缺糖缺氧/复糖复氧对神经细胞损伤及丹酚酸B干预作用机制的研究

【作者】 汪芸；

【导师】 黄启福； 蒋玉凤；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 缺血性脑血管病具有高发病率、高死亡率和高致残率的特点,造成了严重的社会问题和经济负担。因此,深入认识其发病机制,寻找新的治疗靶点是目前亟待解决的问题。缺血性脑血管病的直接原因是脑血流减少,导致氧和能量供应中断,细胞死亡。治疗后脑血流恢复,但往往出现神经系统损伤加重的情况,即缺血再灌注损伤。中医理论认为中风病（这里主要指缺血性脑血管病）的病因与风、火、痰、瘀等毒邪损伤脑络有关。传统中药丹参具有活血化瘀的功效,已广泛应用于缺血性脑血管病的临床治疗。丹酚酸B（Salvianolic acid B）是从传统中药丹参中提取的水溶性有效成分,由3分子丹参素和1分子咖啡酸缩合而成的酚酸类化合物,具有很强的抗氧化、清除自由基作用,但其在脑缺血再灌注损伤过程中的作用机制还不完全清楚。本论文以中医中风病理论为指导,在我们实验室已证明丹酚酸B对小鼠脑缺血性损伤具有治疗作用的基础上,采用原代培养大鼠皮层神经细胞,建立缺糖缺氧/复糖复氧的细胞模型,从兴奋性毒性、钙超载、氧化应激、细胞凋亡的角度研究神经细胞损伤及丹酚酸B干预作用的机制。论文的实验研究包括四个部分:一、缺糖缺氧模型的建立及丹酚酸B有效剂量的确定目的:建立稳定可靠的缺糖缺氧模型,并确定丹酚酸B的有效剂量。方法:体外培养大鼠大脑皮层神经细胞,复制神经细胞缺糖缺氧2h、3h、4h、5h的细胞模型,测氧仪测量培养液中溶解氧浓度,采用MTT法检测细胞活性和存活率。结果:体外培养的神经细胞状态稳定,经免疫组化神经元特异性烯醇化酶（NSE）鉴定阳性细胞比例达70～80%,神经细胞Nissl’s染色显示Nissl’s小体丰富。随缺糖缺氧时间延长,培养液中溶解氧浓度逐渐降低,神经细胞活性、存活率也随之下降,各时间点与正常组比较均有显著性差异（P＜0.01）。选择对细胞损伤较重的缺糖缺氧4 h作为模型时间。神经细胞缺氧缺糖4 h后活性明显下降,与正常组比较有显著性差异（P＜0.01）。丹酚酸B治疗组包括10μg/L,100μg/L,1mg/L,5 mg/L,7.5 mg/L,10 mg/L,25 mg/L和50 mg/L共8个不同剂量,其中10、25、50 mg/L 3个剂量的丹酚酸B明显增强神经细胞活性,且存在量效关系,与缺糖缺氧4 h组相比差异明显（P＜0.01）。将10、25、50 mg/L3个剂量的丹酚酸B加入正常神经细胞,其活性与正常组之间没有显著性差异。选择终浓度10 mg/L作为以下实验丹酚酸B的用药剂量。二、缺糖缺氧对神经细胞损伤及丹酚酸B干预作用机制的研究目的:探讨缺糖缺氧对神经细胞损伤及丹酚酸B干预作用的机制。方法:将培养的神经细胞随机分为正常组、缺糖缺氧4 h组和丹酚酸B治疗组。复制缺糖缺氧4 h的病理模型,MTT法观察神经细胞活性和存活率,比色法检测LDH漏出率和培养液中谷氨酸含量,流式细胞术测定神经细胞线粒体膜电位（MMP）、胞浆内Ca²⁺,倒置相差显微镜、HE染色及透射电镜观察神经细胞形态和超微结构的变化。结果:神经细胞缺糖缺氧4 h后活性、存活率、MMP荧光值明显降低,LDH漏出率、培养液中谷氨酸含量及胞浆内Ca²⁺荧光强度显著升高,与正常组比较有显著性差异（P＜0.01）。丹酚酸B治疗组神经细胞活性、存活率、MMP荧光值均增高,LDH漏出率、培养液中谷氨酸含量、胞浆内Ca²⁺荧光强度下降,与缺糖缺氧4 h组比较有显著性差异（P＜0.05～0.01）。正常组神经细胞胞体呈锥体形,细胞器丰富:缺糖缺氧4 h组大部分神经细胞肿胀,细胞器数目有所减少,可见线粒体肿胀明显;丹酚酸B治疗组神经细胞轻度肿胀,细胞器结构尚完整。三、复糖复氧对神经细胞损伤及丹酚酸B干预作用机制的研究目的:研究复糖复氧对神经细胞的氧化损伤,并在体外模型中证实丹酚酸B的干预作用与其抗氧化能力有关。方法:神经细胞随机分为正常组、复糖复氧组和丹酚酸B治疗组。MTT法检测缺糖缺氧3h/复糖复氧1h、3h、6h、12h、18h、24h共6个时间点神经细胞活性、存活率。缺糖缺氧3h/复糖复氧3h、24h分别代表再灌注早期、晚期,并采用比色法观察神经细胞LDH漏出率,荧光标记和自旋捕集技术检测细胞内ROS水平,比色法测定神经细胞内Mn-SOD、CAT和GSH-PX的活性。倒置相差显微镜观察神经细胞形态变化。结果:缺糖缺氧3h后复糖复氧1h、3h、6h、12h、18h、24h,神经细胞活性、存活率均较正常组降低（P＜0.05～0.01）;除复糖复氧1h、12h外,其他时间点丹酚酸B治疗组神经细胞活性、存活率均高于复糖复氧组（P＜0.05）。神经细胞复糖复氧3h、24h后,LDH漏出率、细胞内ROS均明显高于正常组（P＜0.05～0.01）;而丹酚酸B治疗组神经细胞LDH漏出率、细胞内ROS则低于复糖复氧组（P＜0.05～0.01）。复糖复氧3h、24h神经细胞内Mn-SOD、CAT、GSH-PX的活性均降低,与正常组比较差异明显（P＜0.05～0.01）,丹酚酸B治疗组神经细胞内抗氧化酶的活性明显高于复糖复氧3h、24h组（P＜0.05～0.01）。正常组神经细胞胞体饱满,突起粗大伸展;复糖复氧3h后神经细胞形态发生改变,至24h神经细胞皱缩,部分细胞死亡;丹酚酸B治疗组神经细胞形态变化有所减轻。四、复糖复氧诱导神经细胞凋亡及丹酚酸B抗凋亡作用机制的研究目的:探讨复糖复氧诱导神经细胞凋亡及丹酚酸B抗凋亡作用的机制。方法:神经细胞随机分为正常组、复糖复氧24h组和丹酚酸B治疗组。复制缺糖缺氧3h/复糖复氧24h的细胞模型。激光扫描共聚焦显微镜测定胞浆内Ca²⁺荧光强度,流式细胞仪检测MMP、细胞凋亡率,免疫组化法观察细胞内Bcl-2、Bax的表达水平,Western blot测定细胞色素C释放率,Hoechst 33342染色观察细胞核形态变化,透射电镜观察神经细胞超微结构。结果:缺糖缺氧3h/复糖复氧24h引起胞浆内Ca²⁺荧光强度明显增高,MMP荧光值降低,神经细胞凋亡率明显增高,与正常组相比有显著性差异（P＜0.01）。丹酚酸B治疗组胞浆内Ca²⁺荧光强度降低,MMP荧光值增强,凋亡率下降,与复糖复氧24h组相比亦有显著性差异（P＜0.01）。神经细胞复糖复氧24h,Bcl-2表达减弱,Bax表达升高,与正常组相比有显著性差异（P＜0.01）。丹酚酸B治疗组Bcl-2表达增加,Bax表达降低,与复糖复氧24h组比较差异明显（P＜0.05）。正常组胞浆内细胞色素C含量很低,主要集中于线粒体,复糖复氧24 h后胞浆内细胞色素C含量明显升高,其灰度值与正常组相比有显著性差异（P＜0.01）,丹酚酸B治疗组胞浆内细胞色素C含量低于复糖复氧24 h组（P＜0.05）。复糖复氧24 h组细胞色素C释放率明显高于正常组（P＜0.01）,丹酚酸B治疗组细胞色素C释放率明显降低,两组相比差异明显（P＜0.05）。正常神经细胞经Hoechst33342荧光染色,核呈均匀的蓝色荧光;复糖复氧24 h组则有较多细胞核呈凝聚固缩,荧光明显增强,并有核碎裂;丹酚酸B治疗组细胞核大部分呈淡蓝色或蓝色荧光,接近正常组,仅见个别细胞核浓染呈明亮蓝色荧光。透射电镜观察复糖复氧24 h组神经细胞核染色质凝聚,细胞膜完整,符合凋亡的形态改变;丹酚酸B治疗组神经细胞超微结构变化较复糖复氧24 h组有所减轻。结论:本研究建立了稳定可靠的神经细胞原代培养体系,复制的细胞模型可以实现体外神经细胞缺糖缺氧损伤。缺糖缺氧4 h引起神经细胞培养液中谷氨酸含量增加、细胞内Ca²⁺超载、MMP下降。丹酚酸B通过减少培养液中的谷氨酸含量,抑制神经细胞内Ca²⁺超载,稳定MMP,从而对缺糖缺氧神经细胞起到保护作用。缺糖缺氧3 h/复糖复氧3 h、24 h对神经细胞的损伤与氧化应激有关,丹酚酸B可以清除细胞内ROS,提高抗氧化酶Mn-SOD、CAT、GSH-PX的活性,从而对复糖复氧神经细胞起到保护作用。缺糖缺氧3h/复糖复氧24 h可诱导神经细胞发生凋亡,丹酚酸B通过保护线粒体功能,提高Bcl-2的表达,降低Bax的表达,减少促凋亡物质的释放,起到抗凋亡作用。综上所述,神经细胞缺糖缺氧损伤的机制与兴奋性毒性、细胞内Ca²⁺超载有关,而复糖复氧损伤的机制则涉及到氧化应激、线粒体途径的细胞凋亡。丹酚酸B通过减轻兴奋性毒作用,抑制神经细胞内Ca²⁺超载保护缺糖缺氧的神经细胞,通过改善细胞的氧化-还原状态,保护线粒体功能,拮抗凋亡,对复糖复氧神经细胞起到保护作用。更多还原

【Abstract】 Ischemic cerebrovascular disease has the characteristics of high incidence,high mortality and high deformity.It has caused serious social problems and economic burden.Therefore,to deeply understand its pathogenesis and find a new target is the key issue to be solved.The immediate cause of ischemic cerebrovascular disease is a decrease in the cerebral blood flow,inducing the interruption of oxygen and energy as well as cell death.Cerebral blood flow restores after treatment,however,nervous system damages more seriously,which is called ischemia-reperfusion injury.In Chinese medicine theories,the etiological factors of stroke(here mainly refers to ischemic cerebrovascular disease) is related to the toxin hurts brain collaterals,the toxin is including wind,fire,sputum and stasis.The Chinese traditional herb- Salvia miltiorrhiza has the effects of activating blood circulation and dissipate blood stasis.It has been widely used in clinical treatment of ischemic cerebrovascular disease. Salvianolic acid B(Sal B) is a water-soluble active component extracted from Salvia miltiorrhiza.It is a phenolic acid compound,which is condensated by three molecules tanshinol and one molecule caffeic acid.Sal B exhibits higher antioxidant and radical scavenging activities.Nevertheless,the mechanisms Of its intervening effect during the cerebral ischemia and reperfusion have not yet been explained very clearly.The effects of Sal B on the cerebral ischemic mice have been proved in our laboratory. Under the instruction of Chinese medicine theories and our findings,in the present thesis,we established the oxygen-glucose deprivation/reintroduction model of primary neuron cultures. Then,the mechanisms of neuron injuries and the interventional effects of Sal B were systematically investigated in terms of excitotocitity,calcium overload,oxidant stress and neuron apoptosis.Therefore,the research work is divided into four parts:1、The establishment of the oxygen-glucose deprivation(OGD) model and the determination of effective dose of Sal B.Objective:To establish a stable and reliable OGD model;to determine the effective dose of Sal B.Methods:Primary cortical neuron cultures were preformed by using neonate rats.The neurons which grew for 8～10 d in vitro were treated by OGD for 2 h、3 h、4 h、5 h and the dissolved oxygen concentrations in the cultures media were measured by an oxygen measuring tester.The neurons vitality and survival rate were determined by MTT assay.Results:The primary cultures were in good conditions.The percentage of NSE-positive neurons reached 70～80%,Nissl’s staining showed the Nissl’s bodies were abundant in the neurons.The dissolved oxygen concentrations decreased as the time of OGD prolonged,the neurons vitality and survival rate also reduced.There was significant difference between the control group and the model group at various time points(P＜0.01).Since the cell damages were moreserious at OGD 4 h,we selected it as the duration of OGD in further experiments. After OGD 4 h,the neurons vitality declined(P＜0.01);the Sal B treatment group included eight different doses:10μg/L,100μg/L,1mg/L,5 mg/L,7.5 mg/L,10 mg/L,25 mg/L and 50 mg/L.The neurons vitality in the Sal B treatment group of 10 mg/L,25 mg/L and 50 mg/ L increased obviously compared with that in the OGD 4 h group(P＜0.01).There was a relationship between the dose of Sal B and the neurons vitality.In addition,normal neurons were treated by adding 10 mg/L,25 mg/L and 50 mg/L Sal B,respectively.Their vitalities had no significant difference,as compared with the control group.The final concentration of 10 mg/L was determined as the dosage of Sal B in the following experiments.2、The study of OGD induced neurons injury and the interventional mechanisms of Sal B.Objective:To study OGD induced neurons injury and the interventional mechanisms of Sal B.Methods:Primary cultured rat cortical neurons were randomly divided into the control group,the OGD 4 h group and the Sal B treatment group.The cell model was established by depriving of oxygen and glucose for 4 h.The cortical neurons vitality and survival rate were determined by MTT assay.The leakage rate of lactate dehydrogenase(LDH) and the content of glutamate in neuron medium were measured by chromatometry.The mitochondria membrane potential(MMP),the cytosolic free calcium concentration were quantitatively analyzed by flow cytometry.The neuronal morphous and ultrastructure were observed with an inverted phase contrast microscope,HE staining and a transmission electron microscope (TEM),respectively.Results:After OGD 4 h,the cortical neurons vitality,the survival rate and the fluorescence intensity of MMP reduced,the leakage rate of LDH,the content of glutamate in neuron medium and the cytosolic free calcium increased.There was significant difference between the OGD 4 h group and the control group(P＜0.01).In the Sal B treatment group the cortical neurons vitality,the survival rate and the fluorescence value of MMP were obviously higher than those in the OGD 4 h group,the cytosolic free calcium and the content of glutamate in the medium were significantly lower than those in the OGD 4 h group.Obvious difference was observed between the two groups(P＜0.05～0.01).In addition,the shapes of normal neuronal bodies were pyramidal,cell organs were abundant.In OGD 4 h group,most of the neuronal bodies and mitochondria were markedly swollen,the amount of cell organs decreased.The neuronal bodies of Sal B treatment group were swollen slightly,ultrastructure was still complete.3、The study of OGD-reintroduction(OGD-R) induced neurons injury and the interventional mechanisms of Sal B. Objective:To investigate OGD-R induced neurons oxidative injury and to confirm the interventional mechanisms of Sal B related to its antioxidation in vitro.Methods:Primary neuron cultures were randomly divided into the control group,the OGD-R group and the Sal B treatment group.The cell model was established by depriving of oxygen and glucose for 3 h and reintroducing for 1 h,3 h,6 h,12 h,18 h and 24 h, respectively.The neurons vitality and survival rate were determined by MTT assay.Then,the OGD-R 3 h and OGD-R 24 h were selected as the two timepoints for the following experiments.The leakage rate of LDH was measured by chromatometry.The level of cellular ROS was detected by fluorescent labeling method and spin trapping technique.The activities of neuronal Mn-SOD,CAT and GSH-PX were assaied by chromatometry.The morphologies were observed with an inverted phase contrast microscope.Results:The neurons vitality and the survival rate in the OGD-R group were lower than those in the control group at six different timepoints(P＜0.05～0.01).Except OGD-R 1 h and 12 h,the neurons vitality and the survival rate of Sal B treatment group were obviously higher than those in the OGD-R group(P＜0.05).After OGD-R 3 h、24 h、the leakage rate of LDH, the level of cellular ROS increased obviously compared with the control group(P＜0.05～0.01).In the Sal B treatment group the leakage rate of LDH,the level of cellular ROS were lower than those in the OGD-R group(P＜0.05～0.01).The activities of neuronal Mn-SOD、CAT and GSH-PX reduced in the OGD-R group at the two timepoints,there was obvious difference between the control group and the OGD-R group(P＜0.05～70.01).The activities of these antioxidases in the Sal B treatment group were higher than those in the OGD-R group (P＜0.05～0.01).Moreover,neuronal appearance changed after OGD-R 3 h,neurons shrinked and part of neurons died at OGD-R 24 h.The morphology of neurons in the Sal B treatment group changed slightly.4、The study of OGD-R induced neurons apoptosis and the anti-apoptosis mechanisms of Sal B.Objective:To study OGD-R induced neurons apoptosis and the anti-apoptosis mechanisms of Sal B.Methods:Primary neuron cultures were randomly divided-into the control group,the OGD-R 24 h group and the Sal B treatment group.The cell model was established by depriving of oxygen and glucose for 3 h and reintroducing for 24 h.The cytosolic free calcium was assessed using laser scanning confocal microscopy(LSCM).The MMP and the apoptosis rate were quantitatively analyzed by flow cytometry.The expression of Bcl-2、Bax were observed by immunohistochemical method.The release rate of Cytochrome C was detected by western blot.The morphology of neuronal nuclei was observed by Hoechst 33342 fluorescence staining.The neuronal ultrastructure was observed by TEM.Results:The fluorescence intensity of cytosolic free calcium and the apoptosis rate in the OGD-R 24 h group were higher than those in the control group,the fluorescence value of MMP in the OGD-R 24 h group was lower than that in the control group.There was signiflcant difference between the two groups(P＜0.01).Compared to the OGD-R 24 h group, the fluorescence intensity of cytosolic free calcium and the apoptosis rate in the Sal B treatment group were significantly decreased(P＜0.01),the fluorescence value of MMP in the Sal B treatment group was obviously increased(P＜0.01).The expression level of Bcl-2 decreased and the expression level of Bax increased in the OGD-R 24 h group,there was significant difference compared with the control group(P＜0.01).In the Sal B treatment group,the level of Bcl-2 expression was higher and the level of Bax expression lower than those in the OGD-R 24 h group(P＜0.05).The content of cytosolic Cytochrome C in the control group was very low,Cytochrome C existed in mitochondria mainly.In the OGD-R 24 h group the content of cytosolic Cytochrome C raised markedly compared with the control group(P＜0.01),this index decreased in the Sal B treatment group,there was obvious difference between the two groups(P＜0.05).In the OGD-R 24 h group the release rate of Cytochrome C was higher than that in the control group(P＜0.01),in the Sal B treatment group the release rate was lower than that in the OGD-R 24 h group,there was obvious difference between the two groups(P＜0.05).The nuclei showed adqulis blue fluorescence in the control group.Chromatin condensation,intensive blue fluorescence and nuclear fragmentation were observed in the OGD-R 24 h group.In the Sal B treatment group,most of the nuclei had light blue or blue fluorescence similar to the control group,only individual nuclei were stained densely and had bright blue fluorescence.In the OGD-R 24 h group, shrunken nuclei with condensed and marginated chromatin and the intact plasma membrane were observed by TEM,these showed the morphological changes of apoptosis.In the Sal B treatment group,the neuronal ultrastructures changed slighter,in comparison with these in OGD-R 24 h group.Conclusion:We successfully established a stable and reliable primary neuron cultures system. OGD-induced neuronal injury can be achieved by in vitro model.After OGD 4 h,the content of glutamate in neuron medium increased,cytosolic calcium overload and the fluorescence intensity of MMP reduced.The interventional mechanisms of Sal B in the OGD neurons would be due to the fact that Sal B can diminish the content of glutamate in neuron medium and inhibit calcium overload and maintain the MMP.OGD-R-induced neurons injury is concerned with oxidative stress,Sal B can protect neurons from OGD-R-induced injury through scavenging the cellular ROS and enhancing the activities of Mn-SOD、CAT、GSH-PX. Neurons apoptosis can be induced by OGD-R 24 h,Sal B can reduce neurons apoptosis through conserving the function of mitochondria,elevating the ratio of Bcl-2/Bax, diminishing the release of pro-apoptotic substance.To sum up,the pathogenesis of OGD is related to the excitotocitity,calcium overload, the pathogenesis of OGD-R is involved in oxidant stress and neuron apoptosis by the mitochondrial pathway.Sal B can protect the OGD neurons through relieve excitotocitity, inhibit cytosolic calcium overload,it also has neuroprotective effects on the OGD-R neurons by improving the cellular redox state,preserving mitochondria and resisting apoptosis.更多还原