腦缺血再灌注損傷自由基的改變及其與腦組織損傷的關(guān)系

1、腦缺血再灌註損傷自由基的改變及其與腦組織損傷的關(guān)系【摘要】 目的 探討腦缺血/再灌註損傷自由基的改變及其與組織損傷間的關(guān)系。方法 采用大鼠缺血再灌註動(dòng)物模型，觀察缺血再灌註損傷過程中自由基指標(biāo)、NO的改變，腦組織損傷及腦微循環(huán)障礙的情況，以及中藥羌活石菖蒲的保護(hù)作用。結(jié)果 腦缺血/再灌註損傷過程中脂質(zhì)過氧化增強(qiáng)，LPO明顯升高，SOD明顯降低，腦組織損傷明顯，腦微循環(huán)嚴(yán)重障礙。結(jié)論 腦缺血/再灌註損傷過程中自由基的改變明顯，自由基損傷與組織損傷間關(guān)系密切，中藥羌活石菖蒲對(duì)腦缺血/再灌註損傷有一定保護(hù)作用。為臨床腦外傷等疾病的治療提供實(shí)驗(yàn)依據(jù)?！娟P(guān)鍵詞】 腦缺血/再灌註損傷 自由基 羌活 石菖蒲

2、組織器官的血液供應(yīng)與微循環(huán)的功能狀態(tài)密切相關(guān)，腦微循環(huán)障礙會(huì)導(dǎo)致腦組織細(xì)胞缺血缺氧。通常微循環(huán)障礙可分為三個(gè)階段：一是缺血性障礙，二是無復(fù)流現(xiàn)象，三是再灌註損傷。缺血/再灌註損傷是微循環(huán)障礙的嚴(yán)重階段，常常是患者病情加重甚至死亡的主要原因。腦缺血/再灌註損傷是許多疾病如：腦外傷、休克、腦血栓等疾病中的病理生理過程，本研究觀察腦缺血/再灌註損傷過程中自由基的改變，探討自由基損傷與組織損傷間的關(guān)系。為臨床腦外傷等疾病的治療提供實(shí)驗(yàn)依據(jù)。1 材料和方法1.1 實(shí)驗(yàn)動(dòng)物 標(biāo)準(zhǔn)動(dòng)物SD大鼠30只，隨機(jī)分為三組：（1）對(duì)照組；（2）缺血再灌註組；（3）治療組，每組動(dòng)物各10只。1.2 缺血再灌註動(dòng)物模型的

3、制作（按改良的pullsinell法） 大鼠腹腔麻醉，用雙極電凝凝固雙側(cè)椎動(dòng)脈，然后用動(dòng)脈瘤夾夾閉雙側(cè)頸動(dòng)脈，造成4根動(dòng)脈閉塞全腦缺血（30min）。松開動(dòng)脈瘤夾，為再灌流期（60min）。對(duì)照組動(dòng)物作相同手術(shù)，但不阻斷血管。各組動(dòng)物按時(shí)處死，進(jìn)行相應(yīng)檢測(cè)。1.3 給藥方法 治療組于動(dòng)脈阻斷前30min從尾靜脈註射羌菖註射液，給藥劑量為10g/Kg.其余組註射等量生理鹽水。1.4 腦組織過氧化脂質(zhì)（LPO）測(cè)定 組織勻漿均在生理鹽水中，用組織勻漿器制備。腦組織超氧化物岐化酶(SOD)、過氧化脂質(zhì)（LPO）測(cè)定，均采用北京邦定泰克生物技術(shù)公司試劑盒。SOD為黃嘌呤氧化酶反應(yīng)抑制法，測(cè)定1%腦組織

4、勻漿；LPO為硫代巴比妥酸法，測(cè)定10%腦組織勻漿。1.5 腦組織一氧化氮測(cè)定 采用北京邦定泰克生物技術(shù)公司試劑盒。硝酸還原酶法；測(cè)定10%腦組織勻漿。1.6 腦組織及微循環(huán)超微結(jié)構(gòu)觀察 動(dòng)物處死后，將取下的腦組織迅速放于3的戊二醛固定液內(nèi)，固定24h。用磷酸緩沖液清洗，再用1%鋨酸作后固定.雙蒸水清洗，環(huán)氧樹脂618包埋.半薄切片定位后，LKB作超薄切片，醋酸鈾和枸櫞酸鉛復(fù)染，H-600電鏡下觀察。2 結(jié)果2.1 腦組織過氧化脂質(zhì)（LPO）含量 腦缺血30min再灌流60min組，過氧化脂質(zhì)（LPO）含量較對(duì)照組明顯升高，而SOD含量較對(duì)照組明顯降低，兩組間有顯著差異（P0.05）。治療組過

5、氧化脂質(zhì)（LPO）含量明顯下降而接近對(duì)照組，SOD含量明顯回升，亦接近對(duì)照組，與缺血組之間亦有顯著性差異（P0.05），見表1。 表1 各組大鼠腦組織SOD、LPO測(cè)定結(jié)果（略）*P<0.05; *P<0.012.2 腦組織一氧化氮測(cè)定結(jié)果 腦缺血30min再灌流60min組，NO含量較對(duì)照組明顯下降，兩組間有顯著差異（P0.01），治療組NO含量明顯回升（P0.01），但與對(duì)照組仍有顯著差異(P0.05)，結(jié)果見表2。表2 腦組織NO測(cè)定結(jié)果（略）*P<0.012.3 腦組織超微結(jié)構(gòu)改變 對(duì)照組神經(jīng)元形態(tài)結(jié)構(gòu)正常，核膜清楚核染色質(zhì)分布均勻，胞漿內(nèi)細(xì)胞器較豐富，線粒體和粗面內(nèi)

6、質(zhì)網(wǎng)腫脹等形態(tài)結(jié)構(gòu)完整，突觸小泡分布均勻。腦缺血30min再灌流60min組神經(jīng)元顯示程度不同的腫脹和退行性變，染色質(zhì)分布不均，異染色質(zhì)凝聚且邊集，線粒體和粗面內(nèi)質(zhì)網(wǎng)腫脹，線粒體腔擴(kuò)張，突觸小泡聚集。治療組上述改變明顯減輕。2.4 腦微循環(huán)超微結(jié)構(gòu)改變 腦缺血30min再灌流60min組腦毛細(xì)血管形態(tài)不規(guī)則，管腔畸形。毛細(xì)血管內(nèi)皮細(xì)胞變性，胞漿內(nèi)空泡。內(nèi)皮細(xì)胞胞核固縮，染色質(zhì)邊集，核膜凹陷。胞漿內(nèi)細(xì)胞器減少，線粒體、粗面內(nèi)質(zhì)網(wǎng)（RER）變性。毛細(xì)血管基底膜（BM）欠清晰，毛細(xì)血管管腔中紅細(xì)胞（RBC）變形、聚集。3 討論業(yè)已證明，缺血、缺氧引發(fā)的組織和器官損傷，可以發(fā)生在缺血時(shí)，但更主要是發(fā)生

7、在再灌註時(shí)。對(duì)腦缺血/再灌註損傷的研究日漸深入，認(rèn)為其損傷機(jī)制主要為氧自由基介導(dǎo)的脂質(zhì)過氧化反應(yīng)。當(dāng)腦組織缺血缺氧時(shí)，產(chǎn)生的無氧代謝產(chǎn)物聚集，恢復(fù)血液灌流后，氧分子進(jìn)入腦組織，發(fā)生超氧化物陰離子自由基反應(yīng)，自由基產(chǎn)生量過多和抗氧化系統(tǒng)受損，導(dǎo)致脂質(zhì)過氧化反應(yīng)，造成細(xì)胞膜、線粒體、溶酶體等生物膜損傷，最終導(dǎo)致細(xì)胞腫脹變性、壞死。構(gòu)成再灌註損傷。損傷機(jī)理過程中包含了如下幾個(gè)環(huán)節(jié)：（）缺血再灌註后引起血循環(huán)中氧自由基升高，中性粒細(xì)胞激活、釋放細(xì)胞因子，由此導(dǎo)致血腦屏障開放和腦內(nèi)自由基連鎖反應(yīng)。（）細(xì)胞因子等誘導(dǎo)iNOS表達(dá)。（）自由基通過引發(fā)興奮性神經(jīng)遞質(zhì)過量釋放等機(jī)制導(dǎo)致神經(jīng)元鈣超載，激活NOS。

8、（）過量生成的NO與O-結(jié)合生成毒性更強(qiáng)的ONOO-，導(dǎo)致神經(jīng)蛋白質(zhì)、核酸及脂質(zhì)破壞。（）線粒體不僅作為能量轉(zhuǎn)換器而且涉及到雕亡的調(diào)控，線粒體的某些成分直接參與了雕亡的調(diào)控。腦組織氧和ATP的儲(chǔ)備很少，因而對(duì)缺氧耐受性差。腦細(xì)胞線粒體能量代謝障礙可能在在腦缺血/再灌註損傷中也起了重要作用。 也是引起缺血/再灌註損傷后腦細(xì)胞雕亡的重要原因。本組實(shí)驗(yàn)發(fā)現(xiàn)，缺血/再灌註后實(shí)驗(yàn)組的過氧化脂質(zhì)明顯升高，SOD明顯降低，且與對(duì)照組之間有顯著差異。這表明腦組織缺血缺氧時(shí)無氧代謝產(chǎn)生的大量代謝產(chǎn)物，在再灌註恢復(fù)血運(yùn)后，在氧分子的氧化作用下發(fā)生超氧化反應(yīng)，消耗了大量的SOD，故而血中和腦組織內(nèi)SOD明顯降低，過

9、氧化脂質(zhì)增高。當(dāng)腦缺血再灌註發(fā)生后，細(xì)胞內(nèi)ATP依次分解為AMP腺苷肌苷次黃嘌呤。由于缺血及能量消耗，次黃嘌呤在黃嘌呤氧化酶作用下轉(zhuǎn)化為黃嘌呤，進(jìn)而變?yōu)槟蛩岵a(chǎn)生超氧離子自由基。自由基產(chǎn)生一系列連鎖反應(yīng)使細(xì)胞結(jié)構(gòu)破壞。故本組實(shí)驗(yàn)中，用電鏡觀察腦組織的超微結(jié)構(gòu)，發(fā)現(xiàn)缺血組神經(jīng)元顯示程度不同的腫脹和退行性變，線粒體和粗面內(nèi)質(zhì)網(wǎng)腫脹，線粒體腔擴(kuò)張，突觸小泡聚集等細(xì)胞和膜結(jié)構(gòu)受損的表現(xiàn)。顯示了缺血再灌註中自由基對(duì)腦組織細(xì)胞的損傷。本組實(shí)驗(yàn)研究中，還觀察了腦微循環(huán)受損的情況。在電鏡下發(fā)現(xiàn)，腦的毛細(xì)血管有明顯受損改變。毛細(xì)血管內(nèi)皮細(xì)胞變性，胞漿內(nèi)空泡。內(nèi)皮細(xì)胞胞核固縮，染色質(zhì)邊集，核膜凹陷。胞漿內(nèi)細(xì)胞器減

10、少，線粒體、粗面內(nèi)質(zhì)網(wǎng)（RER）變性。毛細(xì)血管基底膜（BM）變得模煳不清。腦微循環(huán)障礙、毛細(xì)血管內(nèi)皮細(xì)胞變性，又導(dǎo)致功能改變，內(nèi)皮細(xì)胞分泌產(chǎn)生NO的功能障礙，血和腦組織中NO含量降低。這些改變又必然影響腦組織細(xì)胞的血液供應(yīng)，加重腦組織細(xì)胞的缺血缺氧。于是在缺氧條件下，能量代謝障礙，引起抗氧化基團(tuán)形成減少，又可能進(jìn)一步加重氧自由基在損傷中的作用。本組實(shí)驗(yàn)中，觀察了羌菖註射液對(duì)脂質(zhì)過氧化損傷的保護(hù)作用。結(jié)果發(fā)現(xiàn)治療組的LPO明顯低于缺血再灌註組；而治療組的SOD水平明顯高于缺血再灌註組，表明羌菖註射液具有清除超氧化物陰離子自由基、減輕脂質(zhì)過氧化損傷、保護(hù)細(xì)胞的作用。羌活具有抗血栓形成的作用1，增加

11、腦血流量，改善腦血液循環(huán)，改善血液流變性作用4。有文獻(xiàn)報(bào)道羌菖合劑可以防治實(shí)驗(yàn)性腦缺血之腦損傷5；說明羌活、石菖蒲通過對(duì)腦微循環(huán)的影響，保護(hù)腦毛細(xì)血管，從而保護(hù)缺血再灌註過程中的腦組織細(xì)胞，減輕了缺血及再灌註的損傷作用。參考文獻(xiàn) 張明發(fā)，沈雅琴，朱自平，等 羌活的鎮(zhèn)痛抗炎抗血栓形成作用研究J 中醫(yī)藥研究，1996，(6)：51522 趙魯鳴，周恩平，王世軍，等腦脈顆粒劑對(duì)家犬腦循環(huán)及血壓影響的研究J中成藥，1998，20（4）：32333 馮英菊，謝人明羌活對(duì)麻醉動(dòng)物腦循環(huán)的作用J陜西中醫(yī)，1998，19（1）：37384 王 濤，楊 奎，王家葵，等 羌活的藥理作用J中藥藥理臨床，1996，1

12、2（4）：12155 王天然，曾祥元，朱國(guó)標(biāo)，等羌菖合劑對(duì)結(jié)扎右側(cè)頸總動(dòng)脈大鼠腦損傷作用的初步研究J中國(guó)微循環(huán)， 2000，4（4）：221222Cerebral ischemia-reperfusion injury and its radical change in the relationship between brain injuryAbstract Objective To investigate cerebral ischemia / reperfusion injury and its radical change in the relationship between tis

13、sue injury. Methods ischemia - reperfusion animal model to observe the ischemia - reperfusion injury in radical indicators NO change in brain damage and cerebral microcirculation of the situation and the protective effect of traditional Chinese medicine Rhizoma Acorus gramineus. Results cerebral isc

14、hemia / reperfusion injury increased lipid peroxidation, LPO significantly increased, SOD significantly decreased brain obvious tissue damage, cerebral microcirculation serious obstacles. Conclusions Cerebral ischemia / reperfusion injury in radical change significantly, between the free radical dam

15、age and tissue injury is closely related to traditional Chinese medicine Rhizoma shichangpu on cerebral ischemia / reperfusion injury in a certain protection. for the clinical treatment of diseases such as brain injury and provide experimental basis.Keywords: cerebral ischemia / reperfusion injury i

16、n radical Notopterygium gramineusBlood supply to tissues and organs function and microcirculation is closelyrelated to brain microcirculation cause brain tissue ischemia and hypoxia. Usually microcirculation can be divided into three stages: First, ischemic disorders, and second, no-reflow phenomeno

17、n, third, and reperfusion injury. ischemia /reperfusion injury is a serious stage of microcirculation, patients are often sicker or even primary cause of death. ischemia / reperfusion injury in many diseases such as: brain trauma, shock, cerebral thrombosis and other diseases in the pathophysiologic

18、al process, this study was to cerebral ischemia / reperfusion injury in the process of radical change, of free radical damage and therelationship between tissue injury. for the clinical treatment of diseases such as brain injury and provide experimental basis.1 Materials and methodsSD 1.1 standard a

19、nimal laboratory animals 30 rats were randomly divided into three groups: (1 control group, (2 ischemia - reperfusion group, (3 treatment groups, each of the 10 animals.1.2 ischemia - reperfusion animal model (according to the modified method pullsinell intraperitoneal anesthesia, bipolar coagulatio

20、n coagulation withbilateral vertebral artery aneurysm clip and then bilateral carotid artery occlusion, resulting in four full-artery occlusion cerebral ischemia (30min. release the aneurysm clip, as the reperfusion period (60min. control animals for the same surgery, but does not block the blood ve

21、ssels. All the animals were sacrificed on time, make the appropriate testing.1.3 delivery methods artery occlusion in the treatment group 30min before the Qiang-chang from the tail vein injection, administered dose 10g/Kg. The other group was injected with normal saline.1.4 Lipid peroxidation in bra

22、in tissue (LPO were measured in saline homogenates, prepared using tissue homogenizer. Brain tissue superoxide dismutase (SOD), lipid peroxide (LPO determination, all states with Beijing Tektronixbiotechnology company set kit. SOD to xanthine oxidase inhibition,determination of brain tissue 1%, LPO

23、thiobarbituric acid method to determine the 10% of the brain tissue.1.5 Determination of brain nitric oxide bonding with the Tektronix Beijing biotechnology company kit. Nitrate reductase method to determine 10% of the brain tissue.1.6 microcirculation ultrastructural brain tissue and the animals we

24、re then observed, the brain removed quickly put in 3% glutaraldehyde fixative, thefixed-24h. With the phosphate buffer wash, and then after 1% osmium tetroxide for fixed. double distilled water to clean, epoxy 618 embedding. semi-thin section positioning, LKB for ultrathin section, uranyl acetate an

25、d lead citrate stained, H-600 electron microscopy.2 Results2.1 Lipid peroxidation in brain tissue (LPO content of cerebral ischemia and reperfusion 30min 60min group, lipid peroxide (LPO content was significantly higher than the control group, while SOD levels significantly lower than the control gr

26、oup, significant differences between the two groups (P <0.05. thetreatment group lipid peroxide (LPO was significantly decreased and close to the control group, SOD content was recovered, also close to the control group and ischemic group showed significant differences between (P <0.05, Table

27、1. Table 1, the rats brain tissue SOD, LPO assay results (omitted* P <0.05; * P <0.012.2 Determination of brain nitric oxide result of cerebral ischemia and reperfusion 30min 60min group, NO levels were markedly decreased, a significant difference between the two groups (P <0.01, NO group w

28、assignificantly recovered (P <0.01, but with the control group still significantly (P <0.05), results in Table 2.Table 2 Determination of brain tissue NO results (slightly* P <0.012.3 The ultrastructural changes in brain tissue morphology in control neurons in the normal group, the nuclear

29、membrane clearly evenly distributed nuclear chromatin, abundant cytoplasmic organelles, mitochondria and roughendoplasmic reticulum swelling and other morphological integrity, synaptic vesicle distribution . ischemia reperfusion 30min 60min set of neurons show different degrees of swelling and degen

30、eration, the uneven distribution ofchromatin, heterochromatin condensation and margination, mitochondria and swelling of rough endoplasmic reticulum, mitochondria cavity expansion,synaptic vesicle aggregation. the treatment group significantly reduced the above changes.Ultrastructural changes of cer

31、ebral microcirculation 2.4 ischemic reperfusion 60min 30min irregular in brain capillaries, luminal deformity. Capillary endothelial cell degeneration, cytoplasmic vacuoles. Endothelial cell nucleus shrinkage, chromatin margination , nuclear depression. cytoplasmic organellesdecreased, mitochondria,

32、 rough endoplasmic reticulum (RER variability.capillary basement membrane (BM unclear capillary lumen in the red blood cell (RBC deformation and aggregation.3 DiscussionProven, ischemia, hypoxia-induced tissue and organ damage can occur in ischemia, but mostly occurs during reperfusion. On cerebral

33、ischemia / reperfusion injury in increasing depth, that the mechanism of its injury as oxygen free radical-mediated lipid peroxidation. When the brain tissue ischemia and hypoxia, the resulting product of anaerobic metabolism together to restore blood perfusion, the oxygen molecules into the brain t

34、issue, theoccurrence of superoxide anion radical reaction, excessive free radical production and antioxidant system damage, resulting in lipid peroxidation, causing cell membrane, mitochondria, lysosomes and other membrane damage, ultimately leading to cell swelling and degeneration and necrosis. co

35、nstitute reperfusion injury.Damage Mechanism of the process includes the following several aspects: (1ischemia-reperfusion induced increase in blood circulation of oxygen free radicals, neutrophil activation, release of cytokines, leading to blood-brain barrier opening and brain free radical chain r

36、eaction. (2 cytokines induce iNOS expression. (3 radicals caused by excessive release of excitatory neurotransmitters and other mechanisms leading to neuronal calcium overload, activation of NOS. (4excessive NO generated with O-2 generation with the more virulent of ONOO-, caused nerve proteins, nuc

37、leic acids and lipid damage. (5, mitochondria not only as relates to energy conversion and regulation of apoptosis, mitochondrial components directly involved in some regulation of apoptosis. brain tissue reserves of oxygen and ATP very few, so poor tolerance to hypoxia. brainmitochondrial energy me

38、tabolism may be in the ischemia / reperfusion injury also play an important role. is caused by ischemia / reperfusion injury an important cause of brain cells.The experiments showed that ischemia / reperfusion experimental groupsignificantly increased lipid peroxide, SOD significantly decreased in t

39、he control group had significant differences. This indicates that cerebral ischemia produced by anaerobic metabolism during hypoxia a large number of metabolites, blood flow recovery after reperfusion, the oxygen molecules in the oxidation reactionoccur super-oxidized, consuming a large amount of SO

40、D, therefore the blood and brain tissue SOD decreased significantly increased lipid peroxidation. When the brain missing Blood / reperfusion occurred, followed by decomposition ofintracellular ATP AMP adenosine inosine hypoxanthine. as ischemia and energy consumption in the xanthine oxidase hypoxant

41、hine into xanthine under, and then becomes uric acid and generates superoxide anion radicals. a chain reaction of free radicals damage the cell structure. Therefore, this set of experiments, brain tissue ultrastructure by electron microscopy and found that neurons in theischemia group displayed vary

42、ing degrees of swelling and regression of change, mitochondria and swelling of rough endoplasmic reticulum, mitochondria cavity expansion, such as synaptic vesicle aggregation and membrane structure of cell damage performance. shows free radicals on ischemia-reperfusion injury of brain cells. This s

43、et of experiments study, also observed a case of cerebralmicrocirculation damage. In electron microscopy showed significant damage to the brain capillaries to change. capillary endothelial cell degeneration,cytoplasmic vacuoles. endothelial cell nucleus shrinkage, chromatin margination, nuclear depr

44、ession. cytoplasmic organelles decreased, mitochondria, rough endoplasmic reticulum (RER variability. capillary basement membrane (BM blurred. cerebral microcirculation, capillary endothelial cell degeneration, but also lead to functional changes in endothelial cells produce NO dysfunction, blood an

45、d brain tissue NO content was decreased. These changes will inevitably affect the brain cells and the blood supply to brain cells increased ischemia and hypoxia. So in hypoxic conditions, the energy metabolic disorder, caused by the formation of reduced antioxidant group, and may further increase the oxygen free radicals in the injury.This set of experiments, observed Qiang-chang injection of lipidperoxidation injury. LPO was found that the treatment group was significantly lower than the ischemia / reperfusion group, while the SOD