不同粒径二氧化硅对人永生化表皮细胞抗氧化酶基因mRNA表达水平的影响

目的探讨经不同粒径二氧化硅染毒人永生化表皮(HaCaT)细胞后,细胞内氧化应激相关的过氧化氢酶(CAT)、谷胱甘肽还原酶(GSR)和超氧化物歧化酶(SOD-1)的mRNA表达水平的变化。方法对数生长期的HaCaT细胞分别经不同浓度(5,10,15 mg/L)、不同直径(15、30、100nm)二氧化硅粉尘处理24 h后,采用实时荧光定量PCR(RT-Q-PCR)检测CAT、GSR和SOD-1基因mRNA的表达水平。结果与对照组相比,各粒径二氧化硅染毒均能诱导HaCaT细胞CAT、GSR和SOD-1基因的mRNA表达水平发生明显变化。其中,CAT的mRNA表达水平在15 nm组和30 nm组随染毒浓度的增加明显下降;GSR的mRNA表达水平在各颗粒组中均随二氧化硅颗粒浓度的增加呈现明显的下降趋势;SOD-1的mRNA表达水平随二氧化硅颗粒浓度的增加呈现波动趋势。结论在一定剂量范围内,不同粒径二氧化硅染毒可以诱导细胞内CAT、GSR和SOD-1基因mRNA表达水平改变,推测这些改变可能参与二氧化硅的细胞毒性反应。     

纳米二氧化硅对HaCaT细胞谷胱甘肽还原酶表达及活力的影响

目的探讨经不同粒径纳米二氧化硅处理后,HaCaT细胞内谷胱甘肽还原酶(GR)的表达及其活力的变化。方法将对数生长期HaCaT细胞经15μg/ml的15、30、100nm纳米二氧化硅处理24h后,采用实时荧光定量PCR(RT-Q-PCR)检测GR基因mRNA的表达水平,采用Westernblotting检测GR蛋白的表达水平,采用试剂盒检测GR活力变化。结果不同粒径纳米二氧化硅对GR基因的mRNA水平具有明显影响,纳米二氧化硅粒径越小,mRNA表达水平越低,但均低于对照组;GR蛋白表达量随纳米二氧化硅粒径增大而增大,且15、30nm纳米二氧化硅抑制GR表达,100nm纳米二氧化硅促进GR表达;GR活力随纳米二氧化硅粒径的增大而升高。结论纳米二氧化硅可能通过影响GR的表达及酶活力的变化促进氧化应激反应。     

不同粒径二氧化硅致人永生化表皮细胞氧化应激相关指标改变的比较

目的 探讨不同粒径、不同浓度的纳米二氧化硅(nm-SiO2)染毒人永生化表皮(HaCaT)细胞内活性氧簇(reactive oxygen species,ROS)水平以及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活力的变化.方法 将对数生长期HaCaT细胞...     

亚砷酸钠对HaCaT细胞NF-κB蛋白表达的影响

目的 研究不同浓度亚砷酸钠（NaAsO2）对人角质形成细胞株（HaCaT）核转录因子（NF-κB）蛋白表达的影响及其与活性氧（ROS）及过氧化物酶（CAT）的关系。方法 用流式细胞仪检测细胞内ROS水平，用紫外速率直接法测定CAT活性，用蛋白印迹（western blot）方法检测NF-κB蛋白表达。结果 从2．5μmol／L组开始。细胞内ROS水平增高，且呈剂量一反应关系；5μmol／L以上组CAT活性明显下降，5μmol／L以上组NF-κB表达减弱（P〈0．05）。结论 砷诱导HaCaT细胞氧化应激，抑制NF-κB蛋白的表达。     

亚砷酸钠对人皮肤角质形成细胞内过氧化氢酶的影响

目的 探讨亚砷酸钠（NaAsO2）作用于体外培养的人皮肤角质形成细胞（HaCaT）后,细胞内过氧化氢酶（CAT）的活力、mRNA和蛋白表达的变化.方法 对生长至80%融合的HaCaT细胞株,分别加入0.0、2.5、5.0、10.0和20.0 μmol/L的NaAsO2作用24 h.用紫外速率直接法测定细胞内CAT的活力;用逆转录-聚合酶链反应（RT-PCR）检测CAT的mRNA表达水平;用免疫印迹（Western blot）方法检测CAT蛋白表达水平.结果 5.0 μmol/L以上的NaAsO2可明显降低CAT的活力,且呈剂量-反应关系,差异有统计学意义（P＜0.05）;RT-PCR电泳结果可见,5.0 μmol/L以上的NaAsO2作用于细胞后CAT/β-actin吸光度比值与对照组比较明显下降,差异有统计学意义（P＜0.05）;Western blot检测结果与RT-PCR电泳结果一致.结论 NaAsO2可降低HaCaT内CAT基因的mRNA及蛋白表达水平并抑制细胞内CAT活力.     

短期暴露于纳米SiO_2对HaCaT细胞基因组DNA甲基化的影响

目的探讨纳米SiO2对体外培养细胞基因组总体DNA甲基化水平的影响。方法分别以2.5、5、10μg/ml纳米SiO2溶液和10μg/ml微米级SiO2处理人皮肤表皮细胞系(HaCaT)24h;以3μmol/L DNA甲基化转移酶抑制剂5-脱氧杂氮胞苷(DAC)处理48 h的10μg/ml组为阳性对照,并设立溶剂对照组。应用高效毛细管电泳(HPCE)定量分析纳米SiO2处理细胞24h后,基因组DNA总体甲基化的变化,用Q-PCR和Western Blot方法检测甲基化相关蛋白mRNA和蛋白的表达变化,并用DNA甲基化转移酶活性试剂盒检测DNA甲基化转移酶的活性。结果 HPCE定量分析结果显示纳米SiO2可引起HaCaT细胞总体甲基化程度降低,呈剂量依赖性关系;与正常细胞相比,微米SiO2以及2.5、5和10μg/ml纳米SiO2组分别降低37.8%、43.9%、54.9%和52.8%,差异有显著性(P<0.05);对照组5-aza-dC处理后使HaCaT细胞甲基化程度减少27.3%。各组酶的蛋白表达与mRNA表达水平及DNMTs活性变化具有相同的变化趋势,经纳米SiO2处理的HaCaT细胞,DNMT1、DNMT3a及MBD2表达随着纳米SiO2剂量的上升而下降,DAC组表达水平最低。结论纳米SiO2能引起HaCaT细胞整体基因组DNA甲基化水平降低,可能与DNA甲基化转移酶的酶活性降低有关。     

氧化应激损伤对内耳毛细胞内游离钙离子和钙调蛋白表达的影响

目的通过分析氧化应激损伤的内耳毛细胞内游离Ca~(2+)浓度和钙调蛋白(CaM)表达水平,探讨感音性耳聋的氧化应激损伤机制。方法采用不同浓度(50μmol/L、100μmol/L、200μmol/L)双氧水染毒培养内耳毛细胞,24 h后检测细胞过氧化氢酶(CAT)活性,Fluo-3 AM荧光探针定量检测细胞内Ca~(2+)浓度,免疫荧光法分析CaM表达水平。结果氧化损伤的毛细胞CAT活性明显下降,随着染毒浓度的增加下降更明显(F=689.9,P0.01);氧化损伤毛细胞游离Ca~(2+)浓度随着染毒浓度的增加而增加(F=716.107,P0.01);氧化损伤的毛细胞CaM表达水平明显增加,50μmol/L和100μmol/L H_2O_2染毒组随浓度增加表达增强,但200μmol/L H_2O_2染毒组表达水平有所回落(F=732.727,P0.01)。结论氧化应激损伤导致毛细胞内游离Ca~(2+)浓度和CaM表达增加诱发细胞凋亡是多类型感音性耳聋的重要发病机制,严重的氧化应激损伤可抑制细胞CaM表达。     

视网膜色素上皮氧化损伤SIRT1表达改变

目的明确氧化应激对视网膜色素上皮细胞(RPE)中去乙酰化酶1(SIRT1)表达的影响。方法以人RPE细胞为实验对象,不同浓度H_2O_2(0、200、300μmol/L)处理RPE细胞,观察处理后24 h细胞形态的改变情况,检测处理后24 h与72 h细胞中SIRT1的mRNA与蛋白表达情况。结果 H_2O_2作用后,随H_2O_2浓度的增加,RPE细胞的形态受损,有凋亡小体的出现;在氧化应激24 h后细胞内SIRT1的转录水平增加,而在氧化应激72 h后SIRT1的蛋白表达显著下降。结论氧化应激可导致RPE细胞形态改变,SIRT1在RPE细胞内维持着氧化与抗氧化应激系统平衡,因此将SIRT1可作为临床上年龄相关性黄斑变性病(AMD)治疗的靶点。     

纳米二氧化硅致人永生化表皮细胞膜蛋白质组的影响

目的采用蛋白质组学技术探讨15 nm二氧化硅致体外培养的人永生化表皮细胞(HaCaT)膜蛋白质表达的变化。方法分别以2.5、5.0和10.0 mg/L的15nm二氧化硅溶液处理HaCaT细胞24 h,以dd H2O为溶剂对照。应用CCK-8方法检测细胞增殖情况。运用亚细胞蛋白质组提取试剂盒对10.0 mg/L的15 nm二氧化硅溶液处理24 h的HaCaT细胞和正常对照组细胞进行膜蛋白提取,采用差异荧光双向凝胶电泳(2D-DIGE)分析和基质辅助激光解析飞行时间(MALDI-TOF/TOF)质谱鉴定。对差异蛋白质进行GO(gene oncology)功能聚类及跨膜结构域预测分析,采用Western blot免疫印迹验证差异蛋白的表达。结果细胞增殖实验显示,15 nm二氧化硅可以引起HaCaT细胞整体增殖水平下降,呈剂量依赖关系;与正常细胞相比,HaCaT细胞经15 nm二氧化硅以2.5、5.0和10.0 mg/L处理24 h后,活力水平分别为(91.3%±6.1%)、(81.7%±7.0%)和(74.0%±2.6%),差异有统计学意义(P<0.05)。质谱鉴定了10个差异表达蛋白,其中7个差异蛋白具有1个以上的跨膜结构域,GO功能聚类表明这些差异蛋白主要涉及结合活性和结构分子活性。Western blot免疫印迹结果表明G蛋白偶联受体179和L-肌动蛋白表达变化趋势与蛋白质组学分析结果一致。结论 15 nm二氧化硅能够引起HaCaT细胞增殖水平降低,并且导致膜蛋白表达水平的下降。     

人肝细胞氧化应激模型中热休克蛋白90α对细胞周期蛋白B1的降解调控机制研究

目的 观察人肝细胞(L02)氧化应激模型中热休克蛋白90α(heat shock protein 90α, Hsp90α)和相关底物蛋白细胞周期蛋白B1(Cyclin B1)的蛋白水平变化,以及Hsp90α表达下调后对Cyclin B1表达的影响,明确Hsp90α对Cyclin B1功能的影响,为氧化应激相关的肝脏疾病防治提供新思路。方法 以200μM H₂O₂建立氧化应激L02细胞损伤模型;通过CCK-8法观察氧化应激对细胞存活的影响,比色法测定细胞内谷胱甘肽(glutathione, GSH)含量,流式细胞术检测细胞周期,免疫印迹法检测氧化应激对细胞内Hsp90α及Cyclin B1蛋白水平的影响。结果 随着氧化应激刺激时间的延长,GSH含量逐渐减少,抗氧化能力不断降低。从6 h开始,胞内GSH水平较对照组明显下降(均P<0.05);H₂O₂对L02细胞增殖的抑制程度增强;氧化应激后细胞周期检测点G2/M期阻滞明显;氧化应激导致胞内Hsp90α蛋白水平先增加后降低,在刺激24 h时Hsp9...     

锌硒锰镁联合拮抗二氧化硅致肺泡巨噬细胞毒性的作用

[目的]观察葡萄糖酸锌（ZnG）、亚硒酸钠（Na2SeO3）、氯化锰（MnCl2）和硫酸镁（MgSO4）联合作用对二氧化硅（SiO2）致肺泡巨噬细胞（AM）中过氧化氢（H2O2）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH—Px）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）水平变化的影响，寻找4者的最佳剂量组合。[方法]采用大鼠肺灌洗法纯化获得AM（1×10^9个／mL），同时加入SiO2及不同浓度的ZnG＋Na2SeO3＋MnCl2＋MgSO4溶液组合，另设不加组合溶液的SiO2对照组和阴性对照组。37℃，5％CO2培养箱培养18h后，检测上述5种指标。[结果]与SiO2对照组比较，ZnG、Na2SeP3、MnCl2与MgSO4联合使用均可明显降低AM中H2O2、MDA含量，升高GSH—Px、SOD和CAT的活性（P〈0．01），且以1．5mg／L的ZnG、1．0μmol／L的Na2SeO3、1．0mg／L的MnCl2、和0．5μmol／L的MgSO4联合作用效果最好。极差分析结果表明，对GSH—Px、SOD的影响，Na2SeO3的作用强于ZnG、MnCl2和MgSO4；对H2O2、MDA的影响，ZnG的作用强干Na2SeO3、MnCl2与MgSO4；对CAT的影响，MnCl2、ZnG的作用则要强于Na2SeO3与MgSO4。[结论]ZnG、Na2SeO3、MnCl2与MgSO4联合应用可明显拮抗SiO2粉尘所致AM的氧化损伤。     

Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells

Background

Extracts from plants, rich in antioxidants may be used as active ingredients of many preparations, mainly due to their antioxidant, regenerative and anti-aging properties. The work involved a comprehensive evaluation of the Jerusalem artichoke (Helianthus tuberosus L.) leaf and tuber extract as a multifunctional raw material.

Methods

The plant extracts were prepared by using ultrasound-assisted extraction method (UAE).The content of total phenolic and flavonoid compounds of extracts were determined spectrophotometrically using the Folin-Ciocalteu method and aluminium nitrate nonahydrate, respectively. Antioxidant activity of extract was analyzed using DPPH free radical scavenging assay and the effect of the investigated extracts on the proliferation of keratinocytes (HaCaT) and fibroblasts (BJ) was measured. To detect of intracellular reactive oxygen species level in tested cells, the fluorogenic dye H₂DCFDA was used. In the next step, the ability of obtained extracts to regulate the expression of genes (SOD-1, Nox-4) involved in oxidative stress in cells was evaluated.

Results

As a result of the conducted research, it was shown that leaf extract exhibit a higher content of phenols and flavonoids comparing to tuber extracts (5.07 and 7.14 fold higher, respectively). The opposite trend was observed after proliferation assay with Neutral Red test. It was shown that tuber extract in all applied concentrations (25–500?μg·ml^??1) had a positive effect on fibroblast growth. The leaf extract showed proliferative activity only for the smallest tested concentrations (25–100?μg·ml^??1). Similar trends were observed for HaCaT cells. The distinct effect of leaves and tuber extract on the generation of ROS was observed in HaCaT cells. In the present study, it was shown that tuber and leaf extracts may increase the expression of the ROS SOD-1 inactivating enzyme gene in the fibroblast cell line. There were no significant differences in gene expression of the ROS Nox-4 producing enzyme. In the case of keratinocytes, the opposite effect was observed.

Conclusions

The study suggest that Jerusalem artichoke leaves and tubers extracts affect the cell proliferation and can alter the expression of genes related to oxidative stress.

     

The impact of urban environment on oxidative damage (TBARS) and antioxidant systems in lungs and liver of great tits, Parus major

A direct negative link between human health and urban pollution levels generated by increased internal levels of oxyradicals is well established. The impact of urban environment on the physiology of wild birds is however, poorly investigated. Here we compare oxidative damage (i.e., lipid peroxidation, measured as TBARS) and different antioxidant enzymes (glutathione reductase (GR), glutathione-S-transferase (GST), and catalase (CAT)) in lungs of urban and rural great tits, Parus major. In addition, we investigated enzymatic (i.e., CAT) and non-enzymatic (i.e., carotenoids) antioxidant levels in liver tissue. There was no significant difference in lipid peroxidation in lungs between the environments. Among the antioxidant enzymes measured in lungs, only CAT showed a tendency towards increased activity in the urban environment. In contrast, CAT in livers was highly non-significant. However, there was a significantly higher concentration of dietary carotenoids (i.e., lutein (Lut) and zeaxanthin (Zx)) in urban males, along with a sex-specific difference in composition (Lut:Zx ratio) between the environments. Taken together, these results suggest that great tit lungs and livers do not seem to be negatively affected, regarding oxidative stress, by living in an urban environment.     

Protective effect of Indigofera oblongifolia in CCl4-induced hepatotoxicity

The present study aimed at assessing the protective effect of Indigofera oblongifolia on carbon tetrachloride (CCl4-induced hepatotoxicity. Hepatotoxicity was induced in male Wistar rats using CCl4 (1 mL/day at an interval of 72 hours). CCl4-induced animals were treated with I. oblongifolia at different doses. Hepatoprotection was assessed from activities of marker enzymes in serum and antioxidant status in the liver after an experimental period of 10 days. The activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase were significantly (P < .001) increased in serum of CCl4-induced animals when compared with control animals. Antioxidant status was significantly lowered in CCl4-treated animals with a significant (P < .001) increase in the levels of lipid peroxides [thiobarbituric acid-reactive substances (TBARS)], significantly lower levels of glutathione (GSH), and lowered activities of superoxide dismutase (SOD), catalase (CAT), and GSH peroxidase (GPx). The protective effect of I. oblongifolia was evident from lowering of levels of marker enzymes in serum and maintenance of antioxidant status in the liver as seen from lowered levels of TBARS, increased levels of GSH, and increased activities of SOD, CAT, and GPx. These results show the protective effect of I. oblongifolia and suggest the antioxidant property of the extract.     

Antioxidative effects of fermented sesame sauce against hydrogen peroxide-induced oxidative damage in LLC-PK1 porcine renal tubule cells

BACKGROUND/OBJECTIVES

This study was performed to investigate the in vitro antioxidant and cytoprotective effects of fermented sesame sauce (FSeS) against hydrogen peroxide (H₂O₂)-induced oxidative damage in renal proximal tubule LLC-PK1 cells.

MATERIALS/METHODS

1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (^•OH), and H₂O₂ scavenging assay was used to evaluate the in vitro antioxidant activity of FSeS. To investigate the cytoprotective effect of FSeS against H₂O₂-induced oxidative damage in LLC-PK1 cells, the cellular levels of reactive oxygen species (ROS), lipid peroxidation, and endogenous antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) were measured.

RESULTS

The ability of FSeS to scavenge DPPH, ^•OH and H₂O₂ was greater than that of FSS and AHSS. FSeS also significantly inhibited H₂O₂-induced (500 µM) oxidative damage in the LLC-PK1 cells compared to FSS and AHSS (P < 0.05). Following treatment with 100 µg/mL of FSeS and FSS to prevent H₂O₂-induced oxidation, cell viability increased from 56.7% (control) to 83.7% and 75.6%, respectively. However, AHSS was not able to reduce H₂O₂-induced cell damage (viability of the AHSS-treated cells was 54.6%). FSeS more effectively suppressed H₂O₂-induced ROS generation and lipid peroxidation compared to FSS and AHSS (P < 0.05). Compared to the other sauces, FSeS also significantly increased cellular CAT, SOD, and GSH-px activities and mRNA expression (P < 0.05).

CONCULUSIONS

These results from the present study suggest that FSeS is an effective radical scavenger and protects against H₂O₂-induced oxidative damage in LLC-PK1 cells by reducing ROS levels, inhibiting lipid peroxidation, and stimulating antioxidant enzyme activity.     

Effects of antioxidant supplementation and exercise training on erythrocyte antioxidant enzymes

Erythrocytes transport oxygen to tissues and exercise-induced oxidative stress increases erythrocyte damage and turnover. Increased use of antioxidant supplements may alter protective erythrocyte antioxidant mechanisms during training. AIM OF STUDY: To examine the effects of antioxidant supplementation (alpha-lipoic acid and alpha-tocopherol) and/or endurance training on the antioxidant defenses of erythrocytes. METHODS: Young male Wistar rats were assigned to (1) sedentary; (2) sedentary and antioxidant-supplemented; (3) endurance-trained; or (4) endurance-trained and antioxidant-supplemented groups for 14 weeks. Erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities, and plasma malondialdehyde (MDA) were then measured. RESULTS: Antioxidant supplementation had no significant effect (p > 0.05) on activities of antioxidant enzymes in sedentary animals. Similarly, endurance training alone also had no effect (p > 0.05). GPX (125.9 +/- 2.8 vs. 121.5 +/- 3.0 U x gHb(-1), p < 0.05) and CAT (6.1 +/- 0.2 vs. 5.6 +/- 0.2 U x mgHb(-1), p < 0.05) activities were increased in supplemented trained animals compared to non-supplemented sedentary animals whereas SOD (61.8 +/- 4.3 vs. 52.0 +/- 5.2 U x mgHb(-1), p < 0.05) activity was decreased. Plasma MDA was not different among groups (p > 0.05). CONCLUSIONS: In a rat model, the combination of exercise training and antioxidant supplementation increased antioxidant enzyme activities (GPX, CAT) compared with each individual intervention.     

Sexual dimorphism in the hepatic protein response to a moderate trans fat diet in senescence-accelerated mice

Background

Aging is characterized by increases in inflammation and oxidative stress, conditions that are exacerbated by environmental factors such as diet. In this study, we investigated the effects of a trans-fatty acid (TFA) diet on the liver in adult (25 wk) and old (60 wk) senescence-accelerated mice (SAMP8 strain) of both sexes. Our goal was to assess the effects of the diet on protein markers of inflammation and oxidative stress in the liver.

Methods

Male and female mice were placed on life-long diets containing similar amounts of total fat (17%), with differing amounts of TFA: 2% (moderate TFA group) or 0.2% of total energy from TFA (control diet group). At the indicated ages, livers were harvested and evaluated for markers of inflammation and oxidative stress, as well as for enzymes of fat metabolism via immunoblotting. Relative densities of protein bands were determined and compared via a three-factor ANOVA.

Results

Compared to males, females demonstrated significantly lower inflammatory protein expression (ICAM-1, MCP-1, COX-2), along with lower expression of the DNA damage marker, Gadd153, and the oxidative stress marker, HO-1. Female mice demonstrated higher expression of antioxidant enzymes (SOD-1, SOD-2, and Ref-1) and lipogenic enzymes (FASN, ACLY) compared to male mice. While HO-1 was elevated in the female mice fed the TFA diet compared to controls, the diet did not affect other markers of oxidative stress or inflammation. However, the diet was associated with significant increases in FASN and ACLY in adult (25 wk) male mice.

Conclusions

Our results suggest sexually dimorphic protein expression in the liver, with female mice demonstrating lower inflammation and increased oxidative stress defenses. Additionally, considering that FASN and ACLY contribute to hepatic lipogenesis, our results suggest a potential mechanism for the dyslipidemia in adult male mice that is associated with TFA diets.