肢体缺血预适应大鼠血清对HUVEC和H9c2（2-1）细胞H_2O_2损伤的保护作用及机制研究
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摘要
研究背景
细胞内ROS（reactive oxygen species）的产生处于动态平衡状态。缺血再灌注时细胞内产生大量的ROS，造成氧化应激损伤，即缺血再灌注损伤。轻度短暂的组织缺血再灌注可以减轻随后发生的严重的组织缺血再灌注损伤，这种现象称为缺血预适应（Ischemic preconditioning，IPC)。研究发现，创伤小，操作方便的肢体缺血预适应（Limb ischemic preconditioning, LIPC），可以减轻多种动物多个器官的缺血再灌注损伤。血液中ROS增加,易导致内皮损伤,加重缺血再灌注损伤。LIPC可以调节正常内皮功能，对内皮ROS损伤是否有作用目前尚无详细报道。
CAT，SOD，GSH-Px等是细胞内重要的清除ROS的抗氧化酶。细胞内的抗氧化酶受到转录因子Nrf2(NF-E2-related factor2)的调控。Nrf2通过与抗氧化反应元件(Antioxiant response element, ARE)结合，上调与ARE相关的抗氧化基因表达，包括细胞内源性抗氧化酶、Ⅱ相解毒酶等，清除细胞内过多地ROS，维持细胞内氧化还原水平，减轻氧化应激所造成的细胞损伤。受Nrf2调控的蛋白持续表达增加对细胞氧化还原内环境的稳定起到非常关键的作用。
研究目的
本课题主要观察LIPC大鼠的血清是否可以减轻H_2O_2所导致的人脐静脉内皮细胞HUVEC和鼠胚胎心肌细胞H9c2（2-1）的氧化应激损伤，分析Nrf2所调控的抗氧化基因是否参与了该过程，以及有可能所涉及的信号通路。
材料和方法
（1）成年健康SD大鼠，用改良的大鼠尾动脉血压测定仪套管套在大鼠右后肢，加压至200mmHg阻断股动脉造成右后肢短暂缺血，持续5分钟，放气减压恢复血流，持续10分钟，共3个循环。预适应结束后立即腹主动脉无菌采血获得大鼠早期肢体预适应血清。预适应后间隔24小时采血，获得大鼠延迟肢体预适应血清，分装冻存于-80℃。
（2）建立HUVEC和H9c2（2-1）细胞的H_2O_2损伤模型，参考IC50作为后续实验造模浓度。HUVEC和H9c2（2-1）细胞分为5组，对照组（control，C），模型组（model，M），大鼠早期预适应血清组（preconditioning serum, PS），大鼠延迟预适应血清组(delayed preconditioning serum, DPS)，正常大鼠血清组（normal rat serum, NRS），预先用2.5%,5%（V/V）各种血清孵育6小时和12小时，然后用H_2O_2处理2小时，用MTT法检测各种血清预处理对HUVEC和H9c2（2-1）细胞活性的影响。
（3）应用AnnexinV-FITC和PI双染进一步检测细胞的凋亡情况。
（4）细胞处理完成后，用8μM的DHE孵育45分钟，荧光显微镜下检测ROS的含量。
（5）应用LDH，MDA，CK检测试剂盒检测细胞培养上清液中LDH，CK的活性及MDA的浓度。
（6）采用SOD，CAT，GSH-Px检测试剂盒检测细胞培养上清液中SOD，CAT，GSH-Px的活性。
（7）采用免疫荧光法和激光共聚焦显微镜检测各组细胞Nrf2的核转位情况。
（8）采用real-time RT-PCR检测各组细胞中抗氧化基因CAT，HO-1，SOD1，SOD2，GSH-Px1mRNA的水平。
（9）采用Western Blot法检测各组细胞中CAT，HO-1，SOD1，SOD2的蛋白表达情况。
（10）用25μM PI3K的抑制剂LY294002和26μM MEK1/2抑制剂U0126提前1小时处理细胞，观察这两种抑制剂对PS和DPS作用的影响。
（11）各实验组数据采用mean±SD表示，利用SPSS13.0统计软件分析，组间差异比较采用单因素方差分析。P <0.05认为有显著差异。
结果
（1）不同浓度的H_2O_2（100-1200μM）作用2小时后，随H_2O_2浓度增高HUVEC和H9c2（2-1）细胞存活逐渐降低，IC50分别为1099.56±109.11μM和380.98±57.44μM，参考IC50选取1000μM和400μM作为造模浓度。
（2）对于H9c2（2-1）细胞和HUVEC细胞，2.5%PS，DPS，NRS预孵育6小时和12小时，400μM和1000μM H_2O_2处理2小时，MTT570nm处吸光度测定结果显示，M组比C组显著降低（P <0.001），PS，DPS，NRS组的吸光度与M组相比无显著差异（P>0.05）。
（3）5%的各血清预孵育6小时，400μM和1000μM H_2O_2处理2小时，H9c2（2-1）细胞和HUVEC细胞MTT570nm处吸光度测定结果显示，M组比C组显著降低（P <0.001）。PS，DPS，NRS组的吸光度与M组相比无显著差异(P>0.05)。
（4）5%的各血清预孵育12小时，H_2O_2处理2小时，MTT570nm处吸光度测定结果显示，H9c2（2-1）M组吸光度显著低于C组（0.51±0.03vs1.20±0.09，P <0.001），PS组和DPS组吸光度分别为1.14±0.17和1.08±0.23，显著高于M组（P<0.001），PS组和DPS组相比无显著差异(P>0.05)。NRS组吸光度0.53±0.04，与M组相比无显著性差异(P>0.05)。
HUVEC细胞M组吸光度显著低于C组（0.78±0.03vs1.26±0.06，P <0.001），PS组和DPS组吸光度分别为1.09±0.04和1.08±0.04，显著高于比M组（P <0.001），PS组和DPS组相比无显著差异(P>0.05)。NRS组吸光度0.75±0.03，与M组相比无显著性差异(P>0.05)。
（5）5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞PS组、DPS组和M组的早期凋亡率分别为2.74±1.28%、5.22±2.09%和12.28±3.24%（P <0.001，P <0.05）；PS组、DPS组和M组晚期凋亡率分别为6.87±0.88%，8.90±0.65%和36.04±6.22%（P <0.001）；PS组、DPS组和M组总死亡率为9.61±0.93%，14.12±1.43%，48.31±3.33%（P <0.001）。PS组和DPS组相比无显著差异(P>0.05)。NRS组的早期凋亡，晚期凋亡和总死亡率与M组比较无显著差异(P>0.05)。
HUVEC细胞PS组、DPS组的早期凋亡率分别为1.12±0.20%、1.78±0.20%与M组13.24±1.37%相比，显著降低（P<0.01）；PS组和DPS组晚期凋亡率7.54±1.49%和6.87±1.48%，与M组晚期凋亡率25.52±0.86%相比，显著降低（P <0.001，P <0.01）；PS组和DPS组总死亡率为8.65±1.67%和9.65±1.44%，与M组总死亡率38.76±2.23%相比，显著降低（P <0.001）。PS组和DPS组相比无显著差异(P>0.05)。NRS组的早期凋亡率，晚期凋亡率和总死亡率与M组比无显著差异(P>0.05)。
（6）5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞M组荧光亮度与C组比显著升高（96.34±21.23vs23.67±6.5，P <0.01）。PS组和DPS组荧光亮度29.87±5.67和30.67±7.34比M组显著降低（P <0.01）。PS组和DPS组相比无显著差异(P>0.05)。NRS组荧光亮度为88.67±19.21，与M组相比无显著性差异(P>0.05)。
HUVEC细胞M组荧光亮度76.34±19.23比C组20.16±4.5显著升高（P <0.01）。PS组和DPS组荧光亮度22.87±6.78和24.46±8.12比M组显著降低（P<0.01）。PS组和DPS组相比无显著差异(P>0.05)。NRS组荧光亮度为71.34±16.19，与M组比无显著性差异(P>0.05)。
（7）5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞M组MDA浓度从4.42±0.28nmol/ml升高至8.74±0.33nmol/ml（P <0.001）。PS组和DPS组MDA浓度则降至3.48±0.46nmol/ml和3.55±0.68nmol/m（lP <0.001），PS组和DPS组相比无显著差异(P>0.05)。而NRS组的MDA为8.54±1.08nmol/ml，与M组比无显著差异（P>0.05）。
HUVEC细胞M组MDA浓度从4.86±0.28nmol/ml升高至6.67±0.29nmol/ml（P <0.001）。而PS组和DPS组MDA浓度则降至3.44±0.27nmol/ml，3.80±0.42nmol/ml（P<0.001），PS组和DPS组相比无显著差异(P>0.05)。NRS组的MDA浓度为6.25±0.59nmol/ml，与M组比无显著差异（P>0.05）。
5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞的LDH浓度从72.69±6.75U/L升高至191.93±36.52U/L（P <0.001）；PS组和DPS组LDH则降至77.81±13.58U/L，105.75±8.67U/L（P <0.001），PS组和DPS组相比无显著差异(P>0.05)。而NRS组的LDH为173.86±36.17U/L，与M组比无显著差异（P>0.05）。
HUVEC细胞的LDH含量12.58±2.74U/L升高至58.16±5.05U/L（P <0.001）。而PS组和DPS组LDH则降至16.51±4.63U/L和15.54±2.70U/L（P <0.001）。PS组和DPS组相比无显著差异(P>0.05)。而NRS组的LDH为62.13±11.95U/L，与M组比无显著差异（P>0.05）。
400μM H_2O_2处理H9c2（2-1）细胞2小时，细胞培养液中的CK显著升高（15.67±0.91U/L vs30.47±1.76U/L，P <0.001），PS和DPS预先处理12小时，则细胞培养液中的CK降低至20.08±1.26U/L和22.00±1.52U/L（P <0.001）。PS组和DPS组相比无显著差异(P>0.05)。NRS预先处理12小时，则细胞培养液中的CK为29.47±2.26U/L，与M组30.47±1.76U/L相比，无显著差异（P>0.05）。
（8）5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞CAT的活性PS组和DPS组为45.33±3.82U/ml和44.61±5.79U/ml比M组17.30±1.62U/ml显著增高(P <0.01，P <0.05），PS组和DPS组相比无显著差异(P>0.05)。NRS组为17.92±2.17U/ml，与M组比无显著差异（P>0.05）。HUVEC细胞CAT的活性PS组和DPS组为51.25±4.82U/ml和50.5±5.89U/ml比M组21.69±2.02U/ml高(P <0.01,P <0.05），PS组和DPS组相比无显著差异(P>0.05)。NRS组为25.81±4.40U/ml，与M组比无显著差异（P>0.05）。
H9c2(2-1)细胞SOD的活性PS组和DPS组为14.02±1.68U/ml和14.11±0.58U/ml，比M组6.89±0.97U/ml显著增高(P <0.001），PS组和DPS组相比无显著差异(P>0.05)。NRS组为7.56±1.24U/ml，与M组比无显著差异（P>0.05）。HUVEC细胞CAT的活性PS组和DPS组为13.82±0.50U/ml，11.29±3.58U/ml比M组6.43±0.78U/ml显著增高(P <0.001，P <0.05），PS组和DPS组相比无显著差异(P>0.05)。NRS组为7.74±2.02U/ml，与M组比无显著差异（P>0.05）。
H9c2(2-1)细胞GSH-Px的活性PS组和DPS组为43.27±8.56U/ml，37.62±6.00U/ml，比M组15.06±7.09U/ml显著增高(P <0.01），PS组和DPS组相比无显著差异(P>0.05)。NRS组为21.89±3.97U/ml，与M组比无显著差异（P>0.05）。HUVEC细胞GSH-Px的活性PS组和DPS组为52.38±15.75U/ml，41.84±1.66U/ml比M组25.08±8.50U/ml显著增高(P <0.001，P <0.05），PS组和DPS组相比无显著差异(P>0.05)。NRS组为29.34±8.56U/ml，与M组比无显著差异（P>0.05）。
（9）H9c2(2-1)细胞和HUVEC各组细胞Nrf2的分布情况，C组细胞内的绿色荧光（anti–Nrf2-FITC）主要在核周围，呈环状。NRS组和M组只有少量细胞中的Nrf2从细胞浆进入细胞核。而PS组和DPS组中，大量细胞中的Nrf2从细胞浆进入细胞核，PS组和DPS组Nrf2的核转位增多。
（10）H9c2(2-1)细胞M，NRS，PS，DPS组的CAT，HO-1，SOD1，SOD2，GSH-Px1的mRNA水平都比C组增高，但NRS组与M组比无显著差异（P>0.05）。CAT的mRNA水平PS组3.12±0.03和DPS组2.63±0.13比M组1.23±0.04显著增高(P <0.001)。HO-1的mRNA水平PS组4.10±0.26和DPS组3.26±0.21比M组1.30±0.02显著增高(P <0.001)。SOD1的mRNA水平PS组3.74±0.37和DPS组3.89±0.84比M组1.58±0.07显著增高(P <0.001)。SOD2的mRNA水平PS组4.04±0.22和DPS4.34±0.22组比M组1.52±0.11显著增高(P <0.001)。GSH-Px1的mRNA水平PS组3.17±0.17和DPS组4.86±0.71比M组1.58±0.12显著增高(P <0.001)。PS组和DPS组相比无显著差异(P>0.05)。
HUVEC细胞M，NRS，PS，DPS组的CAT，HO-1，SOD1，SOD2，GSH-Px1的mRNA水平都比C组增高，但NRS组与M组比无显著差异（P>0.05）。CAT的mRNA水平PS组3.35±0.03和DPS组2.86±0.13比M组1.46±0.04显著增高(P <0.05)。HO-1的mRNA水平PS组4.51±0.26和DPS组3.67±0.21比M组1.71±0.02显著增高(P <0.001)。SOD1的mRNA水平PS组4.15±0.37和DPS组4.30±0.84比M组1.99±0.07显著增高(P <0.001)。SOD2的mRNA水平PS组3.84±0.22和DPS组4.14±0.22比M组1.32±0.11显著增高(P <0.001)。GSH-Px1的mRNA水平PS组1.97±0.17和DPS组4.66±0.71比M组1.38±0.12显著增高(P <0.05，P<0.001)。PS组和DPS组相比无显著差异(P>0.05)。
Western Blot检测结果，H9c2(2-1)细胞，PS组和DPS组细胞内的抗氧化酶CAT（0.56±0.08,0.53±0.07），HO-1（0.46±0.07,0.44±0.01），SOD1（0.55±0.09,0.61±0.08），SOD（20.47±0.06,0.52±0.07），表达显著增高(M组CAT0.32±0.09，HO-10.29±0.03, SOD10.35±0.05,SOD20.27±0.05，P <0.05)，PS组和DPS组相比无显著差异(P>0.05)。而NRS组抗氧化酶的表达与M相比无显著差异（P>0.05）。
Western Blot检测结果，HUVEC细胞，PS组和DPS组细胞内的抗氧化酶CAT（0.63±0.10,0.62±0.07），HO-1（0.58±0.10,0.60±0.04），SOD1（0.41±0.11,0.54±0.09），SOD2（0.54±0.09,0.51±0.02）表达增高(M组CAT0.33±0.09，HO-10.31±0.05, SOD10.27±0.1,SOD20.28±0.05, P <0.05)，PS组和DPS组相比无显著差异(P>0.05)。NRS组抗氧化酶的表达与M相比无显著差异（P>0.05）。
（11）H9c2(2-1)细胞PS组的吸光度为1.09±0.1，而PS+LY294002组吸光度为0.95±0.15，PS+U0126组吸光度为1.12±0.19，与PS组比无显著差异（P>0.05）。DPS组的吸光度为1.02±0.15，而DPS+LY294002组吸光度为0.99±0.11，DPS+U0126组吸光度为0.95±0.14，与DPS组比无显著差异（P>0.05）。
HUVEC细胞PS组的吸光度为1.58±0.08，而PS+LY294002组吸光度为1.51±0.06，PS+U0126组吸光度为1.45±0.12，与PS比无显著差异（P>0.05）。DPS组的吸光度为1.51±0.06，而DPS+LY294002组吸光度为1.52±0.06，DPS+U0126组吸光度为1.48±0.02，与DPS组比无显著差异（P>0.05）。
LY294002和U0126不影响PS和DPS对H9c2(2-1)细胞和HUVEC细胞H_2O_2损伤的保护作用，不影响Nrf2的核转位。
结论
肢体缺血预适应大鼠早期相和晚期相血清，5%孵育12小时，能够减轻H_2O_2所造成的H9c2(2-1)细胞和HUVEC细胞损伤，增加Nrf2的核转位，从而提高CAT，HO-1，SOD1，SOD2，GSH-Px1的mRNA水平，CAT，HO-1，SOD1，SOD2蛋白表达增加，抗氧化酶CAT，SOD，GSH-Px的活性增高。PI3K/AKT通路和MEK/ERK1/2通路可能不参与其中。
Background
ROS are chemically reactive molecules containing oxygen, including oxygen freeradicals, hydrogen peroxide, lipid peroxide, superoxide anion and etc. In normal cells,reactive oxidants are produced in a controlled manner and some serve useful purposes.Uncontrolled production of ROS results in oxidative stress that impairs cellularfunctions and contributes to the development of ischemia-reperfusion injury. Ischemicpreconditioning is a potent approach to reduce ischemic reperfusion injury. Limbischemic preconditioning is a noninvasive feasibility treatment to protect ischemicreperfusion injury of multiple organs in a variety of animal models, but themechanism is not clear.
CAT, SOD, GSH-Px are important antioxidase to remvove ROS in cell.TheKeap1–Nrf2(NF-E2-related factor2) system is one of the major cellular defensemechanisms against oxidative and/or electrophilic stresses.Transcription factor Nrf2can recognize the antioxidant response element/electrophile responsive element(ARE/EpRE) in the promoter region. It can also regulate the basal and inducibleexpression of numerous antioxidant and detoxifying genes. The elevation expressionof proteins regulated by Nrf2plays a crucial role in the homeostasis of the internalenvironment.
Objective
This study was to observe whether the serum from rats undergoing early limbpreconditioning and delayed limb preconditioning could protect HUVEC andH9c2(2-1) from oxidant stress induced by H2O2. Furtherly, the involvement ofantioxidant gene regulated by Nrf2and signal pathway were explored.
Materials and methods
(1)Male Sprague-Dawley rats were used for the study. A modified noninvasiveblood pressure radiometer cuff was placed around right hindlimb. A pulse sensor wasplaced on the arteria dorsalis pedis. The cuff was inflated to200mmHg for5minutesin order to obtain ischemia and asphygmia also indicated that the arteria femoraliswas blocked. The deflation of the cuff could reperfuse the arteria femoralis for10minand the sphygmus appeared again. The procedure was operated for three cycles.
(2)Firstly, oxidant stress models of HUVEC and H9c2(2-1) induced by H_2O_2were set up, and H_2O_2concentration used in the following experiment was refered tothe IC50. The HUVEC and H9c2(2-1) cells were divided into5groups: controlgroup(C), model group(M), normal rat serum group(NRS)，preconditioning serumgroup(PS), and delayed preconditioning serum group(DPS).The NRS, PS, DPS,groups were incubated with2.5%and5%(V/V) serum for6h and12h respectively,then the cell viability was detected by MTT method.
(3) After treatment, the apoptosis was detected by AnnexinV-FITC and PIdouble staining.
(4) ROS was quantified by fluorescent microscopy incubated with8μM DHEfor45min.
(5) The concentrations of MDA, LDH as well as CK were all determined byusing commercially available kits.
(6) The activity of CAT, SOD and GSH-Px was determined by usingcommercially available kits.
(7) Immunofluorescence staining was used to observe the nucleus translocationof Nrf2by the confocal microscopy.
(8) The mRNA level of CAT, HO-1, SOD1, SOD2, and GSH-Px1wasdetermined by real-time semiquantitative reverse transcription-polymerase chainreaction method.
(9) The protein level of CAT, HO-1, SOD1, and SOD2was detected by westernblot.
(10)The effects of LY294002and U0126on PS and DPS groups were detected aswell.
(11) All data are presented as mean±standard deviation (S.D) and analyzed by SPSS13.0. Differences between mean values of multiple groups were analyzed byone-way analysis of variance. Values of P <0.05were considered to be statisticallysignificant.
Results
(1) Incubation of HUVEC and H9c2(2-1) cells with different concentrations ofH_2O_2(100–1200μM)for2h could decrease the viability of cells in a dose-dependentmanner. The IC50was1099.56±109.11μM and380.98±57.44μM,1000μM and400μM were used as the sample treatment concentration.
(2)H9c2(2-1) and HUVEC cells were pretreated with2.5%, NRS, PS and DPSfor6h and12h, and thereafter H_2O_2treated for2h. MTT results showed that Mgroup had a lower absorbance compared with C group at570nm (P <0.001). Therewere no significant differences in absorbance among NRS, PS, DPS, and M group (P>0.05).
(3) H9c2(2-1) and HUVEC cells were pretreated with5%PS, DPS and NRS for6h, and thereafter H_2O_2for2h. MTT results showed that M group had a lowerabsorbance compared with C group at570nm (P <0.001).The absorbance of Mgroup was not different from those of PS, DPS or NRS group (P>0.05).
(4) For H9c2(2-1) and HUVEC cells, pretreatment with5%PS,DPS and NRSfor12h, and H_2O_2treated2h, M group was lower than C group in MTT570nmabsorbance(P <0.001).
To H9c2(2-1),the absorbance of PS group1.14±0.17and DPS group1.08±0.23was higher than M group0.51±0.03(P <0.001). There were no difference betweenPS and DPS group(P>0.05).But the absorbance of NRS group was no significantdifferences compared with M group (P>0.05).
To HUVEC, the absorbance of PS group1.09±0.04and DPS group1.08±0.04was higher than M group0.78±0.03(P <0.001). Furthermore, there were nodifference between PS and DPS group(P>0.05).But the absorbance of NRS groupwas no significant differences compared with M group (P>0.05).
(5) For H9c2(2-1), pretreatment with5%PS,DPS and NRS for12h, andthereafter H_2O_2treatment for2h, the early apoptosis of PS group and DPS group were2.74±1.28%and5.22±2.09%, which were lower than M group(12.28±3.24%,P <0.001). There were no significant differences between NRS and M group (P>0.05). The late apoptosis of PS group and DPS group were6.87±0.88%and8.90±0.65%, which were lower than M group (36.04±6.22%,P <0.001) There were nosignificant difference between NRS, and M group (P>0.05).The total apoptosis ofPS group and DPS group were9.61±0.93%and14.12±1.43%, which were lowerthan M group(48.31±3.33%,P <0.001). There were no significant differencesbetween NRS and M group (P>0.05).
For HUVEC, pretreatment with5%PS, DPS and NRS for12h followed by H_2O_2treated for2h, the early apoptosis of PS group and DPS group were1.12±0.20%and1.78±0.20%, which were lower than M group (13.24±1.37%,P <0.001). Thelate apoptosis of PS group and DPS group were7.54±1.49%and6.87±1.48%,which were lower than M group (25.52±0.86%,P <0.001,P <0.01). The totalapoptosis of PS group and DPS group were8.65±1.67%and9.65±1.44%, whichwere lower than M group (38.76±2.23%,P <0.001). There were no significantdifferences between NRS and M group (P>0.05).
(6) As expected, the DHE fluorescence in H9c2(2-1) and HUVEC exposed toH_2O_2was strikingly increased compared with control group (P <0.01). Pretreatmentwith5%PS and DPS for12h significantly inhibited DHE fluorescence caused byH_2O_2. Pretreatment with NRS could not restor the DHE fluorescence (P>0.05).
(7) Compared with control group, treatment of H9c2(2-1) cells with400μMH_2O_2for2h caused significantly increase of MDA from4.42±0.28nmol/ml to8.74±0.33nmol/ml (P <0.001).Incubated with5%PS and DPS for12h attenuated MDA(P <0.001).But Incubated with NRS for12h did not affect the level of MDA (P>0.05)
Similarly,treatment of HUVEC with1000μM H_2O_2for2h caused significantlyincrease of MDA from4.86±0.28nmol/ml to6.67±0.29nmol/ml (P <0.001)Incubated with5%PS and DPS for12h attenuated MDA level (P <0.001).Howerverincubated with NRS for12h did not affect the level of MDA (P>0.05).
In addition, treated H9c2（2-1）cells with400μM H_2O_2for2h causedsignificantly increase in LDH level from72.69±6.75nmol/ml to191.93±36.52 nmol/ml (P <0.001). Preincubation with5%PS and DPS for12h markedlydecreased the elevation compared with the M group(P <0.001).However incubatedwith5%NRS for12h did not affect the level of LDH (P>0.05). The phenomenon inHUVEC was similar to H9c2(2-1) cells.
For H9c2(2-1) cells, the CK of PS and DPS group was lower than M group(20.08±1.26U/L and22.00±1.52U/L vs30.47±1.76U/L, P <0.001). NRS groupwas almost equal to M group respectively (P>0.05).
(8) Pretreated H9c2（2-1）cells with5%PS and DPS for12h, the activity of CAT(45.33±3.82U/ml，44.61±5.79U/ml) were higher than that of M group (17.30±1.62U/ml)(P <0.01,P <0.05). The activity of CAT in HUVEC were also raised bytreatment with5%PS and DPS for12h (51.25±4.82U/ml，50.5±5.89U/ml vs21.69±2.02U/ml, P <0.01,P <0.05）There were no notable differences between NRS,and M group (P>0.05).
Pretreated H9c2(2-1) cells with5%PS and DPS for12h, the activity ofSOD(14.02±1.68U/ml,14.11±0.58U/ml) were higher than that of M group (6.89±0.97U/ml,P <0.001). The activity of SOD in HUVEC were also elevated by treatedwith5%PS and DPS for12h(13.82±0.50U/ml，11.29±3.58U/ml vs6.43±0.78U/ml, P <0.01, P <0.05）There were no notable difference between NRS and Mgroup (P>0.05).
Pretreated H9c2(2-1) cells with5%PS and DPS for12h, the activity of GSH-Px(43.27±8.56U/ml，37.62±6.00U/ml) were higher than that of M group (15.07±7.09U/ml)(P <0.01). The activity of GSH-Px of HUVEC were also elvated bytreatment with5%PS and DPS for12h (52.38±5.75U/ml，41.84±1.66U/ml vs25.08±8.50U/ml, P <0.01,P <0.05）There were no notable difference between NRSand M group (P>0.05).
The activity change trends of SOD and GSH-Px were in line with the activity ofCAT among these groups.
(9) Confocal imaging using FITC-conjugated secondary antibody stainingindicated that under normal conditions, Nrf2protein mainly distributed in cytoplasmaround the nucleus, which looked like a ring. In the NRS and M group, only a smallnumber of cells translocated Nrf2into the nucleuses. The number of cells in which Nrf2mainly distributed in nucleus extraordinarily increased in PS and DPS group.
(10)Effects of PS and DPS on mRNA leves of the antioxidant gene CAT, HO-1,SOD1, SOD2, and GSH-Px1were evaluated using real time RT-PCR, with GAPDHas an internal control. In H9c2(2-1) cells treated with5%PS for12h, the mRNAlevels of CAT, HO-1,SOD1,SOD2and GSH-Px1increased3.12±0.03,4.10±0.26,3.74±0.37,4.04±0.22,3.17±0.17fold respectively. Treated with5%DPS for12h,the mRNA levels of CAT, HO-1,SOD1,SOD2and GSH-Px1increased2.63±0.13,3.26±0.21,3.89±0.84,4.34±0.22,4.86±0.71fold respectively. These twogroups were higher than M group(P <0.001). There were not notable differencesbetween NRS and M group (P>0.05).
In HUVEC cells treated with5%PS for12h, the mRNA levels of CAT,HO-1,SOD1,SOD2and GSH-Px1increased3.35±0.03,4.51±0.26,4.15±0.37,3.84±0.22,1.97±0.17fold respectively. Treated with5%DPS for12h, the mRNA levelsof CAT, HO-1,SOD1,SOD2and GSH-Px1increased2.86±0.13,3.67±0.21,4.30±0.84,4.14±0.22,4.66±0.71fold respectively. These two groups were higher than Mgroup(P <0.001, P <0.01). There were not notable differences between NRS and Mgroup (P>0.05).
(11) The protein expression of antioxidant enzymes (CAT, HO-1, SOD-1andSOD-2) was significantly induced.
In H9c2(2-1) cells treated with5%PS for12h, the protein levels of CAT,HO-1,SOD1,SOD2were increased by0.56±0.08,0.46±0.07,0.55±0.09,0.47±0.06fold respectively. Treated with5%DPS for12h, the protein levels of CAT,HO-1,SOD1,SOD2were increased by0.53±0.07,0.44±0.01,0.61±0.08,0.52±0.07fold respectively. These were higher than M group (P <0.05).
In HUVEC cells treated with5%PS for12h, the protein levels of CAT, HO-1,SOD1, SOD2were0.63±0.10,0.58±0.10,0.41±0.11,0.54±0.09fold respectively.Treated with5%DPS for12h, the protein levels of CAT, HO-1, SOD1,SOD2were0.62±0.07,0.60±0.04,0.54±0.09,0.51±0.02fold respectively. These were higherthan M group (P <0.05).
(12)In addition, we treated cells with specific inhibitor of PI3K (LY294002) andMEK (U0126), called PS+LY, PS+U, DPS+LY, DPS+U group.
To H9c2(2-1) cells, the absorbance of PS, PS+LY,PS+U (1.09±0.1,0.95±0.15,and1.12±0.19)were no obvious difference (P>0.05).No differences were observedamong DPS, DPS+LY, DPS+U groups (P>0.05).
To HUVEC cells, the absorbance of PS, PS+LY,PS+U (1.58±0.08,1.51±0.06，and1.45±0.12)were no obvious difference(P>0.05).No differences were observedamong DPS, DPS+LY, DPS+U groups (P>0.05).
Conclusions
Incubation with the media containing5%PS and DPS for12h, decreased theinjury induced by H_2O_2, promoted the nucleus translocation of Nrf2and enhanced themRNA and protein expression of antioxidase, improved the activity of antioxidaseHowerever the PI3K/AKT and MEK/ERK1/2pathway were not involved in thisprotection.
细胞内ROS（reactive oxygen species）的产生处于动态平衡状态。缺血再灌注时细胞内产生大量的ROS，造成氧化应激损伤，即缺血再灌注损伤。轻度短暂的组织缺血再灌注可以减轻随后发生的严重的组织缺血再灌注损伤，这种现象称为缺血预适应（Ischemic preconditioning，IPC)。研究发现，创伤小，操作方便的肢体缺血预适应（Limb ischemic preconditioning, LIPC），可以减轻多种动物多个器官的缺血再灌注损伤。血液中ROS增加,易导致内皮损伤,加重缺血再灌注损伤。LIPC可以调节正常内皮功能，对内皮ROS损伤是否有作用目前尚无详细报道。
CAT，SOD，GSH-Px等是细胞内重要的清除ROS的抗氧化酶。细胞内的抗氧化酶受到转录因子Nrf2(NF-E2-related factor2)的调控。Nrf2通过与抗氧化反应元件(Antioxiant response element, ARE)结合，上调与ARE相关的抗氧化基因表达，包括细胞内源性抗氧化酶、Ⅱ相解毒酶等，清除细胞内过多地ROS，维持细胞内氧化还原水平，减轻氧化应激所造成的细胞损伤。受Nrf2调控的蛋白持续表达增加对细胞氧化还原内环境的稳定起到非常关键的作用。
研究目的
本课题主要观察LIPC大鼠的血清是否可以减轻H_2O_2所导致的人脐静脉内皮细胞HUVEC和鼠胚胎心肌细胞H9c2（2-1）的氧化应激损伤，分析Nrf2所调控的抗氧化基因是否参与了该过程，以及有可能所涉及的信号通路。
材料和方法
（1）成年健康SD大鼠，用改良的大鼠尾动脉血压测定仪套管套在大鼠右后肢，加压至200mmHg阻断股动脉造成右后肢短暂缺血，持续5分钟，放气减压恢复血流，持续10分钟，共3个循环。预适应结束后立即腹主动脉无菌采血获得大鼠早期肢体预适应血清。预适应后间隔24小时采血，获得大鼠延迟肢体预适应血清，分装冻存于-80℃。
（2）建立HUVEC和H9c2（2-1）细胞的H_2O_2损伤模型，参考IC50作为后续实验造模浓度。HUVEC和H9c2（2-1）细胞分为5组，对照组（control，C），模型组（model，M），大鼠早期预适应血清组（preconditioning serum, PS），大鼠延迟预适应血清组(delayed preconditioning serum, DPS)，正常大鼠血清组（normal rat serum, NRS），预先用2.5%,5%（V/V）各种血清孵育6小时和12小时，然后用H_2O_2处理2小时，用MTT法检测各种血清预处理对HUVEC和H9c2（2-1）细胞活性的影响。
（3）应用AnnexinV-FITC和PI双染进一步检测细胞的凋亡情况。
（4）细胞处理完成后，用8μM的DHE孵育45分钟，荧光显微镜下检测ROS的含量。
（5）应用LDH，MDA，CK检测试剂盒检测细胞培养上清液中LDH，CK的活性及MDA的浓度。
（6）采用SOD，CAT，GSH-Px检测试剂盒检测细胞培养上清液中SOD，CAT，GSH-Px的活性。
（7）采用免疫荧光法和激光共聚焦显微镜检测各组细胞Nrf2的核转位情况。
（8）采用real-time RT-PCR检测各组细胞中抗氧化基因CAT，HO-1，SOD1，SOD2，GSH-Px1mRNA的水平。
（9）采用Western Blot法检测各组细胞中CAT，HO-1，SOD1，SOD2的蛋白表达情况。
（10）用25μM PI3K的抑制剂LY294002和26μM MEK1/2抑制剂U0126提前1小时处理细胞，观察这两种抑制剂对PS和DPS作用的影响。
（11）各实验组数据采用mean±SD表示，利用SPSS13.0统计软件分析，组间差异比较采用单因素方差分析。P <0.05认为有显著差异。
结果
（1）不同浓度的H_2O_2（100-1200μM）作用2小时后，随H_2O_2浓度增高HUVEC和H9c2（2-1）细胞存活逐渐降低，IC50分别为1099.56±109.11μM和380.98±57.44μM，参考IC50选取1000μM和400μM作为造模浓度。
（2）对于H9c2（2-1）细胞和HUVEC细胞，2.5%PS，DPS，NRS预孵育6小时和12小时，400μM和1000μM H_2O_2处理2小时，MTT570nm处吸光度测定结果显示，M组比C组显著降低（P <0.001），PS，DPS，NRS组的吸光度与M组相比无显著差异（P>0.05）。
（3）5%的各血清预孵育6小时，400μM和1000μM H_2O_2处理2小时，H9c2（2-1）细胞和HUVEC细胞MTT570nm处吸光度测定结果显示，M组比C组显著降低（P <0.001）。PS，DPS，NRS组的吸光度与M组相比无显著差异(P>0.05)。
（4）5%的各血清预孵育12小时，H_2O_2处理2小时，MTT570nm处吸光度测定结果显示，H9c2（2-1）M组吸光度显著低于C组（0.51±0.03vs1.20±0.09，P <0.001），PS组和DPS组吸光度分别为1.14±0.17和1.08±0.23，显著高于M组（P<0.001），PS组和DPS组相比无显著差异(P>0.05)。NRS组吸光度0.53±0.04，与M组相比无显著性差异(P>0.05)。
HUVEC细胞M组吸光度显著低于C组（0.78±0.03vs1.26±0.06，P <0.001），PS组和DPS组吸光度分别为1.09±0.04和1.08±0.04，显著高于比M组（P <0.001），PS组和DPS组相比无显著差异(P>0.05)。NRS组吸光度0.75±0.03，与M组相比无显著性差异(P>0.05)。
（5）5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞PS组、DPS组和M组的早期凋亡率分别为2.74±1.28%、5.22±2.09%和12.28±3.24%（P <0.001，P <0.05）；PS组、DPS组和M组晚期凋亡率分别为6.87±0.88%，8.90±0.65%和36.04±6.22%（P <0.001）；PS组、DPS组和M组总死亡率为9.61±0.93%，14.12±1.43%，48.31±3.33%（P <0.001）。PS组和DPS组相比无显著差异(P>0.05)。NRS组的早期凋亡，晚期凋亡和总死亡率与M组比较无显著差异(P>0.05)。
HUVEC细胞PS组、DPS组的早期凋亡率分别为1.12±0.20%、1.78±0.20%与M组13.24±1.37%相比，显著降低（P<0.01）；PS组和DPS组晚期凋亡率7.54±1.49%和6.87±1.48%，与M组晚期凋亡率25.52±0.86%相比，显著降低（P <0.001，P <0.01）；PS组和DPS组总死亡率为8.65±1.67%和9.65±1.44%，与M组总死亡率38.76±2.23%相比，显著降低（P <0.001）。PS组和DPS组相比无显著差异(P>0.05)。NRS组的早期凋亡率，晚期凋亡率和总死亡率与M组比无显著差异(P>0.05)。
（6）5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞M组荧光亮度与C组比显著升高（96.34±21.23vs23.67±6.5，P <0.01）。PS组和DPS组荧光亮度29.87±5.67和30.67±7.34比M组显著降低（P <0.01）。PS组和DPS组相比无显著差异(P>0.05)。NRS组荧光亮度为88.67±19.21，与M组相比无显著性差异(P>0.05)。
HUVEC细胞M组荧光亮度76.34±19.23比C组20.16±4.5显著升高（P <0.01）。PS组和DPS组荧光亮度22.87±6.78和24.46±8.12比M组显著降低（P<0.01）。PS组和DPS组相比无显著差异(P>0.05)。NRS组荧光亮度为71.34±16.19，与M组比无显著性差异(P>0.05)。
（7）5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞M组MDA浓度从4.42±0.28nmol/ml升高至8.74±0.33nmol/ml（P <0.001）。PS组和DPS组MDA浓度则降至3.48±0.46nmol/ml和3.55±0.68nmol/m（lP <0.001），PS组和DPS组相比无显著差异(P>0.05)。而NRS组的MDA为8.54±1.08nmol/ml，与M组比无显著差异（P>0.05）。
HUVEC细胞M组MDA浓度从4.86±0.28nmol/ml升高至6.67±0.29nmol/ml（P <0.001）。而PS组和DPS组MDA浓度则降至3.44±0.27nmol/ml，3.80±0.42nmol/ml（P<0.001），PS组和DPS组相比无显著差异(P>0.05)。NRS组的MDA浓度为6.25±0.59nmol/ml，与M组比无显著差异（P>0.05）。
5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞的LDH浓度从72.69±6.75U/L升高至191.93±36.52U/L（P <0.001）；PS组和DPS组LDH则降至77.81±13.58U/L，105.75±8.67U/L（P <0.001），PS组和DPS组相比无显著差异(P>0.05)。而NRS组的LDH为173.86±36.17U/L，与M组比无显著差异（P>0.05）。
HUVEC细胞的LDH含量12.58±2.74U/L升高至58.16±5.05U/L（P <0.001）。而PS组和DPS组LDH则降至16.51±4.63U/L和15.54±2.70U/L（P <0.001）。PS组和DPS组相比无显著差异(P>0.05)。而NRS组的LDH为62.13±11.95U/L，与M组比无显著差异（P>0.05）。
400μM H_2O_2处理H9c2（2-1）细胞2小时，细胞培养液中的CK显著升高（15.67±0.91U/L vs30.47±1.76U/L，P <0.001），PS和DPS预先处理12小时，则细胞培养液中的CK降低至20.08±1.26U/L和22.00±1.52U/L（P <0.001）。PS组和DPS组相比无显著差异(P>0.05)。NRS预先处理12小时，则细胞培养液中的CK为29.47±2.26U/L，与M组30.47±1.76U/L相比，无显著差异（P>0.05）。
（8）5%的各血清预孵育12小时，H_2O_2处理2小时，H9c2(2-1)细胞CAT的活性PS组和DPS组为45.33±3.82U/ml和44.61±5.79U/ml比M组17.30±1.62U/ml显著增高(P <0.01，P <0.05），PS组和DPS组相比无显著差异(P>0.05)。NRS组为17.92±2.17U/ml，与M组比无显著差异（P>0.05）。HUVEC细胞CAT的活性PS组和DPS组为51.25±4.82U/ml和50.5±5.89U/ml比M组21.69±2.02U/ml高(P <0.01,P <0.05），PS组和DPS组相比无显著差异(P>0.05)。NRS组为25.81±4.40U/ml，与M组比无显著差异（P>0.05）。
H9c2(2-1)细胞SOD的活性PS组和DPS组为14.02±1.68U/ml和14.11±0.58U/ml，比M组6.89±0.97U/ml显著增高(P <0.001），PS组和DPS组相比无显著差异(P>0.05)。NRS组为7.56±1.24U/ml，与M组比无显著差异（P>0.05）。HUVEC细胞CAT的活性PS组和DPS组为13.82±0.50U/ml，11.29±3.58U/ml比M组6.43±0.78U/ml显著增高(P <0.001，P <0.05），PS组和DPS组相比无显著差异(P>0.05)。NRS组为7.74±2.02U/ml，与M组比无显著差异（P>0.05）。
H9c2(2-1)细胞GSH-Px的活性PS组和DPS组为43.27±8.56U/ml，37.62±6.00U/ml，比M组15.06±7.09U/ml显著增高(P <0.01），PS组和DPS组相比无显著差异(P>0.05)。NRS组为21.89±3.97U/ml，与M组比无显著差异（P>0.05）。HUVEC细胞GSH-Px的活性PS组和DPS组为52.38±15.75U/ml，41.84±1.66U/ml比M组25.08±8.50U/ml显著增高(P <0.001，P <0.05），PS组和DPS组相比无显著差异(P>0.05)。NRS组为29.34±8.56U/ml，与M组比无显著差异（P>0.05）。
（9）H9c2(2-1)细胞和HUVEC各组细胞Nrf2的分布情况，C组细胞内的绿色荧光（anti–Nrf2-FITC）主要在核周围，呈环状。NRS组和M组只有少量细胞中的Nrf2从细胞浆进入细胞核。而PS组和DPS组中，大量细胞中的Nrf2从细胞浆进入细胞核，PS组和DPS组Nrf2的核转位增多。
（10）H9c2(2-1)细胞M，NRS，PS，DPS组的CAT，HO-1，SOD1，SOD2，GSH-Px1的mRNA水平都比C组增高，但NRS组与M组比无显著差异（P>0.05）。CAT的mRNA水平PS组3.12±0.03和DPS组2.63±0.13比M组1.23±0.04显著增高(P <0.001)。HO-1的mRNA水平PS组4.10±0.26和DPS组3.26±0.21比M组1.30±0.02显著增高(P <0.001)。SOD1的mRNA水平PS组3.74±0.37和DPS组3.89±0.84比M组1.58±0.07显著增高(P <0.001)。SOD2的mRNA水平PS组4.04±0.22和DPS4.34±0.22组比M组1.52±0.11显著增高(P <0.001)。GSH-Px1的mRNA水平PS组3.17±0.17和DPS组4.86±0.71比M组1.58±0.12显著增高(P <0.001)。PS组和DPS组相比无显著差异(P>0.05)。
HUVEC细胞M，NRS，PS，DPS组的CAT，HO-1，SOD1，SOD2，GSH-Px1的mRNA水平都比C组增高，但NRS组与M组比无显著差异（P>0.05）。CAT的mRNA水平PS组3.35±0.03和DPS组2.86±0.13比M组1.46±0.04显著增高(P <0.05)。HO-1的mRNA水平PS组4.51±0.26和DPS组3.67±0.21比M组1.71±0.02显著增高(P <0.001)。SOD1的mRNA水平PS组4.15±0.37和DPS组4.30±0.84比M组1.99±0.07显著增高(P <0.001)。SOD2的mRNA水平PS组3.84±0.22和DPS组4.14±0.22比M组1.32±0.11显著增高(P <0.001)。GSH-Px1的mRNA水平PS组1.97±0.17和DPS组4.66±0.71比M组1.38±0.12显著增高(P <0.05，P<0.001)。PS组和DPS组相比无显著差异(P>0.05)。
Western Blot检测结果，H9c2(2-1)细胞，PS组和DPS组细胞内的抗氧化酶CAT（0.56±0.08,0.53±0.07），HO-1（0.46±0.07,0.44±0.01），SOD1（0.55±0.09,0.61±0.08），SOD（20.47±0.06,0.52±0.07），表达显著增高(M组CAT0.32±0.09，HO-10.29±0.03, SOD10.35±0.05,SOD20.27±0.05，P <0.05)，PS组和DPS组相比无显著差异(P>0.05)。而NRS组抗氧化酶的表达与M相比无显著差异（P>0.05）。
Western Blot检测结果，HUVEC细胞，PS组和DPS组细胞内的抗氧化酶CAT（0.63±0.10,0.62±0.07），HO-1（0.58±0.10,0.60±0.04），SOD1（0.41±0.11,0.54±0.09），SOD2（0.54±0.09,0.51±0.02）表达增高(M组CAT0.33±0.09，HO-10.31±0.05, SOD10.27±0.1,SOD20.28±0.05, P <0.05)，PS组和DPS组相比无显著差异(P>0.05)。NRS组抗氧化酶的表达与M相比无显著差异（P>0.05）。
（11）H9c2(2-1)细胞PS组的吸光度为1.09±0.1，而PS+LY294002组吸光度为0.95±0.15，PS+U0126组吸光度为1.12±0.19，与PS组比无显著差异（P>0.05）。DPS组的吸光度为1.02±0.15，而DPS+LY294002组吸光度为0.99±0.11，DPS+U0126组吸光度为0.95±0.14，与DPS组比无显著差异（P>0.05）。
HUVEC细胞PS组的吸光度为1.58±0.08，而PS+LY294002组吸光度为1.51±0.06，PS+U0126组吸光度为1.45±0.12，与PS比无显著差异（P>0.05）。DPS组的吸光度为1.51±0.06，而DPS+LY294002组吸光度为1.52±0.06，DPS+U0126组吸光度为1.48±0.02，与DPS组比无显著差异（P>0.05）。
LY294002和U0126不影响PS和DPS对H9c2(2-1)细胞和HUVEC细胞H_2O_2损伤的保护作用，不影响Nrf2的核转位。
结论
肢体缺血预适应大鼠早期相和晚期相血清，5%孵育12小时，能够减轻H_2O_2所造成的H9c2(2-1)细胞和HUVEC细胞损伤，增加Nrf2的核转位，从而提高CAT，HO-1，SOD1，SOD2，GSH-Px1的mRNA水平，CAT，HO-1，SOD1，SOD2蛋白表达增加，抗氧化酶CAT，SOD，GSH-Px的活性增高。PI3K/AKT通路和MEK/ERK1/2通路可能不参与其中。
Background
ROS are chemically reactive molecules containing oxygen, including oxygen freeradicals, hydrogen peroxide, lipid peroxide, superoxide anion and etc. In normal cells,reactive oxidants are produced in a controlled manner and some serve useful purposes.Uncontrolled production of ROS results in oxidative stress that impairs cellularfunctions and contributes to the development of ischemia-reperfusion injury. Ischemicpreconditioning is a potent approach to reduce ischemic reperfusion injury. Limbischemic preconditioning is a noninvasive feasibility treatment to protect ischemicreperfusion injury of multiple organs in a variety of animal models, but themechanism is not clear.
CAT, SOD, GSH-Px are important antioxidase to remvove ROS in cell.TheKeap1–Nrf2(NF-E2-related factor2) system is one of the major cellular defensemechanisms against oxidative and/or electrophilic stresses.Transcription factor Nrf2can recognize the antioxidant response element/electrophile responsive element(ARE/EpRE) in the promoter region. It can also regulate the basal and inducibleexpression of numerous antioxidant and detoxifying genes. The elevation expressionof proteins regulated by Nrf2plays a crucial role in the homeostasis of the internalenvironment.
Objective
This study was to observe whether the serum from rats undergoing early limbpreconditioning and delayed limb preconditioning could protect HUVEC andH9c2(2-1) from oxidant stress induced by H2O2. Furtherly, the involvement ofantioxidant gene regulated by Nrf2and signal pathway were explored.
Materials and methods
(1)Male Sprague-Dawley rats were used for the study. A modified noninvasiveblood pressure radiometer cuff was placed around right hindlimb. A pulse sensor wasplaced on the arteria dorsalis pedis. The cuff was inflated to200mmHg for5minutesin order to obtain ischemia and asphygmia also indicated that the arteria femoraliswas blocked. The deflation of the cuff could reperfuse the arteria femoralis for10minand the sphygmus appeared again. The procedure was operated for three cycles.
(2)Firstly, oxidant stress models of HUVEC and H9c2(2-1) induced by H_2O_2were set up, and H_2O_2concentration used in the following experiment was refered tothe IC50. The HUVEC and H9c2(2-1) cells were divided into5groups: controlgroup(C), model group(M), normal rat serum group(NRS)，preconditioning serumgroup(PS), and delayed preconditioning serum group(DPS).The NRS, PS, DPS,groups were incubated with2.5%and5%(V/V) serum for6h and12h respectively,then the cell viability was detected by MTT method.
(3) After treatment, the apoptosis was detected by AnnexinV-FITC and PIdouble staining.
(4) ROS was quantified by fluorescent microscopy incubated with8μM DHEfor45min.
(5) The concentrations of MDA, LDH as well as CK were all determined byusing commercially available kits.
(6) The activity of CAT, SOD and GSH-Px was determined by usingcommercially available kits.
(7) Immunofluorescence staining was used to observe the nucleus translocationof Nrf2by the confocal microscopy.
(8) The mRNA level of CAT, HO-1, SOD1, SOD2, and GSH-Px1wasdetermined by real-time semiquantitative reverse transcription-polymerase chainreaction method.
(9) The protein level of CAT, HO-1, SOD1, and SOD2was detected by westernblot.
(10)The effects of LY294002and U0126on PS and DPS groups were detected aswell.
(11) All data are presented as mean±standard deviation (S.D) and analyzed by SPSS13.0. Differences between mean values of multiple groups were analyzed byone-way analysis of variance. Values of P <0.05were considered to be statisticallysignificant.
Results
(1) Incubation of HUVEC and H9c2(2-1) cells with different concentrations ofH_2O_2(100–1200μM)for2h could decrease the viability of cells in a dose-dependentmanner. The IC50was1099.56±109.11μM and380.98±57.44μM,1000μM and400μM were used as the sample treatment concentration.
(2)H9c2(2-1) and HUVEC cells were pretreated with2.5%, NRS, PS and DPSfor6h and12h, and thereafter H_2O_2treated for2h. MTT results showed that Mgroup had a lower absorbance compared with C group at570nm (P <0.001). Therewere no significant differences in absorbance among NRS, PS, DPS, and M group (P>0.05).
(3) H9c2(2-1) and HUVEC cells were pretreated with5%PS, DPS and NRS for6h, and thereafter H_2O_2for2h. MTT results showed that M group had a lowerabsorbance compared with C group at570nm (P <0.001).The absorbance of Mgroup was not different from those of PS, DPS or NRS group (P>0.05).
(4) For H9c2(2-1) and HUVEC cells, pretreatment with5%PS,DPS and NRSfor12h, and H_2O_2treated2h, M group was lower than C group in MTT570nmabsorbance(P <0.001).
To H9c2(2-1),the absorbance of PS group1.14±0.17and DPS group1.08±0.23was higher than M group0.51±0.03(P <0.001). There were no difference betweenPS and DPS group(P>0.05).But the absorbance of NRS group was no significantdifferences compared with M group (P>0.05).
To HUVEC, the absorbance of PS group1.09±0.04and DPS group1.08±0.04was higher than M group0.78±0.03(P <0.001). Furthermore, there were nodifference between PS and DPS group(P>0.05).But the absorbance of NRS groupwas no significant differences compared with M group (P>0.05).
(5) For H9c2(2-1), pretreatment with5%PS,DPS and NRS for12h, andthereafter H_2O_2treatment for2h, the early apoptosis of PS group and DPS group were2.74±1.28%and5.22±2.09%, which were lower than M group(12.28±3.24%,P <0.001). There were no significant differences between NRS and M group (P>0.05). The late apoptosis of PS group and DPS group were6.87±0.88%and8.90±0.65%, which were lower than M group (36.04±6.22%,P <0.001) There were nosignificant difference between NRS, and M group (P>0.05).The total apoptosis ofPS group and DPS group were9.61±0.93%and14.12±1.43%, which were lowerthan M group(48.31±3.33%,P <0.001). There were no significant differencesbetween NRS and M group (P>0.05).
For HUVEC, pretreatment with5%PS, DPS and NRS for12h followed by H_2O_2treated for2h, the early apoptosis of PS group and DPS group were1.12±0.20%and1.78±0.20%, which were lower than M group (13.24±1.37%,P <0.001). Thelate apoptosis of PS group and DPS group were7.54±1.49%and6.87±1.48%,which were lower than M group (25.52±0.86%,P <0.001,P <0.01). The totalapoptosis of PS group and DPS group were8.65±1.67%and9.65±1.44%, whichwere lower than M group (38.76±2.23%,P <0.001). There were no significantdifferences between NRS and M group (P>0.05).
(6) As expected, the DHE fluorescence in H9c2(2-1) and HUVEC exposed toH_2O_2was strikingly increased compared with control group (P <0.01). Pretreatmentwith5%PS and DPS for12h significantly inhibited DHE fluorescence caused byH_2O_2. Pretreatment with NRS could not restor the DHE fluorescence (P>0.05).
(7) Compared with control group, treatment of H9c2(2-1) cells with400μMH_2O_2for2h caused significantly increase of MDA from4.42±0.28nmol/ml to8.74±0.33nmol/ml (P <0.001).Incubated with5%PS and DPS for12h attenuated MDA(P <0.001).But Incubated with NRS for12h did not affect the level of MDA (P>0.05)
Similarly,treatment of HUVEC with1000μM H_2O_2for2h caused significantlyincrease of MDA from4.86±0.28nmol/ml to6.67±0.29nmol/ml (P <0.001)Incubated with5%PS and DPS for12h attenuated MDA level (P <0.001).Howerverincubated with NRS for12h did not affect the level of MDA (P>0.05).
In addition, treated H9c2（2-1）cells with400μM H_2O_2for2h causedsignificantly increase in LDH level from72.69±6.75nmol/ml to191.93±36.52 nmol/ml (P <0.001). Preincubation with5%PS and DPS for12h markedlydecreased the elevation compared with the M group(P <0.001).However incubatedwith5%NRS for12h did not affect the level of LDH (P>0.05). The phenomenon inHUVEC was similar to H9c2(2-1) cells.
For H9c2(2-1) cells, the CK of PS and DPS group was lower than M group(20.08±1.26U/L and22.00±1.52U/L vs30.47±1.76U/L, P <0.001). NRS groupwas almost equal to M group respectively (P>0.05).
(8) Pretreated H9c2（2-1）cells with5%PS and DPS for12h, the activity of CAT(45.33±3.82U/ml，44.61±5.79U/ml) were higher than that of M group (17.30±1.62U/ml)(P <0.01,P <0.05). The activity of CAT in HUVEC were also raised bytreatment with5%PS and DPS for12h (51.25±4.82U/ml，50.5±5.89U/ml vs21.69±2.02U/ml, P <0.01,P <0.05）There were no notable differences between NRS,and M group (P>0.05).
Pretreated H9c2(2-1) cells with5%PS and DPS for12h, the activity ofSOD(14.02±1.68U/ml,14.11±0.58U/ml) were higher than that of M group (6.89±0.97U/ml,P <0.001). The activity of SOD in HUVEC were also elevated by treatedwith5%PS and DPS for12h(13.82±0.50U/ml，11.29±3.58U/ml vs6.43±0.78U/ml, P <0.01, P <0.05）There were no notable difference between NRS and Mgroup (P>0.05).
Pretreated H9c2(2-1) cells with5%PS and DPS for12h, the activity of GSH-Px(43.27±8.56U/ml，37.62±6.00U/ml) were higher than that of M group (15.07±7.09U/ml)(P <0.01). The activity of GSH-Px of HUVEC were also elvated bytreatment with5%PS and DPS for12h (52.38±5.75U/ml，41.84±1.66U/ml vs25.08±8.50U/ml, P <0.01,P <0.05）There were no notable difference between NRSand M group (P>0.05).
The activity change trends of SOD and GSH-Px were in line with the activity ofCAT among these groups.
(9) Confocal imaging using FITC-conjugated secondary antibody stainingindicated that under normal conditions, Nrf2protein mainly distributed in cytoplasmaround the nucleus, which looked like a ring. In the NRS and M group, only a smallnumber of cells translocated Nrf2into the nucleuses. The number of cells in which Nrf2mainly distributed in nucleus extraordinarily increased in PS and DPS group.
(10)Effects of PS and DPS on mRNA leves of the antioxidant gene CAT, HO-1,SOD1, SOD2, and GSH-Px1were evaluated using real time RT-PCR, with GAPDHas an internal control. In H9c2(2-1) cells treated with5%PS for12h, the mRNAlevels of CAT, HO-1,SOD1,SOD2and GSH-Px1increased3.12±0.03,4.10±0.26,3.74±0.37,4.04±0.22,3.17±0.17fold respectively. Treated with5%DPS for12h,the mRNA levels of CAT, HO-1,SOD1,SOD2and GSH-Px1increased2.63±0.13,3.26±0.21,3.89±0.84,4.34±0.22,4.86±0.71fold respectively. These twogroups were higher than M group(P <0.001). There were not notable differencesbetween NRS and M group (P>0.05).
In HUVEC cells treated with5%PS for12h, the mRNA levels of CAT,HO-1,SOD1,SOD2and GSH-Px1increased3.35±0.03,4.51±0.26,4.15±0.37,3.84±0.22,1.97±0.17fold respectively. Treated with5%DPS for12h, the mRNA levelsof CAT, HO-1,SOD1,SOD2and GSH-Px1increased2.86±0.13,3.67±0.21,4.30±0.84,4.14±0.22,4.66±0.71fold respectively. These two groups were higher than Mgroup(P <0.001, P <0.01). There were not notable differences between NRS and Mgroup (P>0.05).
(11) The protein expression of antioxidant enzymes (CAT, HO-1, SOD-1andSOD-2) was significantly induced.
In H9c2(2-1) cells treated with5%PS for12h, the protein levels of CAT,HO-1,SOD1,SOD2were increased by0.56±0.08,0.46±0.07,0.55±0.09,0.47±0.06fold respectively. Treated with5%DPS for12h, the protein levels of CAT,HO-1,SOD1,SOD2were increased by0.53±0.07,0.44±0.01,0.61±0.08,0.52±0.07fold respectively. These were higher than M group (P <0.05).
In HUVEC cells treated with5%PS for12h, the protein levels of CAT, HO-1,SOD1, SOD2were0.63±0.10,0.58±0.10,0.41±0.11,0.54±0.09fold respectively.Treated with5%DPS for12h, the protein levels of CAT, HO-1, SOD1,SOD2were0.62±0.07,0.60±0.04,0.54±0.09,0.51±0.02fold respectively. These were higherthan M group (P <0.05).
(12)In addition, we treated cells with specific inhibitor of PI3K (LY294002) andMEK (U0126), called PS+LY, PS+U, DPS+LY, DPS+U group.
To H9c2(2-1) cells, the absorbance of PS, PS+LY,PS+U (1.09±0.1,0.95±0.15,and1.12±0.19)were no obvious difference (P>0.05).No differences were observedamong DPS, DPS+LY, DPS+U groups (P>0.05).
To HUVEC cells, the absorbance of PS, PS+LY,PS+U (1.58±0.08,1.51±0.06，and1.45±0.12)were no obvious difference(P>0.05).No differences were observedamong DPS, DPS+LY, DPS+U groups (P>0.05).
Conclusions
Incubation with the media containing5%PS and DPS for12h, decreased theinjury induced by H_2O_2, promoted the nucleus translocation of Nrf2and enhanced themRNA and protein expression of antioxidase, improved the activity of antioxidaseHowerever the PI3K/AKT and MEK/ERK1/2pathway were not involved in thisprotection.
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