亚硒酸钠通过AMPK/mTOR通路调控白血病NB4细胞凋亡

 目的 研究亚硒酸钠对白血病NB4细胞中AMPK及其下游靶蛋白对细胞凋亡的调控作用。方法 分别用亚硒酸钠、AICAR和AMPK干扰序列处理NB4细胞。用Western blot检测细胞AMPK、mTOR及其下游蛋白P70S6K的磷酸化水平及激活和干扰AMPK后AMPK、mTOR及P70S6K的磷酸化水平、流式细胞术检测NB4细胞凋亡率;免疫共沉淀法检测AMPK和mTOR的相互作用。结果 亚硒酸钠可以上调NB4细胞中AMPK的磷酸化水平,下调mTOR及P70S6K的磷酸化水平;AICAR和亚硒酸钠单独处理具有类似的促进NB4细胞凋亡的效果;干扰AMPK后,mTOR及P70S6K的磷酸化水平上升,亚硒酸钠对NB4细胞的促凋亡作用被拮抗;AMPK和mTOR有直接相互作用。结论 亚硒酸钠可通过激活AMPK表达,抑制mTOR及P70S6K,促进NB4细胞凋亡。     

亚硒酸钠对实验性自身免疫性甲状腺炎大鼠甲状腺细胞凋亡的影响

目的 探讨亚硒酸钠对实验性自身免疫性甲状腺炎(EAT)大鼠甲状腺细胞凋亡的影响.方法 Wistar大鼠分为正常对照组、自身免疫性甲状腺炎(EAT)组和硒干预EAT组.制备EAT大鼠模型,硒干预EAT组给予亚硒酸钠灌胃.用放射免疫法(RIA)测定各组大鼠自身抗体水平,HE染色观察甲状腺组织病理改变及TUNEL法标记甲状腺组织中凋亡细胞,观察甲状腺细胞凋亡情况.结果 正常对照组、EAT组、硒干预EAT组的TgAb分别(6.94±1.13)%、(36.24±3.64)%、(17.23±2.90)%,TmAb分别为(5.96±1.40)%、(27.12±5.06)%、(15.98±2.45)%.硒干预EAT组TgAb、TmAb水平较EAT组明显下降(P<0.05).各组大鼠甲状腺组织炎症程度及凋亡程度评分结果比较显示,不同组别的甲状腺组织病变程度间差别有显著性(P<0.001),硒干预EAT组的甲状腺滤泡破坏程度较EAT组减轻,淋巴细胞浸润减少(P<0.05),甲状腺细胞的凋亡数量较EAT组明显减少(P<0.05).结论 亚硒酸钠可能通过抑制甲状腺细胞的凋亡来减轻或抑制自身免疫性甲状腺炎的免疫损伤.     

亚硒酸钠诱导结直肠癌SW480细胞系凋亡

目的探讨亚硒酸钠诱导结直肠癌SW480细胞凋亡的信号通路。方法将SW480细胞分为三组,分别进行亚硒酸钠、Mn TMPy P以及两者联用处理;用DCFH-DA荧光探针法检测细胞内活性氧;Western blot方法检测细胞信号通路JNK和ATF-2的磷酸化,以及c-Myc、c-Jun、c-Fos、cyclin D1、cyclin A2、Bcl-2等相关蛋白表达;流式细胞术检测SW480细胞凋亡率。结果亚硒酸钠可使SW480细胞产生活性氧;抑制JNK和ATF-2的磷酸化(P0.05),下调c-Myc、c-Jun、c-Fos、cyclin D1、cyclin A2、Bcl-2的表达诱导SW480细胞凋亡(P0.05);用活性氧抑制剂Mn TMPy P可减轻亚硒酸钠所致的上述变化(P0.05)。结论亚硒酸钠可通过产生活性氧,抑制JNK和ATF-2的磷酸化,下调c-Myc、c-Jun、c-Fos、cyclin D1、cyclin A2、Bcl-2的表达,促进SW480细胞的凋亡。     

去甲斑蝥素诱导人T淋巴细胞白血病细胞株凋亡

应用TUNEL标记、DNA凝胶电泳以及光镜技术探讨去甲斑蝥素诱导人T淋巴细胞白血病细胞株（6T－CEM）凋亡规律。实验表明：0.625-5mg/L剂量的去甲斑蝥素作用6T－CEM细胞24h及10mg/L剂量的去甲斑蝥素作用6T－CEM细胞18h，台盼兰染色阳性率均在8.5%以下；10－80mg/L去甲斑蝥素作用6T－CEM细胞24h明显抑制6T－CEM细胞生长。TUNEL标记显示：10－40mg/L剂量的去甲斑蝥素作用6T－CEM细胞24h及10mg/L剂量的去甲斑蝥素作用6T－CEM细胞超过18h后即出现典型的细胞凋亡特征。一定剂量范围内的去甲斑蝥素可在直接杀伤作用较小的情况下明显诱导6T－CEM细胞凋亡，不会因细胞坏死而引起炎症反应，这对于去甲斑蝥素应用于临床将有十分重要意义。     

亚硒酸钠对免疫功能影响的实验研究

硒对免疫功能的影响虽已有一些报导,但结果不甚一致。由于天然杀伤细胞(NK)、巨噬细胞(Mφ)等在肿瘤免疫中具有重要作用,笔者对亚硒酸钠(Na_2 SeO_3)在体内、外对小鼠脾细胞NK活性及Mφ毒性作用的影响进行了探讨。     

亚硒酸钠通过线粒体途径诱导人胃癌SGC-7901细胞凋亡的机制

目的通过检测亚硒酸钠诱导人胃癌SGC-7901细胞系凋亡过程中Bax/Bcl-2、线粒体膜电位和细胞色素C(Cyt C)表达的变化,探讨亚硒酸钠诱导胃癌细胞凋亡的作用机制。方法将人胃癌细胞系SGC-7901细胞加入不同浓度亚硒酸钠(2.5、5.0、10.0mol/L)的培养液中培养24h、48h,免疫细胞化学法和免疫印迹法(Western blotting)检测Bax/Bcl-2蛋白的表达;以罗丹明123(rhodamine123)为细胞染色剂,采用流式细胞技术检测细胞的线粒体膜电位的变化;Western blotting检测细胞质Cyt C和线粒体Cyt C蛋白含量的变化。结果免疫细胞化学法和Western blotting检测结果显示,亚硒酸钠能够能够提高Bax蛋白的表达(P0.05)、降低Bcl-2蛋白的表达(P0.05),且呈剂量依懒性;流式细胞术结果显示,亚硒酸钠能够降低细胞线粒体膜电位;提示:亚硒酸钠可以通过改变Bax、Bcl-2蛋白的含量,降低线粒体的膜电位来诱导细胞凋亡。Western blotting检测结果显示,亚硒酸钠能够提高细胞质Cyt C蛋白的表达(P0.05)并降低线粒体内Cyt C蛋白的表达(P0.05),促使线粒体内的Cyt C向细胞质内释放。结论亚硒酸钠通过上调Bax并下调Bcl-2蛋白表达,降低线粒体膜电位,促进Cyt C从线粒体释放到细胞质,通过线粒体途径诱导人胃癌SGC-7901细胞凋亡。     

亚硒酸钠对胃上皮癌细胞增殖和凋亡的影响

胃癌的发生、发展不仅与肿瘤细胞分化异常、增殖过度有关，而且与细胞凋亡减少有关。硒是人体必需的微量元素，具有广泛的生物学功能，特别是对多种肿瘤的生长具有抑制作用。本文通过分裂指数试验、流式细胞术、电镜、TUNEL染色技术等探讨了亚硒酸钠对胃上皮癌细胞增殖和凋亡的影响．进一步阐明亚硒酸钠抑制胃上皮癌细胞生长的机理。     

亚硒酸钠对子宫内膜癌细胞增殖能力的影响

目的: 研究亚硒酸钠(Na₂SeO₃)对子宫内膜癌细胞增殖能力的影响,并探讨其作用机制。方法: 用Na₂SeO₃作用于子宫内膜癌Ishikawa细胞和HEC-1A细胞,MTT法检测细胞增殖能力,流式细胞法测定Na₂SeO₃作用后细胞周期的变化及凋亡情况,Western blotting检测周期蛋白cyclin A的表达。结果: Na₂SeO₃对子宫内膜癌Ishikawa细胞和HEC-1A细胞的增殖均有抑制作用,抑制率在一定浓度范围内与Na₂SeO₃浓度呈正相关,对Ishikawa细胞作用48 h的IC₅₀为3.26 μmol/L,对HEC-1A细胞作用48 h的IC₅₀为4.77 μmol/L。Na₂SeO₃作用后两种细胞G₀/G₁期减少,S期细胞增多,G₂/M期细胞有所增加。作用48 h后两种细胞凋亡率增加。Na₂SeO₃使子宫内膜癌Ishikawa细胞和HEC-1A细胞的cyclin A表达增加。结论: 亚硒酸钠对子宫内膜癌Ishikawa细胞和HEC-1A细胞增殖有抑制作用,其作用机制与上调cyclin A表达,引起细胞周期阻滞和诱导凋亡有关。     

亚硒酸钠对移植于裸鼠人神经胶质瘤的生长抑制作用

应用人脑神经胶质瘤裸鼠移植模型,对亚硒酸钠的抗瘤作用进行初步探讨。结果表明:亚硒酸钠对肿瘤的生长具有显著抑制作用。通过彤态学观察,推测其机理可能与其破坏线粒体结构、阻断能量供应、损害核结构、影响DNA、RNA和蛋白质合成有关。     

SeO2诱导白血病细胞凋亡过程中对细胞内活性氧和Ca2+水平影响

目的：研究SeO2对急性早幼粒细胞白血病细胞株NB4、红白血病细胞K562、急性粒细胞白血病细胞株HL-60的增生、凋亡、活性氧(ROS)及Ca2+水平等的影响。 方法： 采用不同浓度SeO2（3-30 μmol/L）分别处理3种白血病细胞，用流式细胞术测定细胞凋亡率、细胞中ROS和Ca2+的水平。 结果： 10和30 μmol/L SeO2能抑制3种细胞增生，30 μmol/L SeO2作用48 h能使54.0%的NB4细胞、46.5%的K562细胞和49.6%的HL-60细胞发生凋亡。同时下调细胞内ROS和Ca2+水平。NB4和HL-60细胞中ROS阳性细胞比例随SeO2浓度增加而减少，K562中只有30 μmol/L SeO2才能使ROS出现明显下降。10、30 μmol/L SeO2能使NB4和HL-60细胞内Ca2+水平逐步下降。K562中只有30 μmol/L SeO2才能使细胞内Ca2+水平出现明显下降。 结论： SeO2对3种白血病细胞均有诱导凋亡作用，在凋亡过程中涉及细胞内ROS及Ca2+水平下降。     

Different effects of H2O2 treatment on cervical squamous carcinoma cells and adenocarcinoma cells

Introduction

This study aims to compare the antioxidant abilities of cervical squamous carcinoma cells and cervical adenocarcinoma cells and to study the related mechanisms.

Material and methods

Cervical squamous carcinoma and adenocarcinoma cells were treated with H₂O₂. Cell proliferation was determined with the MTT assay. The reactive oxygen species (ROS) level was detected by the 2’,7’-dichlorofluorescein-diacetate (DCFH-DA) method. The 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) method was performed to measure intracellular concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG). The nitrite formation method, the molybdate colorimetric method, and the DTNB colorimetric method were used to determine activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), respectively.

Results

Compared with untreated control cells, cell proliferation of cervical squamous carcinoma cells and cervical adenocarcinoma cells was significantly inhibited by H₂O₂ treatment (p < 0.05). Reactive oxygen species levels and GSSG levels were significantly increased (p < 0.01), whereas GSH levels were significantly decreased (p < 0.05 or 0.01) in both cells after H₂O₂ treatment. Thus the ratio of GSH/GSSG was significantly decreased by H₂O₂ treatment in both cells (p < 0.01). In addition, H₂O₂ treatment significantly increased activities of SOD, CAT, and GPx in both cells (p < 0.05 or 0.01). Furthermore, the above-mentioned changes induced by H₂O₂ treatment were more dramatic in cervical squamous carcinoma cells.

Conclusions

The antioxidant ability of cervical squamous carcinoma cells is lower than that of cervical adenocarcinoma cells, which may be related to the increased ROS levels in cervical squamous carcinoma cells induced by H₂O₂ treatments.     

麦芽酚可抑制活性氧引起的SH-SY5Y细胞凋亡

近年来研究表明，氧化应激在阿尔茨海默病等神经退行性疾病发生发展中具有重要作用。本文研究了麦芽酚对过氧化氢(H2O2)引起的人神经瘤细胞株(SH-SY5Y)凋亡时细胞形态、磷脂酰丝氨酸(PS)外翻和DNA损伤的保护作用。结果发现麦芽酚浓度为2mmol／L，作用2h后，可以保护细胞免受H2O2引起的氧化损伤。实验结果表明麦芽酚可作为一种抗氧化剂，有效保护H2O2引起的SH-SY5Y的凋亡。     

Glutamine protects activated human T cells from apoptosis by up-regulating glutathione and Bcl-2 levels

Glutamine is the most abundant amino acid in the body. A decrease of plasma glutamine concentrations is found in catabolic stress and is related to susceptibility to infections. Glutamine is known to modulate lymphocyte activation; however, little is known about glutamine modulation of cell death of activated human T cells. Using Jurkat T cells, we investigated glutamine modulation of T-cell apoptosis activated by PMA plus ionomycin. We found that glutamine at various concentrations significantly enhanced IL-2 production, cell proliferation, and cell viability of Jurkat T cells. Glutamine also decreased the number of apoptotic cells stimulated with PMA plus ionomycin as demonstrated by flow cytometry. Meanwhile, glutamine down-regulated CD95 and CD95L expression, but up-regulated CD45RO and Bcl-2 expression in activated T cells. Further investigation of CD95-mediated caspase activities revealed that supplementation of glutamine significantly decreased caspase-3 and caspase-8 activities in activated T cells. Since oxidative stress is closely associated with induction of lymphocyte apoptosis, we found that glutamine significantly increased glutathione (GSH), but decreased reactive oxygen species levels in activated T cells. Blockade of intracellular GSH formation enhanced, but exogenous GSH supplementation decreased, activated T-cell apoptosis. Studying normal peripheral lymphoproliferation, we also found that the presence of glutamine increased lymphoproliferation as well as Bcl-2 and CD95 expression; but decreased CD95L and activation-induced T-cell death. Taken together, glutamine appeared to augment lymphoproliferation but suppressed activation-induced T-cell death in both Jurkat T cells and human peripheral T lymphocytes.     

In vitro exposure of human fibroblasts to local anaesthetics impairs cell growth

Lidocaine, bupivacaine or ropivacaine are used routinely to manage perioperative pain. Sparse data exist evaluating the effects of local anaesthetics (LA) on fibroblasts, which are involved actively in wound healing. Therefore, we investigated the effects of the three LA to assess the survival, viability and proliferation rate of fibroblasts. Human fibroblasts were exposed to 0·3 mg/ml and 0·6 mg/ml of each LA for 2 days, followed by incubation with normal medium for another 1, 4 or 7 days (group 1). Alternatively, cells were incubated permanently with LA for 3, 6 or 9 days (group 2). Live cell count was assessed using trypan blue staining. Viability was measured by the tetrazolium bromide assay. Proliferation tests were performed with the help of the colorimetric bromodeoxyuridine assay. Production of reactive oxygen species (ROS) was determined, measuring the oxidation of non‐fluorescent‐2,7′‐dichlorofluorescin. Treatment of cells with the three LA showed a concentration‐dependent decrease of live cells, mitochondrial activity and proliferation rate. Group arrangement played a significant role for cell count and proliferation, while exposure time influenced viability. Among the analysed LA, bupivacaine showed the most severe cytotoxic effects. Increased production of ROS correlated with decreased viability of fibroblasts in lidocaine‐ and bupivacaine‐exposed cells, but not upon stimulation with ropivacaine. This study shows a concentration‐dependent cytotoxic effect of lidocaine, bupivacaine and ropivacaine on fibroblasts in vitro, with more pronounced effects after continuous incubation. A possible mechanism of cell impairment could be triggered by production of ROS upon stimulation with lidocaine and bupivacaine.     

Oxidative stress in normal hematopoietic stem cells and leukemia

Leukemia is developed following the abnormal proliferation of immature hematopoietic cells in the blood when hematopoietic stem cells lose the ability to turn into mature cells at different stages of maturation and differentiation. Leukemia initiating cells are specifically dependent upon the suppression of oxidative stress in the hypoglycemic bone marrow (BM) environment to be able to start their activities. Relevant literature was identified by a PubMed search (2000–2017) of English‐language literature using the terms ‘oxidative stress,’ ‘reactive oxygen species,’ ‘hematopoietic stem cell,’ and ‘leukemia.’ The generation and degradation of free radicals is a main component of the metabolism in aerobic organisms. A certain level of ROS is required for proper cellular function, but values outside this range will result in oxidative stress (OS). Long‐term overactivity of reactive oxygen species (ROS) has harmful effects on the function of cells and their vital macromolecules, including the transformation of proteins into autoantigens and increased degradation of protein/DNA, which eventually leads to the change in pathways involved in the development of cancer and several other disorders. According to the metabolic disorders of cancer, the relationship between OS changes, the viability of cancer cells, and their response to chemotherapeutic agents affecting this pathway are undeniable. Recently, studies have been conducted to determine the effect of herbal agents and cancer chemotherapy drugs on oxidative stress pathways. By emphasizing the role of oxidative stress on stem cells in the incidence of leukemia, this paper attempts to state and summarize this subject.     

IL-6对大鼠急性髓系白血病R2细胞的增殖抑制及分化诱导作用

重组人白细胞介素６（ｒｈＩＬ－６）可抑制大鼠急性髓系白血病（ＡＭＬ）Ｒ２细胞的体外生长（ＩＣ５０１００ｎｇ／Ｌ），并在１０ｎｇ／Ｌ～１μｇ／Ｌ的剂量范围内诱导Ｒ２细胞向终末方向分化。诱导后的Ｒ２细胞具有单核巨噬细胞的形态特征；且非特异性酯酶（ＮＳＥ）及酸性醋酸酯酶（ＡＮＡＥ）的活性增强，而过氧化物酶（ＰＯＸ）的活性却一直很低。经典型的ＤＮＡ梯状条带及凋亡小体证实，≥１μｇ／ＬｒｈＩＬ－６亦可诱导Ｒ２细胞凋亡。我们认为，Ｒ２细胞是研究ＩＬ－６信号转导通路及ＡＭＬ细胞分化机制的一个很好模型。     

Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways

Aim: Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Methods: Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Results: Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Conclusions: Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis.     

Effect of various E. coli LPS chemotypes on apoptosis and activation of human neutrophils

We studied the effects of various Escherichia coli LPS chemotypes on the production of reactive oxygen species and regulation of apoptosis in human neutrophils. A correlation was found between the increase in chemiluminescence (Re-LPS<Ra-LPS<S-LPS) and lengthening of the polysaccharide chain in endotoxins. Shortening of the polysaccharide chain was associated with more pronounced inhibition of neutrophil apoptosis under the influence of endotoxins, which correlated with the increase in hydrophobicity of these chemotypes. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 136–138, August, 2006     

Pseudomonas aeruginosa induces apoptosis in human endothelial cells

Pseudomonas aeruginosa has been shown to enter into human endothelial cells in vitro. To ascertain the effects of bacterial intracellular (IC) infection, endothelial cells were exposed to PAK and PAO-1 strains for 1 h and treated with gentamicin in culture medium for different periods. P. aeruginosa induced a significant production of superoxide and hydrogen peroxide by endothelial cells. Concentrations of IC bacteria were reduced progressively with time and no viable PAO-1 was detected at 24 h after infection. However, IC infection led to killing of 32.2%±2.9 and 51.8%±3.5 of the cells infected with PAK and PAO-1, respectively, as determined by the MTT assay. By three criteria (transmission electron microscopy, DNA electrophoresis and reactivity with annexin V) infected cells exhibited features of apoptosis. Treatment of infected cells with anti-oxidants (catalase, tocopherol and N -acetyl-L-cysteine) significantly decreased the percentage of cell death. In contrast, treatment with aminoguanidine, an inhibitor of inducible NO synthase, increased significantly the killing of PAO-1 infected cells. Based on these results we speculate that in response to P. aeruginosa infection, endothelial cells increase the production of reactive oxygen intermediates to eliminate IC pathogens, but cells do not resist the oxidative stress and die by apoptosis.