Protection Of Serum From Rats Undergoing Limb Ischemic Preconditioning On HUVEC And H9c2（2-1） Cells From H₂O₂-induced Damage

BackgroundROS 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.ObjectiveThis 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 H₂O₂were set up, and H₂O₂concentration 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 ofH₂O₂（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 H₂O₂treated 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 H₂O₂for2h. 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 H₂O₂treated2h, 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 H₂O₂treatment 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 H₂O₂treated 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 toH₂O₂was strikingly increased compared with control group （P <0.01）. Pretreatmentwith5%PS and DPS for12h significantly inhibited DHE fluorescence caused byH₂O₂. Pretreatment with NRS could not restor the DHE fluorescence （P>0.05）.（7） Compared with control group, treatment of H9c2（2-1） cells with400μMH₂O₂for2h 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 H₂O₂for2h 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 H₂O₂for2h 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）.ConclusionsIncubation with the media containing5%PS and DPS for12h, decreased theinjury induced by H₂O₂, 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.