【作者】 李国风；

【导师】 张建新；

【作者基本信息】 河北医科大学 ， 药理学， 2012， 博士

【摘要】 中风是导致残疾的重要原因之一，其损伤的相关危险因素主要包括：兴奋性损害、Ca²⁺超载、自由基及脂质过氧化、线粒体功能障碍、一氧化氮（nitric oxide, NO）、细胞凋亡、细胞因子等。目前，溶栓仍为临床治疗脑缺血的主要手段，但一部分病人在溶栓治疗后由于再灌注等原因导致病情加重。因此，有关脑缺血损伤的发病机制、预防措施及治疗手段成为当今医学研究的热点之一。大脑中动脉闭塞（MCAO）模型因其手术方法简单、动物损伤较小、成功率高而作为局灶性脑缺血损伤模型广泛应用于脑缺血损伤机制的研究。越来越多的证据表明硫化氢（hydrogen sulfide，H₂S）广泛参与机体多种病理生理过程。H₂S已经被认为是继NO和一氧化碳（carbon monoxide, CO）之后的第三种气体信号分子。研究表明，H₂S可通过抗凋亡和抗氧化应激而改善肝缺血再灌注损伤和心肌缺血再灌注损伤。H₂S对脑缺血损伤的影响报道较少，其机制尚不清楚。本研究采用线栓法复制大鼠局灶性脑缺血损伤模型，观察H₂S对局灶性脑缺血损伤的影响，并从氧化应激、凋亡和炎症细胞因子方面探讨H₂S可能的作用机制。第一部分硫化氢对大鼠局灶性脑缺血损伤的影响目的：观察硫化氢（H₂S）对局灶性脑缺血损伤大鼠脑组织硫化氢/3-巯基丙酮酸转硫酶（H₂S/3MST）及脑缺血体积的影响。方法：健康成年雄性SD大鼠（250-280g）共80只，随机分为5组，每组16只，分别为假手术组；缺血模型组；NaHS低剂量组；NaHS中剂量组；NaHS高剂量组。NaHS低、中、高剂量组分别于大鼠脑缺血3h时腹腔注射0.7mg/kg、1.4mg/kg和2.8mg/kg的NaHS，假手术组和缺血模型组大鼠注射等容量的生理盐水。各组大鼠均于缺血24h断头取脑，TTC染色测定脑梗死灶体积，测定脑组织中H₂S含量，改良法测定脑组织3MST的活性。结果：与假手术组比较，缺血模型组大鼠脑梗死体积明显增大（P<0.01），脑组织中H₂S含量、3MST活性明显降低（P<0.01）；与缺血模型组比较，NaHS中、高剂量组大鼠脑梗死体积明显缩小，脑组织中H₂S含量和3MST活性明显升高（P <0.05）。结论：NaHS中、高剂量可明显缩小脑缺血体积，增强脑组织3MST活性，升高脑组织H₂S含量，表明NaHS对大鼠局灶性脑缺血损伤具有一定治疗作用。第二部分硫化氢对局灶性脑缺血损伤大鼠脑组织氧化应激的影响目的：观察H₂S对局灶性脑缺血损伤大鼠脑组织氧化应激的影响，并探讨其作用机制。方法：健康成年雄性SD大鼠（250-280g）共40只，随机分为5组，每组8只，分别为假手术组；缺血模型组；NaHS低剂量组；NaHS中剂量组；NaHS高剂量组。10%的水合氯醛350mg/kg麻醉大鼠，采用线栓法复制大鼠大脑中动脉闭塞模型，假手术组颈部切开，分离颈总动脉、颈内动脉和颈外动脉但不插线栓。NaHS低、中、高剂量组分别于大鼠脑缺血3h时腹腔注射0.7mg/kg、1.4mg/kg和2.8mg/kg的NaHS，假手术组和缺血模型组注射等容量的生理盐水。各组大鼠均于缺血24h时断头取脑，采用硫代巴比妥酸法测定脑组织中丙二醛（MDA）的含量、黄嘌呤氧化酶化学法测定脑组织中超氧化物歧化酶（SOD）的活性以及酶促反应谷胱甘肽消耗法测定脑组织中谷胱甘肽过氧化物酶（GSH-PX）的活性；透射电镜观察脑组织的病理变化。结果：1与假手术组比较，缺血模型组大鼠脑组织中SOD、GSH-PX活性明显降低（P <0.01，P <0.01），MDA含量明显升高（P <0.01）；与缺血模型组比较，NaHS中、高剂量组大鼠脑组织中SOD、GSH-PX活性明显升高（P <0.05，P <0.05），MDA含量明显降低（P <0.05）。2电镜观察结果显示，假手术组大鼠脑组织神经细胞核膜清晰、完整，核大而圆，微管、粗面内质网、高尔基复合体等细胞器清晰可见，线粒体内膜、外膜结构清晰，嵴排列整齐；缺血模型组大鼠可见明显神经元水肿，线粒体膜明显肿胀、线粒体嵴断裂甚至消失，线粒体大量空泡化，细胞器的数量减少等；与缺血模型组比较，NaHS中、高剂量组大鼠可见神经元轻度水肿，线粒体部分肿胀、内外膜结构清晰、部分嵴断裂消失，细胞器数量增多，脑缺血损伤程度明显减轻。结论：局灶性脑缺血损伤后氧化应激状态发生改变，脑组织脂质过氧化物MDA生成增多，抗过氧化物酶SOD和GSH-PX活性减弱；应用NaHS后，脑组织脂质过氧化物MDA生成减少，抗过氧化物酶SOD和GSH-PX活性增强，脑组织损伤明显减轻。第三部分硫化氢对局灶性脑缺血损伤大鼠脑组织神经元凋亡的影响目的：观察H₂S对大鼠局灶性脑缺血损伤所致的神经元凋亡的影响,从神经元凋亡途径探讨H₂S对脑缺血损伤的作用及其机制。方法：健康成年雄性SD大鼠（250-280g）共40只，随机分为5组，每组8只，分别为假手术组；缺血模型组；NaHS低剂量组；NaHS中剂量组；NaHS高剂量组。10%的水合氯醛350mg/kg麻醉大鼠，采用线栓法复制大鼠大脑中动脉闭塞模型，假手术组颈部切开，分离颈总动脉、颈内动脉和颈外动脉但不插线栓。NaHS低、中、高剂量组分别于大鼠脑缺血3h时腹腔注射0.7mg/kg、1.4mg/kg和2.8mg/kg的NaHS，假手术组和缺血模型组注射等容量的生理盐水。各组大鼠均于缺血24h时断头取脑，采用HE染色法观察脑组织形态学变化，Tunel法检测神经细胞凋亡率，免疫组织化学法检测Bcl-2和Bax在脑组织中的定位和表达，Westernblot法分析Caspase-3在脑组织中表达的变化。结果：1HE染色结果显示，假手术组大鼠神经细胞核仁清晰、核圆形，核膜完整，胞浆染色正常，未见任何病理变化。缺血模型组缺血侧脑组织出现严重的神经细胞坏死，细胞肿胀，胞核浓缩，胞浆疏松淡染及空泡化。NaHS中、高剂量组大鼠脑组织上述病理变化较缺血模型组明显减轻。2Tunel检测神经细胞凋亡率结果显示，与假手术组比较，缺血模型组大鼠神经细胞凋亡率明显升高（P<0.01）；与缺血模型组比较，NaHS中、高剂量组大鼠神经细胞凋亡率明显降低（P<0.05）。3免疫组化检测结果显示， Bcl-2和Bax蛋白阳性染色呈棕黄色，主要在神经细胞胞浆表达；假手术组可见少量的Bcl-2和Bax蛋白阳性细胞；与假手术组比较，缺血模型组脑组织Bcl-2阳性细胞表达数目明显减少，Bax阳性细胞表达明显增多（P<0.01）；与缺血模型组比较，NaHS中、高剂量组大鼠脑组织Bax阳性细胞数明显减少，Bcl-2阳性细胞数明显增多（P<0.05）。4Western blot检测结果显示，假手术组仅有少量Caspase-3阳性蛋白表达；与假手术组比较，缺血模型组大鼠脑组织Caspase-3蛋白表达明显升高（P<0.01）；与缺血模型组比较，NaHS中、高剂量组大鼠脑组织Caspase-3蛋白表达明显下降（P<0.01）。结论：H₂S可以明显降低局灶性脑缺血损伤引起的神经元凋亡，其抗凋亡作用可能与降低Bax和Caspase-3蛋白表达、增加Bcl-2蛋白表达有关。第四部分硫化氢对局灶性脑缺血损伤大鼠脑组织炎性细胞因子的影响目的：观察H₂S对局灶性脑缺血损伤大鼠脑组织NF-κB活性及其下游的炎性因子肿瘤坏死因子α（TNF-α）、白介素-1β（IL-1β）和白介素-10（IL-10）的影响，从炎性细胞因子方面探讨H₂S对局灶性脑缺血损伤的作用及其机制。方法：健康成年雄性SD大鼠（250-280g）共40只，随机分为5组，每组8只，分别为假手术组；缺血模型组；NaHS低剂量组；NaHS中剂量组；NaHS高剂量组。10%的水合氯醛350mg/kg麻醉大鼠，采用线栓法复制大鼠大脑中动脉闭塞模型，假手术组颈部切开，分离颈总动脉、颈内动脉和颈外动脉但不插线栓。NaHS低、中、高剂量组分别于大鼠脑缺血3h时腹腔注射0.7mg/kg、1.4mg/kg和2.8mg/kg的NaHS，假手术组和缺血模型组注射等容量的生理盐水。各组大鼠均于缺血24h腹主动脉取血并断头取脑，ELISA法测定血清、脑组织中TNF-α、IL-1β和IL-10的含量。免疫组织化学染色分析脑组织中NF-κB的核移位，Western blot检测脑组织NF-κB的表达。结果：1与假手术组比较，缺血模型组大鼠的血清与脑组织匀浆中IL-1β、TNF-α水平显著升高，IL-10水平显著降低（P <0.01）；与缺血模型组比较，NaHS低、中、高剂量组大鼠的血清中IL-1β、TNF-α水平显著降低，IL-10水平显著升高（P <0.05，P <0.01），NaHS中、高剂量组大鼠脑组织匀浆中IL-1β、TNF-α水平显著降低，IL-10水平显著升高（P <0.01）。2与假手术组比较，缺血模型组大鼠的脑组织中NF-κB表达显著增加（P<0.05），明显从细胞浆移位于细胞核；与缺血模型组比较，NaHS中、高剂量组大鼠脑组织中NF-κB从细胞浆向细胞核的移位被明显抑制（P<0.01），NF-κB的表达也明显降低。结论：H₂S可明显抑制局灶性脑缺血损伤后脑组织NF-κB的核移位，减少其蛋白表达，下调TNF-α、IL-1β，上调IL-10炎症相关细胞因子的表达，从而减轻局灶性脑缺血损伤。小结:1NaHS可明显增强局灶性脑缺血损伤大鼠脑组织3MST活性，增加脑组织H₂S生成，缩小脑梗死体积。提示H₂S可改善局灶性脑缺血损伤。2应用NaHS后局灶性脑缺血损伤大鼠脑组织损伤减轻，同时MDA生成减少，SOD和GSH-PX活性升高。提示H₂S可通过降低脂质过氧化反应减轻局灶性脑缺血损伤。3NaHS可明显降低局灶性脑缺血损伤大鼠神经细胞凋亡率，减弱Bax和Caspase-3蛋白表达，增强Bcl-2蛋白表达，减轻神经元损伤。H₂S可能通过抑制局灶性脑缺血损伤大鼠神经元凋亡改善脑缺血损伤。4大鼠局灶性脑缺血损伤后，NF-κB蛋白表达增强，血清和脑组织中TNF-α、IL-1β含量升高，IL-10含量下降； NaHS可明显抑制局灶性脑缺血损伤发生时NF-κB的活化，下调TNF-α、IL-1β的表达，上调IL-10的表达，从而减轻局灶性脑缺血损伤。更多还原

【Abstract】 Strokes caused by ischemia （lack of blood flow）, blockage or hemorrhageare major causes of mortality and long-term disability. The dangerous factorsof cerebral ischemia injury include Ca2+overload, free radical, nitric oxide（NO）, cell apoptosis, cytokines and so on. Therefore, the prevention,mechanism and treatment of cerebral ischemic injury are the points of medicalresearch at present. Among the various rodent models of stroke, middlecerebral artery occlusion （MCAO） is a simple surgical approach with minimaltrauma and is widely used to reconstruct focal cerebral ischemic model.During the past decade, increasing lines of evidence have shown thathydrogen sulfide （H₂S） has important physiological functions, especially inthe nervous system. Now H₂S is recognized as the third endogenous signalinggasotransmitter, following NO and carbon monoxide （CO）. H₂S has beenshown to attenuate hepatic and myocardial ischemia-reperfusion injuries viaantioxidant and anti-apoptotic signaling. In the present study, we used MCAOas focal cerebral ischemic model to evaluate the effects of H₂S on focalcerebral ischemia in rats and explored the possible mechanisms.Part1Effects of hydrogen sulfide on focal cerebral ischemia injury inratsObjective: To investigate the effects of hydrogen sulfide on focalcerebral ischemia injury in rats.Methods: Eighty male SD rats （weigh250-280g） were randomly dividedinto five groups （n=16）: sham group, ischemia group, NaHS low, middle andhigh dose groups. Rats were anesthetized by10%chloral hydrate at350mg/kg. Focal cerebral ischemia model was reconstructed by inserting craniallya nylon thread with rounded tip into internal carotid artery. Rats were onlyunderwent a surgical operation but without ischemia in the sham group. The NaHS （0.7mg,1.4mg and2.8mg/kg） were respectively administrated at3hafter ischemia in rats. The equal volume of saline was administrated in thesham and the ischemia groups. Rats were sacrificed at24h after ischemia.Half of the rats in each group were used for measurement of the infarctvolumes by TTC staining. The others in each group were used fordetermination of the content of H₂S and the activity of3MST in the braintissue.Results:Compared with those of the group sham, the infarct volume wassignificantly increased, the content of H₂S and the activity of3MST in thebrain were significantly decreased in those of the ischemia group （P<0.01）.Compared with those of the ischemia group, the infarct volume wassignificantly decreased, the content of H₂S and the activity of3MST weresignificantly increased in those of the NaHS middle and high dose groups （P<0.05, P <0.01）.Conclusion: Administration of NaHS could decrease the infarct volume,increase the activity of3MST and the content of H₂S. It could be concludedthat H₂S may play the protective role against focal cerebral ischemic injury.Part2Effects of hydrogen sulfide on oxidative stress in focal cerebralischemic injury in ratsObjective: To investigate the effects of H₂S on focal cerebral ischemicinjury in rats and explore the possible mechanisms.Methods: Forty male SD rats （weigh250-280g） were randomly dividedinto five groups （n=8）: sham group, ischemia group, NaHS low dose, middledose and high dose groups. Rats were anesthetized by10%chloral hydrate at350mg/kg. Focal cerebral ischemia model was reconstructed by insertingcranially a nylon thread with rounded tip into internal carotid artery. Rats wereonly underwent a surgical operation but without ischemia in the sham group.The NaHS （0.7mg,1.4mg and2.8mg/kg） were respectively administrated at3h after ischemia in rats. The equal volume of saline was administrated in thesham and the ischemia groups. Rats were sacrificed at24h after ischemia. The content of malondialdehyde （MDA）, and the activities of superoxidedismutase （SOD） and glutathione peroxidase （GSH-PX） in the brain tissuewere respectively measured. The ultrastructure changes of neurons wereobserved by transmission electron microscope.Results:1The activities of SOD and GSH-PX in the brain tissue weresignificantly decreased, the content of MDA in the brain tissue weresignificantly increased in the ischemia group compared with those of the shamgroup （P<0.01）. The activities of SOD and GSH-PX in the brain tissue weresignificantly increased, the content of MDA in the brain were significantlydecreased in the NaHS middle and high dose groups compared with those ofthe ischemia group （P<0.01）.2Transmission electron microscope showed the neuronal cytoplasm andthe mitochondria are normal in the sham group. The neuronal cytoplasm andthe mitochondria swelled, the cristae of mitochondria disrupted, dissolved ordisappeared, the amounts of organelles are significantly reduced in theischemia group compared with those of the sham group. The above injurieswere significantly ameliorated in the NaHS middle and high dose groupscompared with those of the ischemia group.Conclusion: Administration of NaHS could increase the activities ofSOD and GSH-PX and decrease the content of MDA. It could be concludedthat the protection role of H₂S on focal cerebral ischemic tissue is related todiminishing oxidative stress.Part3Effects of hydrogen sulfide on apoptosis in focal cerebral ischemiainjury in ratsObjective: To study the effects of H₂S on neruronal apoptosis in focalcerebral ischemic injury in rats and explore the possible mechanism.Methods: Forty male SD rats （weigh250-280g） were randomly dividedinto five groups （n=8）: sham group, ischemia group, NaHS low, middle andhigh dose groups. Rats were anesthetized by10%chloral hydrate at350mg/kg. Focal cerebral ischemia model was reconstructed by inserting cranially a nylon thread with rounded tip into internal carotid artery. Rats were onlyunderwent a surgical operation but without ischemia in the sham group. TheNaHS （0.7mg,1.4mg and2.8mg/kg） were respectively administrated at3hafter ischemia in rats. The equal volume of saline was administrated in thesham and the ischemia groups. Rats were sacrificed at24h after ischemia. Thepathological changes of brain tissue were observed with light microscope byHE staining. The neuronal apoptosis were assayed by TUNEL detection. Theexpressions of Bcl-2and Bax in the brain tissue were respectively detected byimmunohistochemisty. The expression of Caspase-3in the brain tissue wasanalyzed by Western blot.Results:1HE staining showed that the neurons are normal in the sham group. Thevascular dilatation, some of neurons cellular swelling, cell nucleusconcentrating，cytoplasmic rarefaction, staining weakly, vacuole formation inthe ischemia group. The pathological alterations were obviously amelioratedin the NaHS middle and high dose groups compared with those of theischemia group.2The apoptotic rate of neurons was significantly increased in theischemia group compared with that of the sham group （P<0.01）. Comparedwith that of the ischemia group, the apoptotic rate of neurons was significantlydecreased in the NaHS middle and high dose groups （P<0.05or P<0.01）.3Immunohistochemisty showed the expression of Bcl-2wassignificantly decreased, the expression of Bax was significantly increased inthe ischemia group compared with those of the sham group （P<0.01）. Theexpression of Bcl-2was significantly increased, the expression of Bax wassignificantly decreased in the NaHS middle and high dose groups comparedwith those of the ischemia group （P<0.05or P<0.01）.4Western blot showed that there was only a small amount of expressionof Caspase-3in the sham group. The expression of Caspase-3wassignificantly increased in the ischemia group compared with that of the shamgroup. The expression of Caspase-3was significantly decreased in the NaHS middle and high dose groups compared with that of the ischemia group.Conclusion: Administration of NaHS could decrease the apoptotic rateof neurons, attenuate the expression of Bax and Caspase-3, and increase theexpression of Bcl-2in brain tissue in focal cerebral ischemic injury in rats.The results showed that H₂S may play a protective role against focal cerebralischemic injury by inhibiting neuronal apoptosis.Part4Effects of hydrogen sulfide on inflammatory factors in focalcerebral ischemia injury in ratsObjective: To investigate the effects of H₂S on focal cerebral ischemicinjury in rats and explore the possible mechanisms.Methods: Forty male SD rats （weigh250-280g） were randomly dividedinto five groups （n=8）: sham group, ischemia group, NaHS low, middle andhigh dose groups. Rats were anesthetized by10%chloral hydrate at350mg/kg. Focal cerebral ischemia model was reconstructed by inserting craniallya nylon thread with rounded tip into internal carotid artery. Rats were onlyunderwent a surgical operation but without ischemia in the sham group. TheNaHS （0.7mg,1.4mg and2.8mg/kg） were respectively administrated at3hafter ischemia in rats. The equal volume of saline was administrated in thesham and the ischemia groups. Rats were sacrificed at24h after ischemia. Thecontents of TNF-α, IL-1β and IL-10were respectively measured byenzyme-linked immunosorbent assay （ELISA）. The transposition of nuclearfactor-κB （NF-кB） in nucleus was detected by immunohistochemisty. Theexpression of NF-кB in the brain tissue was detected by Western blot.Results:1The contents of TNF-α and IL-1β in serum and brain tissue weresignificantly increased in the ischemia group in rats compared with those ofthe sham group （P<0.01）. The contents of IL-10in serum and brain tissuewere significantly decreased in the ischemia group in rats compared with thoseof the sham group （P<0.01）. The contents of TNF-α and IL-1β in serum andbrain tissue were significantly decreased. The content of IL-10in serum andbrain tissue were significantly increased in the NaHS middle and high dose groups compared with those of the ischemia group （P<0.05or P<0.01）.2The NF-κB was significantly translocated from the neurons cytoplasminto the nucleus and the expression of NF-κB was significantly increased inthe ischemia group compared with those of the sham group（P<0.05）. In theNaHS middle and high dose groups, the NF-κB translocation was markedlyinhibited and the expression of NF-κB in the nuclei was obviously decreasedcompared with those of the ischemia group （P﹤0.05）.Conclusion: Administration of NaHS could inhibit NF-κB activation,decrease the expression of TNF-α and IL-1β, increase the expression of IL-10,which may be one of the molecular mechanisms of its neuroprotection.Summary:1Administration of NaHS could decrease the infarct volume, increase theactivity of3MST and the content of H₂S. H₂S may play the protective roleagainst focal cerebral ischemic injury.2Administration of NaHS could increase the activities of SOD andGSH-PX and decrease the MDA content. The protection role of H₂S on focalcerebral ischemic tissue may be related to diminishing oxidative stress.3Administration of NaHS could decrease the apoptotic rate of neurons,attenuate the expression of Bax and Caspase-3, and increase the expression ofBcl-2in brain tissue in focal cerebral ischemic injury in rats. H₂S may play aprotective role against focal cerebral ischemic injury by inhibiting neuronalapoptosis.4Administration of NaHS could inhibit NF-κB activation, decrease theexpression of TNF-α and IL-1β, increase the expression of IL-10, which maybe one of the molecular mechanisms of its neuroprotection.更多还原