二羟异黄酮和三羟异黄酮对CHO细胞抗氧化活性的比较研究

目的:研究二羟异黄酮(Daidzein)和三羟异黄酮(Genistein)对H2O2诱导的中国仓鼠卵巢细胞(CHO)氧化损伤的保护作用及探讨其抗氧化活性的作用机制。方法:H2O2体外培养CHO细胞建立细胞氧化损伤模型,预先加入三羟异黄酮和二羟异黄酮(0.25～20μmol/L)作用24h作为保护后再加入100μmol/LH2O2作用3h,显微镜下观察CHO细胞形态改变,MTT法检测活细胞数,LDH法检测细胞的损伤情况,MDA生成量和SOD活性的测定检测细胞膜脂质过氧化反应程度。结果:H2O2处理细胞3h后,细胞形态发生明显改变,胞体变小、突起缩回;MTT吸光值减小,活细胞数减少;MDA生成量和LDH释放量明显增多,SOD活性下降,与正常对照组比较有显著性差异(P<0.01)。Daidzein处理组和Genistein处理组细胞形态明显改善,MTT吸光值显著增加,MDA生成量和LDH释放量明显减少,与H2O2处理组相比有显著性差异(P<0.01)。结论:与三羟异黄酮相比,二羟异黄酮一样能显著抑制H2O2对CHO细胞的氧化损伤,且具有较强的保护作用。     

氯离子通道阻断剂对氧化剂诱导的肾小管上皮细胞损伤的保护作用

目的研究氯离子（Cl-）通道阻断剂在氧化剂诱导的肾小管上皮细胞损伤中的作用。方法以H2O2诱导肾小管上皮细胞株（LLC-PK1）损伤，观察Cl-通道阻断剂对受损细胞LDH释放量、ATP含量和DNA降解程度的影响。结果Cl-通道阻断剂可使受损细胞的LDH释放量下降、ATP含量回升和DNA降解程度减轻。结论Cl-通道参与了氧化剂对细胞损伤的病理过程，Cl-通道阻断剂对肾小管上皮细胞具有保护作用。     

缬沙坦预处理对乳鼠心肌细胞缺氧/复氧损伤的保护作用

目的:探讨缬沙坦预处理对乳鼠心肌细胞缺氧/复氧损伤的保护作用及其机制.方法:常规方法培养心肌细胞,培养72小时后,分为三组:正常对照组、心肌细胞缺氧/复氧损伤组、缬沙坦预处理后心肌细胞缺氧/复氧损伤组.分别观察心肌细胞结构,测定心肌细胞存活率、乳酸脱氢酶(LDH)、丙二醛(MDA)及超氧化物歧化酶(SOD)水平.结果:与对照组比心肌细胞缺氧/复氧损伤组细胞存活率明显下降、培养液中LDH、MDA含量明显增高,SOD含量明显下降;与心肌细胞缺氧/复氧损伤组比,缬沙坦预处理组心肌细胞存活率明显提高,培养液中LDH、MDA含量明显下降、S0D含量明显上升.结论缬沙坦通过抗氧化、抗脂质过氧化反应,清除氧自由基对乳鼠心肌细胞缺氧/复氧损伤有保护作用.     

还原型谷胱甘肽在离体肝脏保存中应用的实验研究

目的:研究UW液中加入还原型谷胱甘肽在离体肝脏保存中对肝细胞的保护作用。方法:封闭群雄性Wistar大鼠,采用原位灌注切取肝脏,分别用UW液、改良UW液(加入谷胱甘肽0.95g/L)0～4℃保存12h、24h、36h后行Krebs-Henseleit液离体灌注,记录肝脏胆汁分泌总量,并检测灌注流出液AST、LDH含量。结果:随着保存时限延长,灌洗液中AST、LDH值升高,胆汁分泌总量呈下降趋势。12h、24h,两组之间均无统计学差异(P>0.05)。保存36h,两组差异均无统计学意义(P<0.05)。离体灌注液生化指标显示改良UW液组长时间保存时肝脏细胞损伤程度较低。结论:在离体肝脏保存过程中,UW液中加入还原型谷胱甘肽对肝脏长期保存(36h)有保护作用,但24h内对肝脏保存效果影响不大,临床需在24小时内完成肝移植手术,故保存不需要添加还原型谷胱甘肽。     

还原型谷胱甘肽对梗阻性黄疸大鼠肾脏线粒体保护作用的研究

目的:探讨氧自由基在梗阻性黄疸引发肾脏损害中的作用以及还原型谷胱甘肽对肾脏线粒体的保护作用.方法:将54只Wistar大鼠随机分为假手术对照组(A组),胆总管结扎组(8组),胆总管结扎+药物治疗组(C组).采用胆总管结扎法建立大鼠梗阻性黄疸模型,C组采用还原型谷胱甘肽每天150mg/kg腹腔注射治疗21 d.3组大鼠分别于术后第7、14、21天处死,每组每次处死6只.检测血清胆红素(BIL)、肌酐(Cr)、尿素氮(BUN)以及肾脏线粒体脂质过氧化产物丙二醛(MDA)和线粒体膜胆固醇含量的变化,并观察肾脏病理组织学改变.结果:B组及C组大鼠肾脏线粒体膜胆固醇及MDA含量高于A组(P<0.05),但C组增高的程度低于B组(P<0.05),并且病理改变也较轻.结论:氧自由基所引发的脂质过氧化作用是梗阻性黄疸引起肾脏损害的重要原因之一,应用还原型谷胱甘肽可减少脂质过氧化作用对肾脏的损害作用,对梗阻性黄疸时的肾脏损害具有保护作用.     

PKC在缺氧预处理和去甲肾上腺素预处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用

目的 评价蛋白激酶C(PKC)在缺氧预处理和去甲肾上腺素预处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用.方法 原代培养乳鼠心肌细胞,随机分为6组(n=25):对照组(Ⅰ组)常规培养;缺氧复氧组(Ⅱ组)细胞缺氧3 h,复氧1 h;缺氧预处理组(Ⅲ组)缺氧20 min,复氧20 min后制备缺氧复氧模型;去甲肾上腺素预处理组(Ⅳ组)细胞经终浓度为10-7 mol/L去甲肾上腺素孵育30 min后,去除去甲肾上腺素,再行缺氧复氧;H7+缺氧预处理组(Ⅴ组)细胞经终浓度为5×10-5 mol/L的H7孵育10 min后,去除H7,其余操作同Ⅲ组;H7+去甲肾上腺素预处理组(Ⅵ组)细胞经终浓度为5×10-5 mol/L的H7(PKC活性抑制剂)孵育10 min后,去除H7,其余操作同Ⅳ组.复氧结束后,测定心肌细胞存活率、培养液乳酸脱氢酶(LDH)、肌酸激酶(CK)活性和心肌细胞MDA含量和SOD活性.结果 与Ⅰ组比较,Ⅱ组细胞存活率和SOD活性降低,LDH、CK的活性及MDA含量升高(P＜0.01).与Ⅱ组比较,Ⅲ组和Ⅳ组细胞存活率和SOD活性升高,LDH、CK活性及MDA含量降低(P＜0.01).与Ⅲ组比较,Ⅴ组细胞存活率和SOD活性降低,LDH、CK活性及MDA含量升高(P＜0.01).与Ⅳ组比较,Ⅵ组细胞存活率和SOD活性降低,LDH、CK活性及MDA含量升高(P＜0.05).结论 PKC激活参与了缺氧预处理与去甲肾上腺素预处理减轻乳鼠心肌细胞缺氧复氧损伤.     

异氟醚对肺泡Ⅱ型上皮细胞Na^＋—K^＋—ATP酶活性的影响

目的 通过原代培养的正常及受（H2O2）损伤的肺泡Ⅱ型上皮细胞,探讨异氟醚（Iso)对肺泡Ⅱ型上皮细胞Na^ -K^ -ATP酶活性的影响。方法 肺泡Ⅱ型上皮细胞分离、纯化、培养24h后,分为六组：对照组（不加任何药物）,0．28mmol/L Iso组,2．8mmol/L Iso组,75μmol/L H2O2组,75μmol/L H2O2 0.28mmol/L Iso组和75μmol/L h2o2 2.8mmol/L Iso组,继续培养3h。分别测定肺泡Ⅱ型上皮细胞Na^ -K^ -ATP酶活性以及培养液中乳酸脱氢酶（LDH）活性和丙二醛（MDA）含量。结果 Iso降低正常肺泡Ⅱ型上皮细胞Na^ -K^ -ATP酶活性并加重H2O2引起的肺泡Ⅱ型上皮细胞Na^ -K^ ATP酶活性的降低;Iso对正常培养的肺泡Ⅱ型上皮细胞培养液中LDH活性和MDA含量无明显影响,但2．8mmol/L Iso可使受H2O2损伤的肺泡Ⅱ型上皮细胞培养液中LDH活性和MDA含量增加。结论 Iso可以降低肺泡Ⅱ型上皮细胞Na^ -K^ -ATP酶活性,尤其是在过氧化环境中可加重肺泡Ⅱ型上皮细胞的损伤。     

枸橼酸减轻草酸钙晶体对大鼠肾小管上皮细胞损伤作用的实验研究

目的 通过研究枸橼酸能否减轻一水草酸钙（calcium oxalate monohydrate，COM）晶体对体外培养的Wistar大鼠肾小管上皮细胞造成的损伤，以探讨枸橼酸在尿路结石防治中的作用及其机制。方法分离、培养正常雄性Wistar大鼠的肾小管上皮细胞，在原代培养第5天时，将细胞分成3组（空白对照组、COM组及枸橼酸组），分别于各组培养液中培养2h后用比色法测定各组细胞培养基中丙二醛（malonaldehyde，MDA）的含量及细胞的钙镁ATP酶（Ca`2＋-Mg`2＋-ATPase）和钠钾ATP酶（Na`2＋K`＋-ATPase）的活性。结果①COM组和枸橼酸组细胞Ca2＋-Mg`2＋ATPase和Na`＋＋K`＋-ATPase活性均明显低于对照组（P〈0．05）；②与枸橼酸组相比，COM组细胞Ca`2＋-Mg`2＋-ATPase和a`＋＋K`＋-ATPase活性下降更加明显（P〈O．05）；③COM组和枸橼酸组细胞培养基中MDA的含量均明显高于对照组（P〈O．05）；④与枸橼酸组相比，COM组细胞培养基中MDA的含量更高（P〈O．05）。结论外源性枸橼酸能够减轻一水草酸钙晶体对体外培养的Wistar大鼠肾小管上皮细胞造成的脂质过氧化损伤。     

金天格胶囊对H2O2诱导的小鼠成骨细胞MC3T3-E1氧化应激损伤及炎症因子的作用

目的 研究金天格胶囊对骨质疏松中氧化应激损伤和炎症因子释放的改善作用。方法体外培养MC3T3-E1小鼠成骨细胞,分别加入10、20、50和100 μg/mL金天格胶囊溶液,测定药物作用不同时间后MC3T3细胞存活率,和细胞培养上清液中ALP和PICP含量。利用1 mmol/L H2O2建立MC3T3细胞诱导损伤模型,给予不同浓度金天格胶囊,测定细胞培养上清液中SOD、GSH、CAT和MDA含量和TNF-α、IL-6及IL-1β水平,然后测定MC3T3细胞中Tnfa、Il-6、Il-1b mRNA相对表达。结果 与空白组比较,100 μg/mL金天格胶囊能显著提高MC3T3-E1细胞存活率,50、100 μg/mL金天格胶囊能显著增加细胞培养上清中ALP和PICP含量。在H2O2刺激MC3T3-E1细胞损伤模型中,50、100 μg/mL金天格胶囊能显著提高H2O2诱导损伤后细胞存活率,并能明显增加细胞培养上清液中SOD、GSH、CAT活力,降低MDA水平;此外,与H2O2组相比,50、100 μg/mL金天格胶囊能够显著降低MC3T3细胞培养上清中TNF-α、IL-6含量,显著降低细胞中Tnfa、Il-6 mRNA相对表达,100 μg/mL金天格胶囊能降低Il-1b mRNA相对表达。结论 金天格胶囊能促进小鼠成骨细胞MC3T3-E1增殖,并抑制由H2O2引起的炎症基因表达和炎症因子释放。     

异氟醚对肺泡Ⅱ型上皮细胞Na~ -K~ -ATP酶活性的影响

目的　通过原代培养的正常及受 (H2 O2 )损伤的肺泡Ⅱ型上皮细胞 ,探讨异氟醚 (Iso)对肺泡Ⅱ型上皮细胞Na K ATP酶活性的影响。方法　肺泡Ⅱ型上皮细胞分离、纯化、培养 2 4h后 ,分为六组 :对照组 (不加任何药物 ) ,0 2 8mmol/LIso组 ,2 8mmol/LIso组 ,75 μmol/LH2 O2 组 ,75 μmol/LH2 O2 0 2 8mmol/LIso组和 75 μmol/LH2 O2 2 8mmol/LIso组 ,继续培养 3h。分别测定肺泡Ⅱ型上皮细胞Na K ATP酶活性以及培养液中乳酸脱氢酶 (LDH)活性和丙二醛 (MDA)含量。结果　Iso降低正常肺泡Ⅱ型上皮细胞Na K ATP酶活性并加重H2 O2 引起的肺泡Ⅱ型上皮细胞Na K ATP酶活性的降低 ;Iso对正常培养的肺泡Ⅱ型上皮细胞培养液中LDH活性和MDA含量无明显影响 ,但 2 8mmol/LIso可使受H2 O2 损伤的肺泡Ⅱ型上皮细胞培养液中LDH活性和MDA含量增加。结论　Iso可以降低肺泡Ⅱ型上皮细胞Na K ATP酶活性 ,尤其是在过氧化环境中可加重肺泡Ⅱ型上皮细胞的损伤     

促红细胞生成素抑制过氧化氢诱导的肾小管细胞凋亡

目的探讨促红细胞生成素（erythropoietin,EPO）是否可以抑制过氧化氢（H2O2）诱导的氧化应激和细胞凋亡及其抑制凋亡的相关机制。方法传代培养大鼠近端肾小管上皮细胞（NRK-52E）,分为正常对照组（NC组）、H1组（1mmol／L H2O2）、H2组（5mmol／LH2O2、E组（H2U＋EPO组）。细胞免疫荧光检测NRK-52E细胞是否表达EPO受体（erythropoietin receptor,EPOR）。荧光探针氯甲基二氯二氢荧光素二乙酯（CM—H2DCFDA）标记检测细胞内活性氧（reactive oxygen species,ROS）水平。流式细胞仪AnnexinV-FITC／PI双染法检测细胞凋亡指数。RTPCR检测凋亡相关基因B细胞淋巴瘤／白血病-2（B-cell lymphoma／L eukemia-2,Bcl-2）家族中Bcl-2和BaxmRNA的表达。结果NRK52E细胞表达EPOR。H2O2引起NRK-52E细胞内ROS水平升高,上调BaxmRNA表达、下凋Bcl-2mRNA表达,诱导NRK-52E细胞凋亡。EPO可以抑制H2O2诱导的ROS水平升高、Bcl2mRNA表达下调、BaxmRNA表达上调及NRK-52E细胞凋亡。结论EPO可以缓解H2O2诱导的氧化应激、上调Bcl-2mRNA表达、下调BaxmRNA表达,抑制H2O2诱导的NRK52E细胞凋亡,发挥肾小管细胞保护效应。     

氧化交联淀粉浆料HD-2的研制及应用

用过氧化氢氧化淀粉，控制一定的氧化深度，进一步与交联剂反应合成了一种新型上浆剂．分析了过氧化氢的用量、反应pH值对氧化深度的影响，选择了一种新型交联剂进行交联反应．测试结果表明，该氧化交联淀粉上浆性能优良。     

针刺对光老化大鼠皮肤组织CAT、GSH-Px和H2O2影响的实验研究

目的:研究针刺对UV照射引起的皮肤光老化大鼠皮肤组织中过氧化氢酶(CAT)、谷胱甘肽氧化酶(GSH-Px)和过氧化氢(H2O2)的含量的变化,探讨针刺对抗皮肤光老化的作用机理。方法:将大鼠随机分成4组,除正常组外,其余各组模拟日光中UV(UVA+UVB)照射,造成皮肤光老化模型,每次造模前,针刺组给予电针刺激,阳性对照组采用VE涂抹造模部位,15周后对比各组生化指标结果。结果:模型组与正常组比较大鼠皮肤组织中CAT、GSH-Px活性明显降低,H2O2含量明显增加(P<0.05);针刺组与模型组比较CAT、GSH-Px活性增加,H2O2含量明显降低(P<0.05),与VE组比较无统计学意义(P>0.05)。结论:针刺可延缓UV引起的大鼠皮肤光老化,能增强皮肤组织中CAT、GSH-Px活性,降低H2O2含量。     

H2O2氧化应激诱导兔椎间盘髓核细胞衰老的研究

目的:研究H2O2不同浓度对兔椎间盘髓核细胞形态、活力、增值、周期等的影响.方法:新西兰大白兔(2～3 kg,雌)10只,无菌条件下酶消化法分离髓核细胞.含15％FBS的DMEM/F12(1:1)培养液培养,细胞90％融合后传第1代.按照H2O2不同浓度(0μmol/L、130 μmol/L、216 μmol/L、360 μmol/L、600 μmol/L、1 000 μmol/L)分组,0 μmol/L H2O2为空白对照组.在细胞对数生长期,不同浓度H2O2处理1h后原培养液继续培养48 h,通过检测,分析比较各组髓核细胞与空白对照组间形态、活力、增值、周期的差异性.结果:与空白对照组比较,当H2O2浓度为130 μmol/L、216 μmol/L时,髓核细胞生物学特性未见显著性改变(P＞0.05);当H2O2浓度为360 μmol/L、600 μmol/L、1 000 μmol/L时,髓核细胞出现衰老改变:细胞质减少并出现空泡、增值减慢、衰老相关-β-半乳糖苷酶蓝染率增加、细胞周期阻滞于G1期而进入S期减少(P＜0.05).随着H2O2浓度的增高,衰老程度不断升高.结论:一定浓度的H2O2能够导致髓核细胞提前衰老,引起髓核细胞生物学特性的改变.     

过氧化脲漂白剂的配制与标定

目的：配制不同增稠载体及浓度的过氧化脲（carbamide peroxide,CP）漂白剂。方法：以卡波姆（carbopol）、聚乙烯吡咯烷酮（PVP）或泊洛沙姆（Poloxamer）作为漂白剂的增稠载体,分别配制含有10%、15%和20%CP的漂白凝胶,并通过0.02mol/LKMnO4标准溶液对其所含过氧化氢（hydrogen peroxide,HP）含量进行标定。结果：以Carbopol、PVP或Poloxamer为增稠载体配制的10%CP中HP含量分别为3.4%、3.6%和3.2%;15%CP中HP含量分别为5.2%、5.3%和5.0%;20%CP中HP含量分别为6.7%、6.6%和7.0%。结论：以不同增稠载体配制的CP漂白剂HP含量均在10%以下,符合家庭漂白剂的含量标准。     

Enhanced detection of reactive oxygen species produced by human spermatozoa with 7-dimethyl amino-naphthalin–1, 2-dicarbonic acid hydrazide

A new chemiluminescence technique has been assessed for the detection of reactive oxygen species generated by purified populations of human sperm. This revised protocol involves the use of horse-radish peroxidase (HRP) in combination with a luminol analogue, 7-dimethyl amino-naphthalin-1,2-dicarbonic acid hydrazide (DNDH), that exhibits two-three times the quantal efficiency of luminol itself. The chemiluminescent signal generated with these reagents was significantly (P less than 0.001) greater than that obtained with the conventional luminol-based methodology for both the steady-state situation and following stimulation of the sperm with PMA and A23187. Dose-response analyses indicated that the DNDH/HRP chemiluminescence system could give linear standard curves with hydrogen peroxide concentrations into the nmol l-1 range. In contrast, the exponential rise in chemiluminescence recorded with luminol was not observed until hydrogen peroxide concentrations exceeded 10 mumol l-1. It is concluded that the enhanced sensitivity of the DNDH/HRP system to low levels of hydrogen peroxide should facilitate the application of chemiluminescent techniques to the diagnosis of oxidative stress in cases of male infertility.     

Effects of H2O2 exposure on human sperm motility parameters,reactive oxygen species levels and nitric oxide levels

Research has revealed that reactive oxygen species (ROS) negatively affect sperm function, both in vivo and in vitro. Sperm preparation techniques for assisted reproductive technologies (ART) are potential causes for additional ROS production. This study aimed to correlate the concentration of exogenous H₂O₂ with sperm motility parameters and intracellular ROS and nitric oxide (NO) levels to reiterate the importance of minimising ROS levels in ART. Human spermatozoa from 10 donors were incubated and exposed to different exogenous H₂O₂ concentrations (0, 2.5, 7.5 and 15 μm ). Subsequently, motility was determined using computer‐aided semen analysis, while ROS (2,7‐dichlorofluorescin diacetate) and NO (diaminofluorescein‐2/diacetate) were analysed using fluorescence‐activated cell sorting. Results showed that H₂O₂ did affect the sperm parameters. Exogenous H₂O₂ was detrimental to motility and resulted in a significant increase in overall ROS and NO levels. A significant increase in static cells was seen as well. It is important to elucidate the mechanisms between intracellular ROS levels with sperm motility parameters. While this experiment demonstrated a need to reduce exogenous ROS levels during ART, it did not illustrate the cause and effect relationship of intracellular ROS and NO levels with sperm motility. Further research needs to be conducted to define a pathological level of ROS.     

Hydrogen peroxide prevents vascular calcification induced ROS production by regulating Nrf-2 pathway

Background: Although vascular calcification in end-stage renal disease (ESRD) represents a ubiquitous human health problem, effective therapies with limited side effects are still lacking, and the precise mechanisms are not fully understood. The Nrf-2/ARE pathway is a pivotal to regulate anti-oxidative responses in vascular calcification upon ESRD. Although Nrf-2 plays a crucial role in atherosclerosis, pulmonary fibrosis, and brain ischemia, the effect of Nrf-2 and oxidative stress on vascular calcification in ESRD patients is still unclear. The aim of this research was to study the protective role of hydrogen peroxide in vascular calcification and the mechanism of Nrf-2 and oxidative stress on vascular calcification.

Materials and methods: Here we used the rat vascular smooth muscle cell model of β-glycerophosphate-induced calcification resembling vascular calcification in ESRD to investigate the therapeutic effect of 0.01?mM hydrogen peroxide on vascular calcification and further explores the possible underlying mechanisms.

Results: Our current report shows the in vitro role of 0.01?mM hydrogen peroxide in protecting against intracellular ROS accumulation upon vascular calcification. Both hydrogen peroxide and sulforaphane pretreatment reduced ROS production, increased the expression of Nrf-2, and decreased the expression of Runx2 following calcification.

Conclusion: Our study demonstrates that 0.01?mM hydrogen peroxide can effectively protect rat aortic vascular smooth muscle cells against oxidative stress by preventing vascular calcification induced ROS production through Nrf-2 pathway. These data might define an antioxidant role of hydrogen peroxide in vascular calcification upon ESRD.     

Protective effect of paraoxonase 1 of high-density lipoprotein in type 2 diabetic patients with nephropathy

Aim:   Paraoxonase 1 (PON1) is an important antioxidative enzyme associated with high-density lipoproteins (HDL). Recent data suggest that HDL antioxidative ability may be altered in type 2 diabetic patients. The aim of this study was, therefore, to investigate whether HDL-PON1 activity and HDL antioxidative ability were related to the presence and severity of diabetic nephropathy.
Methods:   Sixty type 2 diabetic patients, who were subdivided into normoalbuminuria, microalbuminuria and macroalbuminuria, and 20 age- and sex-matched healthy controls were recruited. HDL was then isolated to measure PON1 activity, lipid peroxide and its ability in protecting low-density lipoprotein (LDL) from oxidation.
Results:   HDL-PON1 activity and HDL antioxidative ability in protecting LDL from oxidation was preserved in diabetic patients with normoalbuminuria, but significantly decreased in those with microalbuminuria or macroalbuminuria. HDL peroxide was comparable between healthy controls and normoalbuminuric patients, but significantly increased in those with microalbuminuria or macroalbuminuria. On multiple analyses, urinary protein was an independent negative determinant of HDL-PON1 activity, and HDL-PON1 activity was positively associated with HDL antioxidative ability, and negatively associated with HDL peroxide.
Conclusion:   HDL-PON1 activity is decreased in type 2 diabetic patients with incipient or overt nephropathy, which relates to the reduced HDL antioxidative ability in protecting LDL from oxidation and increased peroxide in HDL.