调控对氧磷酶1基因对急性敌敌畏中毒小鼠肝氧化损伤的保护作用
조공대양린매1기인대급성활활외중독소서간양화손상적보호작용
The protective effect of regulation of paraoxonase 1 gene on liver oxidative stress injury induced by dichlorvos poisoning in mice
  • 万方数据
    中华危重病急救医学 중화위중병급구의학
    Chinese Critical Care Medicine
    2015年 4期 285-290 ,共6页

    郭瑞娟%陈隆望%连洁%吴斌%洪广亮%葛赟%刘瑶%邱俏檬%卢中秋

    곽서연%진륭망%련길%오빈%홍엄량%갈빈%류요%구초몽%로중추

    对氧磷酶%中毒%敌敌畏%肝损伤%氧化应激 對氧燐酶%中毒%敵敵畏%肝損傷%氧化應激
    대양린매%중독%활활외%간손상%양화응격
    Paraoxonase%Poisoning%Dichlorvos%Liver injury%Oxidative stress
    目的：探讨调控对氧磷酶1（PON1）基因对急性敌敌畏中毒小鼠肝组织氧化损伤的保护作用。方法实验1：将12只雄性Balb/c小鼠按随机数字表法分为对照组、对照绿色荧光蛋白慢病毒（Lv-GFP）组、重组PON1慢病毒（Lv-PON1）组3组，每组4只。经尾静脉分别注射转染2×107 TU的Lv-GFP或Lv-PON1慢病毒，对照组给予等量生理盐水。分别于0、1、3、5、7、9 d取眼底静脉血，检测血清PON1活性；转染3 d取肝组织，分别用反转录-聚合酶链反应（RT-PCR）和蛋白免疫印迹试验（Western Blot）检测PON1 mRNA和蛋白表达。实验2：另将96只雄性Balb/c小鼠按随机数字表法分为对照组、敌敌畏染毒组、Lv-GFP干预组、Lv-PON1干预组4组，每组24只。经尾静脉分别注射转染Lv-GFP或Lv-PON1慢病毒后3 d，腹腔注射敌敌畏溶液9 mg/kg染毒，对照组给予等量生理盐水。各组分别于染毒后6、12、24、48 h麻醉处死6只小鼠取肝组织，RT-PCR检测PON1 mRNA、核转录因子E2相关因子2（Nrf2） mRNA表达；Western Blot检测PON1蛋白表达；化学比色法检测丙二醛（MDA）、谷胱甘肽（GSH）含量；双抗夹心酶联免疫吸附试验（ELISA）检测超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性。结果实验1：Lv-PON1转染正常小鼠后，血清PON1活性逐渐升高，自3 d起维持在较高水平；而对照组和Lv-GFP组血清PON1活性均处于正常低水平状态。转染3 d肝组织PON1 mRNA和蛋白表达均显著高于对照组和Lv-GFP组。实验2：与对照组相比，敌敌畏染毒组染毒后6 h肝组织PON1 mRNA和蛋白表达、 Nrf2 mRNA表达、GSH、SOD、CAT水平即明显降低〔PON1 mRNA（灰度值）：0.237±0.075比0.674±0.011，PON1蛋白（灰度值）：0.602±0.086比0.998±0.124，Nrf2 mRNA（灰度值）：0.089±0.012比0.126±0.010，GSH（mg/g）：3.84±0.33比5.52±0.40，SOD（μg/g）：0.383±0.040比0.564±0.052， CAT（ng/g）：7.32±1.28比12.46±1.54，均P＜0.05〕，MDA含量明显升高（nmol/g：7.78±0.41比2.34±0.25， P＜0.05）；随时间延长，PON1 mRNA和蛋白表达、Nrf2 mRNA表达、GSH、SOD、CAT水平逐渐升高，MDA含量逐渐降低，至24 h时Nrf2 mRNA表达已升至对照组水平（0.133±0.019比0.126±0.009，P＞0.05），48 h时已明显高于对照组（0.206±0.028比0.124±0.010，P＜0.05）。与敌敌畏染毒组比较，Lv-PON1干预组6 h时肝组织PON1 mRNA和蛋白表达、 Nrf2 mRNA表达、 GSH、 SOD、 CAT水平即明显升高〔PON1 mRNA（灰度值）：0.726±0.021比0.237±0.075， PON1蛋白（灰度值）：0.739±0.050比0.602±0.086，Nrf2 mRNA（灰度值）：0.158±0.007比0.089±0.012，GSH（mg/g）：4.30±0.22比3.84±0.33，SOD（μg/g）：0.454±0.062比0.383±0.040， CAT（ng/g）：8.98±1.02比7.32±1.28，均P＜0.05〕，MDA含量即明显下降（nmol/g：6.56±0.44比7.78±0.41，P＜0.05）。结论调控PON1能够降低敌敌畏中毒小鼠肝组织MDA含量，增强SOD、 CAT活性，升高GSH含量，并可能通过启动Nrf2抗氧化程序增强抗氧化能力，对急性敌敌畏中毒小鼠肝损伤发挥保护效应。
    목적：탐토조공대양린매1（PON1）기인대급성활활외중독소서간조직양화손상적보호작용。방법실험1：장12지웅성Balb/c소서안수궤수자표법분위대조조、대조록색형광단백만병독（Lv-GFP）조、중조PON1만병독（Lv-PON1）조3조，매조4지。경미정맥분별주사전염2×107 TU적Lv-GFP혹Lv-PON1만병독，대조조급여등량생리염수。분별우0、1、3、5、7、9 d취안저정맥혈，검측혈청PON1활성；전염3 d취간조직，분별용반전록-취합매련반응（RT-PCR）화단백면역인적시험（Western Blot）검측PON1 mRNA화단백표체。실험2：령장96지웅성Balb/c소서안수궤수자표법분위대조조、활활외염독조、Lv-GFP간예조、Lv-PON1간예조4조，매조24지。경미정맥분별주사전염Lv-GFP혹Lv-PON1만병독후3 d，복강주사활활외용액9 mg/kg염독，대조조급여등량생리염수。각조분별우염독후6、12、24、48 h마취처사6지소서취간조직，RT-PCR검측PON1 mRNA、핵전록인자E2상관인자2（Nrf2） mRNA표체；Western Blot검측PON1단백표체；화학비색법검측병이철（MDA）、곡광감태（GSH）함량；쌍항협심매련면역흡부시험（ELISA）검측초양화물기화매（SOD）、과양화경매（CAT）활성。결과실험1：Lv-PON1전염정상소서후，혈청PON1활성축점승고，자3 d기유지재교고수평；이대조조화Lv-GFP조혈청PON1활성균처우정상저수평상태。전염3 d간조직PON1 mRNA화단백표체균현저고우대조조화Lv-GFP조。실험2：여대조조상비，활활외염독조염독후6 h간조직PON1 mRNA화단백표체、 Nrf2 mRNA표체、GSH、SOD、CAT수평즉명현강저〔PON1 mRNA（회도치）：0.237±0.075비0.674±0.011，PON1단백（회도치）：0.602±0.086비0.998±0.124，Nrf2 mRNA（회도치）：0.089±0.012비0.126±0.010，GSH（mg/g）：3.84±0.33비5.52±0.40，SOD（μg/g）：0.383±0.040비0.564±0.052， CAT（ng/g）：7.32±1.28비12.46±1.54，균P＜0.05〕，MDA함량명현승고（nmol/g：7.78±0.41비2.34±0.25， P＜0.05）；수시간연장，PON1 mRNA화단백표체、Nrf2 mRNA표체、GSH、SOD、CAT수평축점승고，MDA함량축점강저，지24 h시Nrf2 mRNA표체이승지대조조수평（0.133±0.019비0.126±0.009，P＞0.05），48 h시이명현고우대조조（0.206±0.028비0.124±0.010，P＜0.05）。여활활외염독조비교，Lv-PON1간예조6 h시간조직PON1 mRNA화단백표체、 Nrf2 mRNA표체、 GSH、 SOD、 CAT수평즉명현승고〔PON1 mRNA（회도치）：0.726±0.021비0.237±0.075， PON1단백（회도치）：0.739±0.050비0.602±0.086，Nrf2 mRNA（회도치）：0.158±0.007비0.089±0.012，GSH（mg/g）：4.30±0.22비3.84±0.33，SOD（μg/g）：0.454±0.062비0.383±0.040， CAT（ng/g）：8.98±1.02비7.32±1.28，균P＜0.05〕，MDA함량즉명현하강（nmol/g：6.56±0.44비7.78±0.41，P＜0.05）。결론조공PON1능구강저활활외중독소서간조직MDA함량，증강SOD、 CAT활성，승고GSH함량，병가능통과계동Nrf2항양화정서증강항양화능력，대급성활활외중독소서간손상발휘보호효응。
    ObjectiveTo investigate the protective effect of paraoxonase 1 (PON1) gene against liver oxidative stress injury in mice due to dichlorvos poisoning.Methods Experiment 1: 12 male Balb/c mice were randomly divided into three groups, with 4 mice in each group: control group, green fluorescent protein lentivirus control&nbsp;group (Lv-GFP group), and recombinant PON1 lentivirus group (Lv-PON1 group). 2×107 TU of Lv-GFP or Lv-PON1 was transfected via tail vein, while normal saline was given to those in control group. Blood was collected on 0, 1, 3, 5, 7, 9 days via fundus venous plexus for the assay of serum PON1 activity. PON1 mRNA and protein expression levels were respectively determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot on the 3rd post-lentivirus transfection day. Experiment 2: according to the random number table method, another 96 male Balb/c mice were divided into four groups of 24 mice in each control group, dichlorvos group, Lv-GFP intervention group, and Lv-PON1 intervention group. Lv-GFP or Lv-PON1 was transfected via tail vein followed by intraperitoneal injection of dichlorvos 9 mg/kg, while those in control group were given normal saline. Six mice in each group were sacrificed respectively at 6, 12, 24, 48 hours, and liver tissue was collected. PON1 mRNA and nuclear factor E2-related factor 2 (Nrf2) mRNA expression levels were determined by RT-PCR, and PON1 protein level was determined by Western Blot. The content of malondialdehyde (MDA) and glutathione (GSH) in the liver tissue were determined by chemical colorimetry. The activity of superoxide dismutase (SOD) and catalase (CAT) were measured by double antibody sandwich enzyme linked immunosorbent assay (ELISA).Results Experiment 1: after Lv-PON1 was transfected to normal mice, PON1 activity in serum gradually increased and maintained a high level on 3rd day, while that of the control group and Lv-GFP group showed a normal low level. On the 3rd post-lentivirus transfection day, PON1 mRNA and PON1 protein expressions in liver were significantly higher than those of control group and Lv-GFP group. Experiment 2: compared with control group, the mice in dichlorvos group showed significant decreases in PON1 mRNA, PON1 protein, Nrf2 mRNA as well as GSH, SOD, CAT levels at 6 hours [PON1 mRNA (gray value):0.237±0.075 vs. 0.674±0.011, PON1 protein (gray value): 0.602±0.086 vs. 0.998±0.124, Nrf2 mRNA (gray value): 0.089±0.012 vs. 0.126±0.010, GSH (mg/g): 3.84±0.33 vs. 5.52±0.40, SOD (μg/g): 0.383±0.040 vs. 0.564±0.052, CAT (ng/g): 7.32±1.28 vs. 12.46±1.54, allP< 0.05], and remarkable increase in MDA content (nmol/g: 7.78±0.41 vs. 2.34±0.25,P< 0.05). With the extension of time, PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels gradually increased, MDA content gradually decreased, Nrf2 mRNA expression level had risen to the level of control group at 24 hours (0.133±0.019 vs. 0.126±0.009,P> 0.05), and it was higher than that of the control group at 48 hours (0.206±0.028 vs. 0.124±0.010,P< 0.05). Compared with that of the dichlorvos group, Lv-PON1 intervention group showed a significant increase in PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels [PON1 mRNA (gray value): 0.726±0.021 vs. 0.237±0.075, PON1 protein (gray value): 0.739±0.050 vs. 0.602±0.086, Nrf2 mRNA (gray value): 0.158±0.007 vs. 0.089±0.012, GSH (mg/g): 4.30±0.22 vs. 3.84±0.33, SOD (μg/g): 0.454±0.062 vs. 0.383±0.040, CAT (ng/g): 8.98±1.02 vs. 7.32±1.28, allP< 0.05], and a decrease in MDA content (nmol/g: 6.56±0.44 vs. 7.78±0.41,P< 0.05).Conclusion Regulation of PON1 gene can reduce MDA content, enhance SOD and CAT activities, increase GSH content, and it may also up-regulate Nrf2 mRNA expression to play a protective effect against oxidative stress of liver injury induced by dichlorvos poisoning.
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