中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2015年
11期
911-915
,共5页
袁佳%王迪芬%刘颖%陈先俊%张海玲
袁佳%王迪芬%劉穎%陳先俊%張海玲
원가%왕적분%류영%진선준%장해령
核因子E2相关因子2%富氢水%创伤性脑损伤%氧化应激
覈因子E2相關因子2%富氫水%創傷性腦損傷%氧化應激
핵인자E2상관인자2%부경수%창상성뇌손상%양화응격
Nuclear factor erythroid 2-related factor 2%Hydrogen rich water%Traumatic brain injury%Oxidative stress
目的 探讨富氢水对创伤性脑损伤(TBI)大鼠核因子E2相关因子2(Nrf2)表达及氧化应激损伤的影响.方法 将90只雄性SD大鼠按随机数字表法分为假手术组、TBI组、富氢水干预组,每组30只.采用改良Feeney法自由落体撞击制作TBI模型;假手术组开颅窗后不撞击.富氢水干预组撞击致脑损伤后腹腔注射富氢水5 mL/kg,每日1次,共5d;假手术组和TBI组给予等量生理盐水.各组分别于术后6、12、24、48 h和5d时取6只大鼠进行神经功能缺损评分(NSS),活杀后取损伤灶周边皮质脑组织备检.分光光度法检测过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量;实时荧光定量反转录-聚合酶链反应(RT-qPCR)和蛋白质免疫印迹试验(Western Blot)检测Nrf2 mRNA和核蛋白表达;苏木素-伊红(HE)染色观察脑组织病理学改变.结果 ①NSS评分:富氢水干预组术后12、24、48 h和5d时NSS评分(分)较TBI组明显下降(12 h:9.83±2.32比13.17±2.71,24 h:9.83±2.79比13.50±2.43,48h:7.50±2.07比11.83±2.14,5 d:5.50±1.87比10.50±2.43,均P＜ 0.05).②TBI术后CAT、GSH-Px明显低于假手术组,MDA明显高于假手术组,以24h时最为明显[CAT (kU/g):1.080±0.312比3.571±0.758,GSH-Px(kU/g):9.195±3.173比32.385±10.619,MDA(μmol/g):12.282±2.896比4.349±1.511,均P＜0.01];富氢水干预组各项指标均明显改善,接近假手术组水平.③RT-qPCR:3组各时间点Nrf2 mRNA表达差异均无统计学意义.④Western Blot:TBI组术后脑组织Nrf2核蛋白表达(灰度值)较假手术组明显上升,24 h达高峰(0.703±0.262比0.238±0.120,P＜0.05);富氢水干预组术后Nrf2核蛋白表达明显高于TBI组,以24h时最为明显(1.110±0.372比0.703±0.262,P＜0.05).⑤HE染色:假手术组脑组织结构完整;TBI组各时间点出现不同程度的脑损伤,以24h时损伤最为明显;富氢水干预组损伤较TBI组明显减轻.结论 富氢水可上调TBI大鼠脑组织Nrf2表达,减轻脑组织氧化应激损伤,其机制可能与Nrf2诱导其下游抗氧化物酶表达有关.
目的 探討富氫水對創傷性腦損傷(TBI)大鼠覈因子E2相關因子2(Nrf2)錶達及氧化應激損傷的影響.方法 將90隻雄性SD大鼠按隨機數字錶法分為假手術組、TBI組、富氫水榦預組,每組30隻.採用改良Feeney法自由落體撞擊製作TBI模型;假手術組開顱窗後不撞擊.富氫水榦預組撞擊緻腦損傷後腹腔註射富氫水5 mL/kg,每日1次,共5d;假手術組和TBI組給予等量生理鹽水.各組分彆于術後6、12、24、48 h和5d時取6隻大鼠進行神經功能缺損評分(NSS),活殺後取損傷竈週邊皮質腦組織備檢.分光光度法檢測過氧化氫酶(CAT)、穀胱甘肽過氧化物酶(GSH-Px)活性及丙二醛(MDA)含量;實時熒光定量反轉錄-聚閤酶鏈反應(RT-qPCR)和蛋白質免疫印跡試驗(Western Blot)檢測Nrf2 mRNA和覈蛋白錶達;囌木素-伊紅(HE)染色觀察腦組織病理學改變.結果 ①NSS評分:富氫水榦預組術後12、24、48 h和5d時NSS評分(分)較TBI組明顯下降(12 h:9.83±2.32比13.17±2.71,24 h:9.83±2.79比13.50±2.43,48h:7.50±2.07比11.83±2.14,5 d:5.50±1.87比10.50±2.43,均P＜ 0.05).②TBI術後CAT、GSH-Px明顯低于假手術組,MDA明顯高于假手術組,以24h時最為明顯[CAT (kU/g):1.080±0.312比3.571±0.758,GSH-Px(kU/g):9.195±3.173比32.385±10.619,MDA(μmol/g):12.282±2.896比4.349±1.511,均P＜0.01];富氫水榦預組各項指標均明顯改善,接近假手術組水平.③RT-qPCR:3組各時間點Nrf2 mRNA錶達差異均無統計學意義.④Western Blot:TBI組術後腦組織Nrf2覈蛋白錶達(灰度值)較假手術組明顯上升,24 h達高峰(0.703±0.262比0.238±0.120,P＜0.05);富氫水榦預組術後Nrf2覈蛋白錶達明顯高于TBI組,以24h時最為明顯(1.110±0.372比0.703±0.262,P＜0.05).⑤HE染色:假手術組腦組織結構完整;TBI組各時間點齣現不同程度的腦損傷,以24h時損傷最為明顯;富氫水榦預組損傷較TBI組明顯減輕.結論 富氫水可上調TBI大鼠腦組織Nrf2錶達,減輕腦組織氧化應激損傷,其機製可能與Nrf2誘導其下遊抗氧化物酶錶達有關.
목적 탐토부경수대창상성뇌손상(TBI)대서핵인자E2상관인자2(Nrf2)표체급양화응격손상적영향.방법 장90지웅성SD대서안수궤수자표법분위가수술조、TBI조、부경수간예조,매조30지.채용개량Feeney법자유락체당격제작TBI모형;가수술조개로창후불당격.부경수간예조당격치뇌손상후복강주사부경수5 mL/kg,매일1차,공5d;가수술조화TBI조급여등량생리염수.각조분별우술후6、12、24、48 h화5d시취6지대서진행신경공능결손평분(NSS),활살후취손상조주변피질뇌조직비검.분광광도법검측과양화경매(CAT)、곡광감태과양화물매(GSH-Px)활성급병이철(MDA)함량;실시형광정량반전록-취합매련반응(RT-qPCR)화단백질면역인적시험(Western Blot)검측Nrf2 mRNA화핵단백표체;소목소-이홍(HE)염색관찰뇌조직병이학개변.결과 ①NSS평분:부경수간예조술후12、24、48 h화5d시NSS평분(분)교TBI조명현하강(12 h:9.83±2.32비13.17±2.71,24 h:9.83±2.79비13.50±2.43,48h:7.50±2.07비11.83±2.14,5 d:5.50±1.87비10.50±2.43,균P＜ 0.05).②TBI술후CAT、GSH-Px명현저우가수술조,MDA명현고우가수술조,이24h시최위명현[CAT (kU/g):1.080±0.312비3.571±0.758,GSH-Px(kU/g):9.195±3.173비32.385±10.619,MDA(μmol/g):12.282±2.896비4.349±1.511,균P＜0.01];부경수간예조각항지표균명현개선,접근가수술조수평.③RT-qPCR:3조각시간점Nrf2 mRNA표체차이균무통계학의의.④Western Blot:TBI조술후뇌조직Nrf2핵단백표체(회도치)교가수술조명현상승,24 h체고봉(0.703±0.262비0.238±0.120,P＜0.05);부경수간예조술후Nrf2핵단백표체명현고우TBI조,이24h시최위명현(1.110±0.372비0.703±0.262,P＜0.05).⑤HE염색:가수술조뇌조직결구완정;TBI조각시간점출현불동정도적뇌손상,이24h시손상최위명현;부경수간예조손상교TBI조명현감경.결론 부경수가상조TBI대서뇌조직Nrf2표체,감경뇌조직양화응격손상,기궤제가능여Nrf2유도기하유항양화물매표체유관.
Objective To investigate the effects of hydrogen rich water on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and oxidative stress in rats with traumatic brain injury (TBI).Methods Ninety healthy male Sprague-Dawley (SD) rats were randomly divided into sham operation group, TBI group and hydrogen rich water treatment group (HW group), with 30 rats in each group.TBI model was reproduced by the modified Feeney weight dropping method.The skulls of rats in sham operation group underwent only craniotomy without direct hit.The rats in HW group received brain injury by hitting after craniotomy, followed by injection of hydrogen rich water (5 mL/kg) intraperitoneally once a day for 5 days after successful reproduction of the model.The rats in sham operation group and TBI group were given an equal amount of normal saline in same manner.Six rats from each group were sacrificed at 6, 12, 24, 48 hours and 5 days after evaluating neurological severity scores (NSS).The brain tissue in injured ipsilateral cortex was harvested.The activity of catalase (CAT), glutathione peroxidase (GSH-Px), and content of malondialdehyde (MDA) were determined by spectrophotometry.The expressions of mRNA and nucleoprotein of Nrf2 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) and Western Blot.The pathological changes were observed with microscopy after hematoxylin and eosin (HE) staining.Results ① NSS score:compared with TBI group, NSS in HW group at 12, 24, 48 hours and 5 days were significantly decreased (12 hours: 9.83±2.32 vs.13.17±2.71, 24 hours: 9.83±2.79 vs.13.50±2.43, 48 hours: 7.50±2.07 vs.11.83±2.14, 5 days:5.50 ± 1.87 vs.10.50 ± 2.43, all P ＜ 0.05).② Compared with sham operation group, the activity of GSH-Px and CAT in TBI group were markedly declined after operation, while the MDA content was elevated significantly, especially at 24 hours [CAT (kU/g): 1.080±0.312 vs.3.571 ±0.758, GSH-Px (kU/g): 9.195±3.173 vs.32.385± 10.619, MDA (μmol/g): 12.282±2.896 vs.4.349± 1.511, all P ＜ 0.01].Compared with TBI group, the parameters in HW group were improved, and they were similar as sham operation group.③ RT-qPCR: no significant difference was found in the expression of Nrf2 mRNA at each time point in three groups.④ Western Blot: the expression of Nrf2 nucleoprotein (gray value) in TBI group was apparently higher than that in sham operation group, and peaked at 24 hours (0.703 ± 0.262 vs.0.238 ± 0.120, P ＜ 0.05), and the expression in HW group was obviously higher than that in TBI group, especially at 24 hours (1.110 ± 0.372 vs.0.703 ± 0.262, P ＜ 0.05).⑤ HE staining: the brain structure in sham operation group was found to be intact.However, there were different degrees of pathological changes at each time in TBI group, especially at 24 hours.The pathological damage of brain tissue in HW group was significantly milder.Conclusions Hydrogen rich water can up-regulate the expression of Nrf2, and reduce oxidative damage of traumatic brain injury in rats.Nrf2 can up-regulate the expression of its downstream antioxidant enzymes, which may be the mechanism of the upregulation expression of Nrf2 in the study.
