绞股蓝皂苷减轻H2O2诱导大鼠成骨细胞氧化应激损伤的机制

文题释义：绞股蓝皂苷：来源于葫芦科植物绞股蓝的全草,具有抗氧化、神经保护、降血压和降血脂等作用。研究发现,绞股蓝皂苷对多种细胞的氧化损伤具有保护作用,还可以通过调节NF-κB、AKT和MAPK信号通路抑制RANKL诱导的破骨细胞的形成,抑制骨量丢失,有可能成为治疗骨质疏松等骨代谢相关疾病的潜在药物。 氧化应激：机体受到刺激时氧化和抗氧化系统的平衡被破坏,对细胞和组织产生氧化损伤,氧化应激被认为和机体衰老密切相关。研究发现,绝经后骨质疏松症的发生也与氧化应激有关,是目前骨代谢疾病研究的新热点。 背景：绞股蓝皂苷具有抗氧化作用,多应用于降血压、抗衰老、抗肿瘤等,但其具体保护机制尚不明确,对氧化应激损伤的成骨细胞增殖、分化的影响亦未知。 目的：探讨绞股蓝皂苷减轻大鼠成骨细胞氧化应激损伤的机制,以及对氧化损伤的成骨细胞的增殖、分化的影响。 方法：采用分离乳鼠颅骨细胞的单层培养法培养成骨细胞,实验分为3组,以普通生长培养基为空白组,以普通生长培养基+氧化损伤为对照组,以含绞股蓝皂苷的培养基+氧化损伤为实验组。实验组和对照组成骨细胞经含150 μmol/L H₂O₂的生长培养基诱导氧化损伤模型。干预3,5 d CCK8法检测绞股蓝皂苷对氧化损伤的成骨细胞活性的影响;在诱导后第7天采用碱性磷酸酶染色检测成骨细胞碱性磷酸酶的活性;诱导第21天采用茜素红染色观察成骨细胞矿化情况;Western blot检测NOX4、骨形态发生蛋白2和Smad4的蛋白表达量。实验方案经广州中医药大学动物实验伦理委员会批准。 结果与结论：①绞股蓝皂苷可促进氧化损伤的成骨细胞增殖;②碱性磷酸酶染色和茜素红染色结果表明绞股蓝皂苷对受氧化应激损伤的成骨细胞的分化有一定促进作用;③与对照组相比,绞股蓝皂苷可下调实验组NOX4蛋白的表达,上调实验组骨形态发生蛋白2和Smad4蛋白的表达(P < 0.05);④结果说明,绞股蓝皂苷对H₂O₂诱导的成骨细胞氧化应激损伤具有一定的保护作用,并且对损伤的成骨细胞有促进增殖分化的作用,其机制可能与抑制NOX4蛋白表达并激活BMP/Smad通路有关。 ORCID: 0000-0002-0713-8120(林燕平) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

雌激素对H2O2诱导的氧化应激的保护作用及机制

为了探讨雌激素17β-estradiol对H2O2诱导的氧化应激的影响及其可能机制。本研究将H2O2作用于大鼠皮层神经元,利用四甲基偶氮唑盐(MTT)检测单独H2O2或者17β-estradiol存在时其对细胞活力和乳酸脱氢酶(LDH)释放的影响;检测单独H2O2或者17β-estradiol存在时其对糖原合成激酶-3β(GSK-3β)活性的影响及其对磷酸化和非磷酸化GSK-3β表达的影响;检测GSK-3β抑制剂LiCl对H2O2诱导的细胞活力的影响;检测雌激素受体抑制剂ICI-182780存在时17β-estradiol对GSK-3β表达的影响。结果显示:(1)H2O2作用于大鼠皮层神经元后显著降低细胞的活力,各种浓度的17β-estradiol预处理细胞后均能部分阻断H2O2对细胞活力的影响;(2)H2O2作用后显著增加细胞乳酸脱氢酶的释放,而17β-estradiol则部分拮抗此种作用;(3)H2O2增加了GSK-3β的活性,而提前加入17β-estradiol则可以降低H2O2诱导的GSK-3β活性增加;(4)H2O2降低了GSK-3β的磷酸化,而17β-estradiol则部分拮抗了这种作用;(5)GSK-3β抑制剂LiCl也可以拮抗H2O2诱导的细胞活力下降;(6)与对照组相比,17β-estradiol本身亦可增加GSK-3β的磷酸化,此作用可被雌激素受体抑制剂ICI-182780部分拮抗,而ICI-182780本身对GSK-3β的磷酸化无显著影响。以上结果提示,17β-estradiol可以拮抗H2O2诱导的氧化应激作用,17β-estradiol通过与其受体结合而抑制GSK-3β的活性可能是其发挥保护作用的机制之一。     

萝卜硫素对H2O2诱导人肝细胞氧化应激损伤模型的保护作用

背景:氧化应激损伤在慢性肝炎发生发展中起着重要的作用,而天然产物萝卜硫素具有较好的抗氧化活性。目的:研究萝卜硫素对H2O2诱导人肝细胞(LO2)氧化应激损伤的保护作用,并探讨其可能的作用机制。方法:体外培养LO2细胞,采用H2O2诱导LO2细胞造模,并同时将细胞分为对照组、H2O2诱导组、H2O2+低剂量萝卜硫素组(10μmol/L)、H2O2+中剂量萝卜硫素组(20μmol/L)及H2O2+高剂量萝卜硫素组(40μmol/L)。采用100μg/L的H2O2处理人肝细胞24 h,诱导细胞氧化应激损伤;H2O2+萝卜硫素组将H2O2与不同浓度萝卜硫素同时处理细胞24 h。采用CCK8法分析萝卜硫素对细胞存活率的影响,分析细胞匀浆中的氧化应激产物水平,Western Blot法分析Nrf2、GCLC、NQO1及血红素加氧酶1蛋白表达水平。研究方案的实施符合恩施土家族苗族自治州中心医院的相关伦理要求。结果与结论:①H2O2能诱导LO2细胞氧化应激损伤;②相比于H2O2模型组,各浓度萝卜硫素能诱导LO2细胞增殖、抑制丙二醛和一氧化氮的产生及促使超氧化物歧化酶活性升高(P<0.05);③Western Blot结果显示萝卜硫素能诱导Nrf2向细胞核内转移,诱导GCLC、NQO1和血红素加氧酶1蛋白表达(P<0.05);④结果说明,萝卜硫素能改善H2O2诱导的LO2细胞氧化应激损伤,其药理学作用与Nrf2信号传导活化有关。     

Salidroside protects human fibroblast cells from premature senescence induced by H(2)O(2) partly through modulating oxidative status

Although salidroside and salidroside-like compounds are considered as most critical constitutes needed and responsible for multiple therapeutic benefits of Rhodiola rosea L., including anti-aging, direct demonstration regarding the role of salidroside in anti-aging process is still deficient. In this study, we selected the H(2)O(2)-induced premature senescence model in human fetal lung diploid fibroblasts to investigate the protection of salidroside against aging in vitro and associated molecular mechanisms. We found that salidroside considerably reversed senescence-like phenotypes in the oxidant challenged model, including alterations of morphology, cell cycle, SA-β-gal staining, DNA damage, as well as related molecules expression such as p53, p21 and p16. The protection occurred in a dose-dependent manner, with 5μM offering best efficacy. The proposed antioxidant property of the compound was confirmed in this cellular system, and thus at least partially accounted for the protection of the compound against premature senescence. Similar protection of salidroside against replicative senescence was observed as well. Interestingly, the regulation of senescence-related molecules by salidroside involved ROS-irrelevant mechanisms in both models. This finding presents salidroside as an attractive agent with potential to retard aging and attenuate age-related diseases in humans.     

Chemoprotective effect of all-trans retinoic acid (ATRA) on oxidative stress and lung metastasis induced by benzo(a)pyrene

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of cancer. All-trans retinoic acid (ATRA) is an active metabolite of vitamin A under the family retinoids, derived by irreversible oxidation of retinol (vitamin A), the parent compound for all natural retinoids. The aim of the present study is to divulge the chemopreventive and chemoprotective nature of ATRA during benzo(a)pyrene (B(a)P) induced lung cancer development in BALB/c mice. Administration of B(a)P (50?mg/kg body weight) to mice resulted in increased lipid peroxides (LPO), lipid hydroperoxides (LOOH) and nitric oxide (NO) with concomitant decrease in the levels of tissue anti-oxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and vitamin C. ATRA supplementation (0.585?mg/kg body weight) attenuated all these alterations, which indicates the anti-cancer effect that was further confirmed by histopathological analysis. Overall, the above data show that the anti-cancer effect of ATRA is more pronounced when used as an chemopreventive agent against B(a)P-induced lung carcinogenesis.     

热休克蛋白70对H2O2诱发大鼠心肌细胞凋亡的保护作用

目的:探讨热休克蛋白70(HSP₇₀)对心肌细胞凋亡的保护作用。方法:体外培养大鼠心肌细胞同时温度诱导细胞产生HSP₇₀,用过氧化氢(H₂O₂)造成心肌细胞损伤。采用免疫组化、DNALadder、流式细胞仪、细胞色素C氧化酶及琥珀酸脱氢酶比活力的测定、电镜观察等作为检测指标的方法。结果:温度诱导后HSP₇₀在胞浆中大量表达,H₂O₂损伤组与HSP₇₀保护组凋亡率(12.18±1.94,6.42±0.86,P<0.01)、细胞色素C氧化酶比活力(5.824±1.949,10.375±2.513)及琥珀酸脱氢酶比活力(0.884±0.152,1.174±0.214,P<0.01)有显著差异。电镜观察损伤组细胞膜、细胞器损伤明显,并出现凋亡小体。结论:HSP₇₀可延迟细胞凋亡,对心肌细胞具有保护作用。     

硫化氢对培养的大鼠主动脉平滑肌细胞增殖的抑制作用

目的:探讨硫化氢(H₂S)对内皮素-1(ET-1)诱导的血管平滑肌细胞(VSMC)增殖的影响及丝裂原激活的蛋白激酶(MAPK)信号转导途径的作用。方法:体外培养雄性SD大鼠主动脉VSMC, 将细胞分成对照组、血清组、内皮素组、NaHS组、血清+NaHS组和内皮素+NaHS组进行研究, 以不同浓度梯度NaHS处理VSMC, 观察对VSMC[³H]-TdR掺入和MAPK活性的影响。结果:加入5×10^-5-5×10^-4mol/LNaHS可明显浓度依赖性地抑制由内皮素诱导的VSMC增殖, 其[³H]-TdR掺入量减低, 抑制率分别为16.8%-37.4%(P＜0.01), 其MAPK活性明显减低, 抑制率为7.4%-33.6%(P＜0.05或P＜0.01)。结论:H₂S对内皮素诱导的VSMC增殖有抑制作用, 同时使MAPK活性下调。推测H₂S对VSMC增殖的抑制效应可能由MAPK信号途径所介导。     

芒果苷对H_2O_2诱导的BRL细胞氧化损伤的保护作用

目的探讨芒果苷对过氧化氢诱导的正常大鼠肝BRL细胞氧化损伤的保护作用。方法选取正常大鼠肝细胞株BRL,通过过氧化氢诱导法制备细胞氧化损伤模型,实验设对照组、H_2O_2组、芒果苷组。HE染色、台盼蓝染色观察细胞形态变化和存活率变化;比色法检测细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量;Western blot检测细胞中Bcl-2、Bax和Caspase-3蛋白表达变化。结果对照组BRL细胞贴壁生长良好,排列紧密。H_2O_2诱导后细胞形态不规则,胞膜皱缩,贴壁能力下降;细胞存活率降低;抗氧化酶SOD、GSH-Px活性降低,MDA含量升高(P0.05);Bax和Caspase-3蛋白表达升高(P0.05)、Bcl-2蛋白表达降低(P0.05)。与H_2O_2组相比,芒果苷可显著改善H_2O_2引起的细胞形态变化,提高细胞存活率;提高SOD、GSH-Px活性,降低MDA含量(P0.05);抑制Bax和Caspase-3蛋白表达(P0.05),上调Bcl-2蛋白表达(P0.05)。结论芒果苷对BRL细胞氧化损伤具有保护作用,其作用机制可能与其提高细胞清除氧自由基能力和抑制细胞凋亡有关。     

Effect of Medium on H(2)O(2) Levels and Peroxidase-Like Activity by Mycoplasma pneumoniae

H₂O₂ levels accumulated by Mycoplasma pneumoniae can be influenced by carbon source, different horse sera, yeast extract, thallium acetate, or growth in a simplified, dialyzed medium. Thus, increased levels of H₂O₂ were detected by growth in glycerol, by omission of thallium acetate, and by the use of dialyzed medium. The ability of M. pneumoniae in the presence of glucose, but not with fructose, mannose, or glycerol, to remove both endogenous and exogenous H₂O₂ suggests an inducible peroxidase similar to bacterial enzymes in streptococci. This peroxidase-like activity is in turn influenced by the complex interplay of medium components, explaining perhaps some of the variability of H₂O₂ accumulations observed with glucose. A survival value has been suggested for this “peroxidase-like” activity.     

CYP2E1 induced by ethanol causes oxidative stress, proteasome inhibition and cytokeratin aggresome (Mallory body-like) formation

The role of oxidative stress in alcoholic liver disease and cytokeratin aggresome formation is the focus of this in vitro study. HepG2 cells transduced to over express CYP2E1 (E47) and control HepG2 cells (C34) were first treated with arachidonic acid, then Fe-NAT, and finally with ethanol. In the E47 ethanol-treated cells, CYP2E1 was induced and a higher level of reactive oxygen species and carbonyl proteins were generated. The proteasome activity decreased significantly in the E47 ethanol-treated cells. This inhibition was prevented when CYP2E1 was inhibited by DAS. Microarray analysis showed gene expression down regulation of the proteasome subunit, as well as ubiquitin pathway proteins in the E47 ethanol-treated cells. 4-Hydroxynonenal (4-HNE) adducts were increased in the E47 cells treated with ethanol. Furthermore, the immunoprecipitated 4-HNE modified proteins from these cells stained positive with antibodies to the proteasome subunit alpha 6. These results indicate that the ethanol induced CYP2E1 generates oxidative stress that is responsible for the decrease in proteasome activity. Cytokeratin 8 and 18 were induced by ethanol treatment of E47 cells and polyubiquitinated forms of these proteins were found in the polyubiquitin smear upon Western blots analysis. Cytokeratin aggresomes and Mallory body-like inclusions formed in the ethanol-treated E47 cells, indicating that the ubiquitinated cytokeratins accumulated as a result of the inhibition of the proteasome by ethanol treatment when oxidation of ethanol induced oxidative stress. This is the first report where ethanol caused Mallory body-like cytokeratin inclusions in transformed human liver cells in vitro.     

Genetic and biochemical characterization of glycerol uptake in mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence

Highly virulent strains of Mycoplasma mycoides subsp. mycoides SC belonging to the African cluster contain an operon with the genes gtsA, gtsB, and gtsC, encoding membrane ATP binding cassette transporter proteins GtsA, GtsB, and GtsC, which are involved in glycerol transport. Strain Afadé from the African cluster incorporated [U-(14)C]glycerol with a time-dependent increase. The less virulent strain L2 of the European cluster, which lacks gtsB and gtsC, failed to incorporate glycerol. Antibodies against GtsB noncompetitively inhibited glycerol uptake. L-alpha-Glycerophosphate was not transported by M. mycoides subsp. mycoides SC. It is postulated to be synthesized by phosphorylation of glycerol during transport and subsequently metabolized further to dihydroxyacetone phosphate accompanied by release of H(2)O(2). Peroxide production in glycerol-containing growth medium was high for the African strain Afadé but very low for the European strain L2. Virtually no H(2)O(2) was produced by both strains without glycerol. Hence, the efficient glycerol uptake system found in the virulent strain of the African cluster leads to a strong release of peroxide, a potential virulence factor which is lacking in the less virulent European strains. M. mycoides subsp. mycoides SC might have adopted, as a strategy for virulence, a highly efficient uptake system for glycerol which allows the production of an active metabolic intermediate that damages host cells.     

In vitro study of the effect of bisphosphonates on mineralization induced by a composite material: poly 2(hydroxyethyl) methacrylate coupled with alkaline phosphatase

We have immobilized the mineralizing agent alkaline phosphatase (AlkP) in a hydrophilic polymer (poly 2(hydroxyéthyl) methacrylate) (pHEMA) in a copolymerization technique. Histochemical study on polymer sections revealed that AlkP has retained its biological activity. The image analysis of sections using a tessellation method showed a lognormal distribution of the area of the tiles surrounding AlkP particles thus confirming a homogeneous distribution of the enzyme in the polymer. Pellets of pHEMA-AlkP were incubated with a synthetic body fluid containing organic phosphates (beta-glycerophosphate). Mineral deposits with a rounded shape (calcospherites) were obtained in about 17 days. We have investigated the effects of three bisphosphonates (etidronate, alendronate and tiludronate) on this system. Bisphosphonates at a concentration of 10(-2) M totally inhibited AlkP in solution at a concentration of 10(-4) mg/ml. Inhibition has been reported being due to the chelation of a metal cofactor (Zn2+). Etidronate and alendronate appeared to inhibit the calcospherite deposition onto the pHEMA-AlkP material in a similar way. Both bisphosphonates possess three sites for mineral complexion. On the other hand, tiludronate having only two sites was associated with a reduced inhibitory effect on mineralization. When used in microgravity conditions, mineralization was impaired with etidronate and larger crystals were obtained with tiludronate. However, these effects were obtained in non-physiological conditions (a 20 degrees C temperature was used during the STS80 flight of the space shuttle). The pHEMA-AlkP material provides an interesting method to study the effects of pharmacological compounds and environmental factors on the bone and cartilage mineralization process.     

Hyperglycemia potentiates H(2)O(2) production in adipocytes and enhances insulin signal transduction: potential role for oxidative inhibition of thiol-sensitive protein-tyrosine phosphatases

Insulin signal transduction in adipocytes is accompanied by a burst of cellular hydrogen peroxide (H(2)O(2)) that facilitates insulin signaling by inhibiting thiol-dependent protein-tyrosine phosphatases (PTPs) that are negative regulators of insulin action. As hyperglycemia is associated with increased cellular reactive oxygen species, we postulated that high glucose conditions might potentiate the H(2)O(2) generated by insulin and modulate insulin-stimulated protein phosphorylation. Basal H(2)O(2) generation was increased threefold in differentiated 3T3-L1 adipocytes by growth in 25 mM glucose versus 5 mM glucose. High glucose increased the sensitivity of the insulin-stimulated H(2)O(2) signal to lower concentrations of insulin. Basal endogenous total PTP activity and the activity of PTP1B, a PTP implicated in the negative regulation of insulin signaling, were reduced in high glucose conditions, and their further reduction by insulin stimulation was more enhanced in high versus low glucose medium. Phosphorylation of the insulin receptor, IRS-1, and Akt in response to insulin was also significantly enhanced in high glucose conditions, especially at submaximal insulin concentrations. In primary rat adipocytes, high glucose increased insulin-stimulated H(2)O(2) production and potentiated the oxidative inhibition of total PTP and PTP1B activity; however, insulin signaling was not enhanced in the primary cells in high glucose apparently due to cross-regulation of insulin-stimulated protein phosphorylation by activation of protein kinase C (PKC). These studies indicate that high glucose can enhance insulin stimulated H(2)O(2) generation and augment oxidative PTP inhibition in cultured and primary adipocytes, but the overall balance of insulin signal transduction is determined by additional signal effects in high glucose, including the activation of PKC.     

IL-6预处理保护H2O2致心肌细胞损伤作用机制初探

目的初步探讨IL-6预处理对H2O2致心肌细胞氧化应激损伤的作用机制。方法采用心肌细胞原代培养方法,以H2O2刺激心肌细胞,建立细胞氧化应激模型;采用MTT法检测细胞活力;AnnexinV-FITC染色法和流式细胞术检测细胞凋亡率;检测心肌细胞内谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、丙二醛（MDA）的表达情况。结果 H2O2可降低心肌细胞存活率并能增加其凋亡,IL-6预处理后能显著改善细胞活力及凋亡情况,与模型组相比差异有统计学意义（P〈0.05）。低浓度IL-6能明显增加细胞内GSH与SOD的水平,并降低MDA的含量,随着IL-6浓度的增加,这种效应逐渐消失。结论 IL-6预处理能保护H2O2致心肌细胞损伤作用,这可能与IL-6调节细胞GSH、SOD、MDA表达有关。     

The oxidative stress induced in vivo by Shiga toxin‐2 contributes to the pathogenicity of haemolytic uraemic syndrome

Typical haemolytic uraemic syndrome (HUS) is caused by Shiga toxin (Stx)‐producing Escherichia coli infections and is characterized by thrombotic microangiopathy that leads to haemolytic anaemia, thrombocytopenia and acute renal failure. Renal or neurological sequelae are consequences of irreversible tissue damage during the acute phase. Stx toxicity and the acute inflammatory response raised by the host determine the development of HUS. At present there is no specific therapy to control Stx damage. The pathogenic role of reactive oxygen species (ROS) on endothelial injury has been largely documented. In this study, we investigated the in‐vivo effects of Stx on the oxidative balance and its contribution to the development of HUS in mice. In addition, we analysed the effect of anti‐oxidant agents as therapeutic tools to counteract Stx toxicity. We demonstrated that Stx induced an oxidative imbalance, evidenced by renal glutathione depletion and increased lipid membrane peroxidation. The increased ROS production by neutrophils may be one of the major sources of oxidative stress during Stx intoxication. All these parameters were ameliorated by anti‐oxidants reducing platelet activation, renal damage and increasing survival. To conclude, Stx generates a pro‐oxidative state that contributes to kidney failure, and exogenous anti‐oxidants could be beneficial to counteract this pathogenic pathway.     

Protective effect of the cruciferous vegetable mustard leaf (Brassica campestris) against in vivo chromosomal damage and oxidative stress induced by gamma-radiation and genotoxic chemicals

We evaluated the possible protective effect of the popular Indian cruciferous vegetable mustard leaf (Brassica campestris) against chromosomal damage and oxidative stress induced by gamma-radiation, cyclophosphamide (CPH) and urethane (URE), in mice. In vivo bone marrow micronucleus test was performed to assess chromosomal damage, and oxidative stress was monitored by estimating the changes in lipid peroxidation and the status of glutathione (GSH) as well as redox cycle antioxidants. Pretreatment with 50-250 mg/kg body wt of mustard leaf extract (MLE) for seven days significantly reduced the frequencies of micronuclei induced by gamma-radiation, CPH and URE. The protective effect against chromosomal damage was associated with modulation of lipid peroxidation as well as an increase in GSH and the GSH-dependent enzyme glutathione S-transferase (GST). Mass spectral analysis showed the presence of glucosinolates in MLE used for the pretreatment of mice. These findings indicate that intake of the green leafy cruciferous vegetable mustard leaf can lead to protection against in vivo genotoxicity and oxidative stress.