT assay, which consists of hypodiploid cells with propidium iodide labeling and intracellular reactive oxygen species levels using 2',7'-dichlorofluorescein diacetate as the probe by flow cytometry. Results HcyT (250-2000μM) induced HUVECs apoptosis in a time- and concentration-dependent manner. Reactive oxygen species levels rose in response to increasing HcyT concentrations at 24-h incubation. The reduction of cell apoptosis by N-acetylcysteine, vitamin E, or pyrrolidine dithiocarbamate, occurred simultaneously with a significant decrease in intracellular reactive oxygen species levels. Conclusion HcyT exerts its cytotoxic effects on endothelial cells through an apoptotic mechanism involving cellular reactive oxygen species production. The capacity of N-acetylcysteine, vitamin E, and pyrrolidine dithiocarbamate to scavenge HcyT-induced cellular reactive oxygen species correlates well with their efficiency to protect against HcyT-promoted apoptotic damage. The protective effect of pyrrolidine dithiocarbamate on cell apoptosis indicates HcyT-generated hydrogen peroxide may provoke cell apoptosis via activating nuclear factor-kappa binding protein.     

Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence

Reactive oxygen species (ROS) appear to play a role in limiting both cellular and organismic lifespan. However, because of their pleiotropic effects, it has been difficult to ascribe a specific role to ROS in initiating the process of cellular senescence. We have studied the effects of oxidative DNA damage on cell proliferation, believing that such damage is of central importance to triggering senescence. To do so, we devised a strategy to decouple levels of 8-oxoguanine, a major oxidative DNA lesion, from ROS levels. Suppression of MTH1 expression, which hydrolyzes 8-oxo-dGTP, was accompanied by increased total cellular 8-oxoguanine levels and caused early-passage primary and telomerase-immortalized human skin fibroblasts to rapidly undergo senescence, doing so without altering cellular ROS levels. This senescent phenotype recapitulated several salient features of replicative senescence, notably the presence of senescence-associated beta-galactosidase (SA beta-gal) activity, apparently irreparable genomic DNA breaks, and elevation of p21^Cip1, p53, and p16^INK4A tumor suppressor protein levels. Culturing cells under low oxygen tension (3%) largely prevented the shMTH1-dependent senescent phenotype. These results indicate that the nucleotide pool is a critical target of intracellular ROS and that oxidized nucleotides, unless continuously eliminated, can rapidly induce cell senescence through signaling pathways very similar to those activated during replicative senescence.     

Replicative senescence of human fibroblasts: the role of Ras-dependent signaling and oxidative stress

Replicative senescence of human fibroblasts is a widely used cellular model for human aging. While it is clear that telomere erosion contributes to the development of replicative senescence, it is assumed that additional factors contribute to the senescent phenotype. The free radical theory of aging suggests that oxidative damage is a major cause of aging; furthermore, the expression of activated oncogenes, such as oncogenic Ras, can induce premature senescence in primary cells. The functional relation between the various inducers of senescence is not known. The present study was guided by the hypothesis that constitutive activation of normal, unmutated Ras may contribute to senescence-induced growth arrest in senescent human fibroblasts. When various branches of Ras-dependent signaling were investigated, constitutive activation of the Ras/Raf/MEK/ERK pathway was not observed. To evaluate the role of oxidative stress for the senescent phenotype, we also investigated stress-related protein kinases. While we found no evidence for alterations in the activity of p38, we could detect an increased activity of Jun kinase in senescent fibroblasts. We also found higher levels of reactive oxygen species (ROS) in senescent fibroblasts compared to their younger counterparts. The accumulation of ROS in senescent cells may be related to the constitutive activation of Jun kinase.     

Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes

Replicative senescence of human diploid fibroblasts (HDFs) or melanocytes is caused by the exhaustion of their proliferative potential. Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses including H(2)O(2), hyperoxia, or tert-butylhydroperoxide. Cells in replicative senescence share common features with cells in SIPS: morphology, senescence-associated beta-galactosidase activity, cell cycle regulation, gene expression and telomere shortening. Telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative damage. SIPS could be a mechanism of accumulation of senescent-like cells in vivo. Melanocytes exposed to sublethal doses of UVB undergo SIPS. Melanocytes from dark- and light- skinned populations display differences in their cell cycle regulation. Delayed SIPS occurs in melanocytes from light-skinned populations since a reduced association of p16(Ink-4a) with CDK4 and reduced phosphorylation of the retinoblastoma protein are observed. The role of reactive oxygen species in melanocyte SIPS is unclear. Both replicative senescence and SIPS are dependent on two major pathways. One is triggered by DNA damage, telomere damage and/or shortening and involves the activation of the p53 and p21(waf-1) proteins. The second pathway results in the accumulation of p16(Ink-4a) with the MAP kinase signalling pathway as possible intermediate. These data corroborate the thermodynamical theory of ageing, according to which the exposure of cells to sublethal stresses of various natures can trigger SIPS, with possible modulations of this process by bioenergetics.     

Oxidative stress decreases in the trophocytes and fat cells of worker honeybees during aging

Trophocytes and fat cells of honeybees have been used for cellular senescence studies, but their oxidative stress and antioxidant enzyme activities with aging in workers is unknown. Here, we assayed reactive oxygen species and the activities of antioxidant enzymes in the trophocytes and fat cells of young and old workers. Young workers had higher reactive oxygen species levels, higher superoxide dismutase and thioredoxin reductase activities as well as lower catalase and glutathione peroxidase activities compared to old workers. Adding these results up, we propose that oxidative stress decreases with aging in the trophocytes and fat cells of workers.     

Reactive oxygen species and hematopoietic stem cell senescence

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism through self-renewal, proliferation, differentiation, and mobilization. Their functions can be affected by reactive oxygen species (ROS) that are produced endogenously through cellular metabolism or after exposure to exogenous stress. At physiological levels, ROS function as signal molecules which can regulate a variety of cellular functions, including HSC proliferation, differentiation, and mobilization. However, an abnormal increase in ROS production occurs under various pathological conditions, which can inhibit HSC self-renewal and induce HSC senescence, resulting in premature exhaustion of HSCs and hematopoietic dysfunction. This review aims to provide a summary of a number of recent findings regarding the cellular sources of ROS in HSCs and the mechanisms of action whereby ROS induce HSC senescence. In particular, we highlight the roles of the p38 mitogen-activated protein kinase (p38)-p16^Ink4a (p16) pathway in mediating ROS-induced HSC senescence.