【作者】 刘玲；

【导师】 董强； 任惠民； 肖保国；

【作者基本信息】 复旦大学 ， 神经病学， 2009， 博士

【摘要】 组织激肽释放酶对离体缺血／缺氧性损伤的影响及其机制研究激肽释放酶-激肽系统（kallikrein-kinin system，KKS）是机体重要的炎性调节系统，广泛分布于人及其他哺乳动物的中枢神经系统内，在脑血管病、中枢神经系统变性、炎症、肿瘤等多种疾病中发挥作用。组织激肽释放酶（tissue kallikrein，TK）是KKS的重要组成部分，主要通过其效应物质缓激肽（bradykinin，BK）激活Bl受体（bradykinin Blreceptor，BlR）和B2受体（bradykinin B2 receptor，B2R）发挥病理生理作用。近年来越来越多的研究报道TK在脑缺血缺氧性损伤中起保护作用，但除了明确它的保护作用主要涉及到B2R及相关的信号通路外，其具体的神经保护机制并不清楚。目前认为脑缺血缺氧性损伤的核心是各种途径诱发的细胞内Ca²⁺超载。引起缺血缺氧损伤细胞内Ca²⁺超载的机制包括谷氨酸依赖的和酸敏感离子通道（acid sensing ion channels，ASICs）等介导的谷氨酸非依赖的Ca²⁺毒性损伤途径。那么，TK与缺血缺氧损伤中谷氨酸依赖的和非依赖的Ca²⁺毒性损伤机制的关系如何呢?本研究利用体外培养的大鼠皮层神经元，分别建立离体谷氨酸损伤模型、酸中毒、缺氧-酸中毒损伤模型，模拟在体缺血缺氧／再灌注过程，分别观察TK对谷氨酸介导的神经毒性损伤和ASICs介导神经元毒性的影响，并初步探索其机制。第一部分组织激肽释放酶对谷氨酸诱导的神经毒性损伤的影响及其机制一组织激肽释放酶对谷氨酸诱导的神经毒性损伤的影响目的本研究在无Mg²⁺的条件下，将离体培养的大鼠皮层神经元，暴露于100μmol/L谷氨酸（glutamate，Glu）和10μmol／L甘氨酸（Glycine，Gly）1 h，然后再恢复正常培养，建立离体神经元Glu毒性损伤模型，模拟在体缺血／再灌注过程。观察高浓度Glu对离体神经元的损伤作用及TK干预对其影响。方法1．通过检测Glu暴露后恢复正常培养不同时间（2 h，4 h，6 h及24 h）时，释放到上清液里的LDH量，了解Glu对神经细胞的毒性作用；2．采用CCK-8活细胞计数试剂盒检测神经元的存活率，了解Glu对神经细胞活力的影响。我们通过检测不同浓度TK、谷氨酸受体（GluR）拮抗剂、B2R激动剂和抑制剂干预组在Glu损伤后24 h时的LDH释放率和存活率，比较它们对Glu毒性作用的影响；3．采用免疫荧光法标记成熟神经元标记物MAP-2，观察TK和GluR阻断剂对Glu暴露1h后恢复培养3 h时神经元形态学的影响；4．用Hoechest 33342荧光核染液和TUNEL法检测Glu暴露后24 h时不同干预对神经元凋亡的影响。结果1．LDH测定结果显示：暴露于Glu 1 h后在恢复正常培养过程中，LDH释放率进行性增加，TK可浓度依赖性抑制Glu暴露后24 h时LDH的释放，激活B2R和阻断GluR也有类似作用。2．TK处理、阻断GluR、激活B2R均能提高Glu损伤后24 h时神经元的活力。3．MAP-2免疫荧光染色显示TK和GluR抑制剂干预均能显著抑制Glu暴露后恢复培养3 h对神经元形态的破坏，抑制胞体肿胀、轴突串珠样变或断裂等。4．Hoechest核染色和TUNEL染色的研究显示TK、B2R激动剂和GluR阻断剂均能降低Glu暴露后24 h时神经细胞核的固缩率和TUNEL阳性细胞率。上述TK的作用均能够被B2R抑制剂所阻断。结论短暂高浓度Glu暴露后恢复培养（再灌注）能够诱导严重的神经元损伤，TK预处理、激活B2R以及阻断GluR（NMDA和．AMPA受体）均能拮抗Glu诱导的毒性损伤，而抑制B2R能够阻断TK的作用。提示TK通过B2R，能够拮抗Glu诱导的神经毒性损伤。二组织激肽释放酶对谷氨酸诱导的氧化／硝基化应激反应的影响目的在离体Glu毒性损伤模型上，观察TK对Glu诱导的氧化／硝基化应激反应的影响。方法培养8～10 d的大鼠皮层神经元，随机分为正常对照、TK、B2R激动剂和抑制剂以及GluR抑制剂组，分别暴露于含Glu（100μmol／L，）和Gly（10μmol／L）的无Mg²⁺的细胞液中，并在Glu处理前以及处理0．5 h，1 h，2 h，5 h以及6 h时：1．采用DCFH-DA荧光探针标记细胞内活性氧（ROS），在荧光显微镜下直接观察细胞内DCF绿色荧光，同时采用荧光酶标仪上，在激发波长488 nm，发射波长525 nm处检测细胞内DCF的相对荧光单位（Relative fluorescent unit，RFU），根据RFU计算出细胞内ROS的相对含量；2．采用DAF-FM／DA荧光探针标记细胞内一氧化氮（NO），采用荧光酶标仪上在激发波长495 nm，发射波长515 nm处检测细胞内DAF-FM的RFU，根据RFU计算出细胞内NO的相对产量；此外，在Glu处理2 h时，提供足量一氧化氮合酶（NOS）的反应底物L-精氨酸，然后用DAF-FM／DA荧光探针检测出细胞内NO的含量，以此代表总NOS的活性，分别测定出Ca²⁺存在与不存在时细胞内NO含量，以两者之差异表示nNOS的相对活性。3．采用荧光染料Rhodamine 123（Rh 123）标记活细胞的线粒体，用荧光酶标仪检测出激发波长507 nm，发射波长529 nm处Rh 123的RFU，根据RFU计算出线粒体膜电位水平（MMP）的相对水平；4．采用Ca²⁺敏感的荧光探针Fluo-3／AM标记细胞内的游离Ca²⁺，并用荧光酶标仪检测出激发波长488 nm，发射波长525 nm处细胞内Fluo-3的RFU，根据RFU计算出细胞内游离Ca²⁺水平。结果1．与正常对照相比，Glu暴露（0．5 h，1 h，2 h，5 h，6 h）能够时间依赖地诱导细胞内ROS和NO合成增加和游离Ca²⁺水平升高，MMP水平降低；2．TK、BK及GluR抑制剂可显著降低Glu损伤细胞内ROS和NO产量和游离Ca²⁺水平，对Glu暴露2 h时上述指标的影响最显著；3．Glu暴露2 h能够显著提高细胞内nNOS的活性，TK、BK及GluR抑制剂干预均能抑制nNOS活性升高；4．TK的作用能够部分被B2R抑制剂阻断。结论TK，通过B2R，能够拮抗Glu诱导氧化／硝基化应激性损伤，降低细胞内ROS和NO的产量、抑制nNOS的活性、延缓线粒体膜去极化以维持线粒体膜电位水平稳定并防止细胞内Ca²⁺超载。三组织激肽释放酶抗谷氨酸毒性作用的细胞内信号机制目的在离体Glu毒性损伤模型上，观察TK对Glu损伤细胞内MAPK、PI3K/Akt及NF-κB信号通路的磷酸化蛋白表达的影响，探索TK保护作用可能涉及的细胞内信号通路。方法1．应用免疫细胞化学法检测TK和GluR抑制剂干预对Glu暴露后恢复培养1 h时磷酸化ERK1／2（p-ERK1／2）表达的影响。2．采用Western blot定量检测Glu暴露后0 h和1 h时磷酸化的和总的ERK1／2、磷酸化Akt（p-Akt）、磷酸化JNK（p-JNK）、磷酸化P38（p-P38）以及磷酸化IKKα／β（p-IKKα／β）蛋白的表达水平，并比较TK、B2R激动剂和抑制剂、GluR抑制剂及ERK通路阻断剂PD98059干预对这些蛋白表达水平的影响。结果1．免疫细胞化学染色结果显示正常神经元、Glu损伤神经元和TK预处理的Glu损伤神经元，均可见观察到明显的p-ERKl／2红色荧光，但以TK预处理组的荧光强度最强。2．Western blot定量检测结果显示：①各干预对p-ERK1和2表达水平的影响不同：Glu暴露1 h能够极显著提高p-ERK1和2的表达水平至正常对照的153．7和82．4倍，Glu暴露后恢复培养1 h时p-ERK1和2的表达水平迅速降至正常对照的6．8和20．39倍；ERK抑制剂PD98059和GluR阻断剂几乎能够完全阻断Glu暴露及暴露后1 h时p-ERK1／2蛋白的表达；TK预处理极显著地增加Glu暴露神经元p-ERK1的表达水平，在暴露后0 h和损伤后1 h时p-ERK1的表达水平分别为正常对照的566．8和51．06倍；阻断B2R能够抑制TK对p-ERK1的表达水平的影响；B2R激动剂仅能增强Glu暴露后恢复1 h时p-ERK1的表达；TK和BK干预均不影响Glu暴露诱导p-ERK2蛋白表达的增加。②PI3K／Akt通路的Akt蛋白磷酸化水平在Glu暴露时受到抑制，而恢复正常培养后逐渐p-Akt表达逐渐恢复；虽然TK和B2R激动剂对Glu暴露后0 h及1 h神经元p-Akt蛋白水平的提高作用并不显著，但阻断B2R能够显著降低TK预处理组Glu暴露后1 h时的p-Akt蛋白的表达水平。③正常培养的大鼠皮层神经元p-P38的表达量极低，Glu暴露能显著提高神经细胞内p-P38蛋白表达水平，恢复正常培养后，其表达水平逐渐恢复；TK和BK对p-P38蛋白表达水平的抑制作用不明显。④Glu暴露能够提高p-JNK蛋白的表达水平，而暴露后恢复培养1h时，p-JNK表达水平基本回降至正常水平；TK和BK干预均能降低Glu暴露诱导的p-JNK蛋白表达增加；⑤正常培养的大鼠皮层神经元p-IKKα／β蛋白表达水平很低，暴露于Glu后p-IKKα／β表达水平明显提高；TK预处理能促进正常神经元和Glu暴露神经元表达p-IKKα/β蛋白，阻断ERK1／2信号通路可显著降低Glu损伤后1h时p-IKKα/β蛋白的表达水平。⑥此外，激活B2R或阻断GluR对上述通路磷酸化蛋白表达水平影响与TK干预相似，而阻断B2R则能够显著抑制TK对这些磷酸化蛋白表达的影响。结论1．高浓度Glu暴露能够抑制PI3K/Akt通路激活、而促进MAPK信号通路家族和NF-κB信号通路激活。2．TK对这些信号通路有不同的影响：显著促进NF-κB通路和ERK1／2信号途径尤其是ERK1激活，轻微提高PI3K／Akt信号通路的活化水平，显著抑制JNK通路而不影响P38通路的活化。激活B2R能够产生与TK类似的作用，阻断B2R能够显著抑制TK的作用。提示，TK主要通过激活B2R，影响多条信号通路的活化状态，而产生抗Glu神经毒性作用。第二部分组织激肽释放酶对酸敏感离子通道介导的神经毒性损伤的影响及机制一酸敏感离子通道激活对离体皮层神经元的影响目的采用培养的大鼠皮层神经元，建立各种离体酸中毒损伤模型，通过观察这些模型对神经元的损伤作用，了解酸敏感离子通道（ASICs）激活对神经元的影响。方法采用原代大鼠皮层神经元，在GluR抑制剂MK801、CNQX和Ca通道阻断剂尼莫地平以及Na通道阻断剂TTX存在的情况下，建立缺氧复氧（Oxygen and Glucose Deprivation，OGD）、缺氧-酸中毒后再灌注（OGD-Acidosis，OGD-Ac）、持续酸中毒（continuous acidosis，C-Ac）及酸中毒再灌注（acidosis，Ac）损伤模型。通过检测LDH释放率，了解上述模型对神经细胞的毒性作用；采用CCK-8活细胞计数试剂盒检测细胞存活率，了解上述模型对神经细胞活力的影响；采用免疫细胞荧光法标记成熟神经元标记物MAP-2，观察暴露于OGD和OGD-Ac 90 min再灌注4 h后神经元的形态变化。结果在MK801、CNQX、尼莫地平以及TTX存在的条件下：1．神经元暴露于pH6．0的酸性细胞液2 h后在恢复正常培养（再灌注）过程中，其LDH释放率进行性升高、存活率进行性降低，表明随再灌注时间延长神经元损伤进行性加重，至再灌注24 h时，LDH释放率为45．6％，存活神经元约58．6%。2．C-Ac暴露诱导更为严重的神经元损伤，LDH释放率升高和神经元存活率降低更显著，暴露24 h时存活的神经元仅10％左右；ASICs广谱阻断剂阿米洛利（Amiloride）或ASIC1a特异性阻断剂狼蛛毒素1（PcTX1）均能够抑制C-Ac暴露引发的LDH释放增加与神经元活力下降。3．神经元经OGD处理90 min后在复氧（再灌注）的过程中，出现进行性损伤，表现为时间依赖地LDH释放率升高和神经元存活率降低；而OGD-Ac处理90 min后再灌注对神经元的损伤较单纯OGD处理后再灌注诱发的损伤严重，在同一灌注时间点其LDH释放率更高，神经元存活率更低；ASIC1a特异性和非特异性阻断剂PcTX1和Amiloride均能够显著抑制OGD-Ac后再灌注过程LDH释放率的升高与神经元活力的降低。4．免疫荧光双标法标记神经元MAP-2和ASIC1a的研究结果显示：与OGD损伤神经元相比，OGD-Ac损伤神经细胞的形态改变更显著，破坏更严重，神经突起基本全部断裂，残存的突起也呈串珠样变，MAP-2阳性细胞减少更显著。结论尽管采用MK801、CNQX、尼莫地平以及TTX阻断了亲离子型谷氨酸受体、电压依赖的Ca²⁺和Na⁺通道，阻断了这些通道激活后诱发的细胞损伤，酸性细胞外环境（pH6．0）不仅本身能够诱导神经元损伤，而且可显著加重缺氧复氧神经元损伤，阻断ASICs尤其是ASIC1 a具有神经保护作用，能够减轻神经元损伤，促进神经元存活。提示我们建立的各种离体酸中毒模型是有效的，ASICs尤其是ASIC1a激活介导了酸中毒对神经元的损伤，产生了Glu非依赖的神经毒性损伤，而阻断ASIC1a激活有神经保护作用。二组织激肽释放酶对酸中毒诱导的离体神经元损伤的影响目的通过观察TK对各种离体酸中毒损伤模型引发的神经元损伤的影响，了解TK对ASICs尤其是ASIC1a介导的谷氨酸非依赖的神经毒性作用有无影响。方法采用原代大鼠皮层神经元，在MK801、CNQX、尼莫地平以及TTX存在的情况下，建立OGD、OGD-Ac、C-Ac及Ac后再灌注损伤模型，通过检测LDH释放率、神经元存活率以及神经元死亡率（PI/Hoechst染色检测），比较TK、B2R激动剂和ASIC1a各种阻断剂对各种模型诱发的神经损伤的影响；采用免疫荧光双标法标记神经元MAP-2和ASIC1a，观察TK对C-Ac处理3 h和OGD-Ac后再灌注4 h对神经元形态和ASIC1a表达的影响；采用TUNEL染色观察并比较TK和PcTX1对OGD-Ac损伤神经元凋亡情况的影响。结果1．LDH释放和细胞活力检测结果显示TK能够浓度依赖地降低对Ac后再灌注、C-Ac处理及OGD-Ac后再灌注损伤诱导的LDH释放增加和细胞活力下降。2．PI／Hoechst染色结果提示TK处理能够降低上述损伤诱导的PI阳性细胞率升高，即降低神经元死亡率。3．ASICs广谱阻断剂、ASIC1a特异性阻断剂及B2R激动剂干预对上述模型产生神经损伤的保护作用与TK处理相似，而B2R抑制剂HOE140能够降低TK的保护效应。4．TUNEL染色的结果提示TK和PcTX1均能显著减少OGD-Ac后再灌注6 h时神经元的凋亡。5．免疫荧光双标法的研究结果显示：TK能够减轻C-Ac处理3 h和OGD-Ac 90 min后再灌注4 h对神经细胞形态的破坏，但对ASIC1a的表达量无显著影响。结论TK能有效抑制各种单纯酸中毒和缺氧-酸中毒对离体神经元的损伤、减少神经元死亡和凋亡并促进神经元存活，具有ASICs阻断剂相类似的抗酸毒性损伤的神经保护作用。三组织激肽释放酶对神经元酸敏感离子通道各亚基表达的影响目的观察TK对正常神经元和缺氧后复氧不同时间的损伤神经元ASICs各亚基的基因水平和蛋白水平表达的影响。方法在OGD前和OGD后复氧不同时间（复氧1 h、2 h、3 h、6 h及12 h）时用RT-PCR检测ASIC1a、2a和2b mRNA表达水平；采用Western blot检测ASIC1a、2a和2b蛋白的表达情况，同时观察TK处理24 h对正常神经元和OGD后复氧不同时间的神经元上述指标表达的影响。结果1．RT-PCR检测结果显示：正常培养的大鼠皮层神经元上均有ASIC1a、2a和2b mRNA表达，暴露于OGD 90 min后在复氧过程，ASIC1a mRNA表达水平在复氧前6 h缓慢下降，6 h以后快速下降，至12 h时达最低；ASIC2b mRNA表达水平在复氧过程缓慢进行性降低至12 h时达最低；ASIC2a mRNA表达在复氧初期有所增加，复氧2 h后也逐渐降低。TK处理能够降低正常培养神经元和OGD损伤神经元在复氧不同时间时ASICs各亚基mRNA的表达水平。2．Westernblot结果显示：ASIC1a蛋白表达，在复氧0～6 h间轻微下降，在6 h以后，迅速下降，至12 h达最低后出现升高；TK预处理能显著降低复氧0～6 h和12～24 h期间ASIC1a蛋白的表达水平。ASIC2a蛋白表达水平在复氧早期出现下降，2 h时明显升高，复氧3 h以后逐渐升高，12 h后再度下降。虽然TK处理能够提高ASIC2a蛋白表达水平，但与OGD损伤组相比，差别不具有统计学意义。ASIC2b蛋白表达在复氧过程进行性降低，与其mRNA表达水平的变化趋势基本一致；TK干预不影响ASIC2b蛋白的表达。结论1．OGD后复氧过程中ASIC1a、2a和2b mRNA表达均出现降低，但其蛋白表达的变化却不尽相同。2．TK处理显著降低ASICs各亚基mRNA表达水平，但对各亚基蛋白表达水平的影响却不尽相同：在复氧初期（0～6 h）和后期（12～24 h）显著抑制ASIC1a蛋白表达，轻度促进ASIC2a蛋白表达，而不影响ASIC2b蛋白表达。提示TK抗酸毒性作用，在一定程度上可能与其抑制ASIC1a蛋白同时促进ASIC2a蛋白表达有关。四TK对缺氧-酸中毒损伤神经元保护作用的细胞内信号机制目的在OGD-Ac损伤模型上，观察细胞内PI3K／Akt和MAPK家族各信号通路的活化情况，通过检测TK处理对这些信号通路活化状态的影响，了解TK对缺氧-酸中毒损伤保护作用涉及的信号通路。方法采用MK801、CNQX、尼莫地平以及TTX阻断亲离子型谷氨酸受体、电压依赖的Ca²⁺和Na⁺通道后，建立OGD-Ac再灌注皮层神经元损伤模型。1．免疫荧光分别检测正常和OGD-Ac 90 min后再灌注1 h时神经元内p-Akt、P-JNK、P-P38和p-ERK 1／2蛋白的表达。2．采用Western blot定量检测正常神经元和OGD-Ac90 min后再灌注0 h和1 h时的神经细胞内p-Akt、p-JNK、p-P38和p-ERK1／2蛋白及相应的总蛋白的表达情况，并观察TK和ASIC1a抑制剂干预对上述指标的影响。3．采用CCK-8活细胞计数试剂盒，通过检测神经元存活率，了解ERK1／2通路阻断剂对TK促OGD-Ac损伤神经元存活效应的影响。结果1．免疫荧光检测结果显示：p-Akt、p-JNK、p-P38和p-ERK1／2蛋白在正常神经元上均有表达，在荧光显微镜下可见到相应标记蛋白的红色荧光，OGD-Ac 90 min后再灌注1 h时，上述蛋白表达有所增加，在荧光显微镜下可观察到更强的红色荧光。2．Western blot定量检测结果显示：①OGD-Ac损伤可抑制Akt蛋白磷酸化，而损伤后再灌注可促进Akt蛋白磷酸化；②TK对OGD-Ac损伤及损伤后再灌注神经细胞内p-Akt蛋白表达的促进作用不显著；③正常培养的大鼠皮层神经元均有p-JNK、p-P38和p-ERK1／2蛋白表达，OGD-Ac暴露90min可提高p-JNK蛋白的表达水平，而对p-P38和p-ERK1／2蛋白的表达水平影响不明显；OGD-Ac后再灌注1 h时p-JNK和p-ERK1／2的表达水平均显著提高，而p-P38表达水平依然无显著改变。④TK预处理对OGD-Ac后再灌注损伤神经细胞内各MAPK信号通路的影响不尽相同：TK干预显著提高OGD-Ac损伤及损伤后再灌注1 h时ERK1／2蛋白的磷酸化水平，抑制JNK蛋白的磷酸化，而不影响p-P38蛋白的表达。ASIC1a阻断剂能够提高p-Akt的表达水平，降低p-JNK的表达水平，并不显著影响ERK1／2的磷酸化水平。3．CCK-8检测结果显示：TK和PCTX1处理均可提高OGD-Ac后再灌注4 h时神经元的活力；ERK1／2通路抑制剂PD98059，能够显著抑制TK的促存活作用。结论1．OGD-Ac 90 min处理能够抑制PI3K／Akt、促进JNK和P38信号通路激活，OGD-Ac后再灌注能够激活PI3K／Akt、JNK和ERK1／2信号通路，对P38通路无明显影响。2．TK处理能够强烈促进ERK1／2信号通路激活并显著抑制JNK通路激活，对PI3K／Akt和P38通路无明显影响。提示TK可能通过影响ERK1／2和JNK信号通路的活化状态，减轻OGD-Ac诱导神经元损伤、促进神经元存活、抑制神经元死亡和凋亡，从而减轻ASICs介导的缺氧-酸中毒损伤。1．组织激肽释放酶（TK），具有抗Glu神经毒性作用的特性，主要通过B2R，抑制Glu诱导的氧化／硝基化应激反应和凋亡、促进神经元存活。TK的作用与促进ERK1／2通路，尤其是ERK1，PI3K／Akt和NF-κB通路活化，抑制JNK通路活化有关。2．TK能够产生ASICs阻断剂类似的神经保护作用，能够减轻各种离体单纯酸中毒模型以及缺血-酸中毒再灌注模型对神经元的损伤，抑制神经元死亡、促进损伤神经元存活。TK的作用可能与增强ERK1／2信号通路激活、抑制JNK通路激活，以及抑制ASIC1a和促进ASIC2a蛋白表达有关。3．总之，根据我们的研究结果，我们认为TK作为一种神经保护剂，在某种程度上，可能是通过影响Glu依赖的和非依赖的Ca²⁺神经毒性作用机制对缺血／缺氧性损伤产生保护效应的。更多还原

【Abstract】 Tissue kallikrein （TK）,an important component of the kallikrein/kinin system（KKS）,is a serine proteinase capable of cleaving low molecular weight kininogen torelease vasoactive kinins.Kinins in turn enable to tigger a series of biological effectsmainly by binding to bradykinin B2 （B2R） and B 1 receptor （B 1R）.All components ofthe KKS are expressed and widely distributed throughout many mammalian tissuesand are up-regulated by ischemic stroke.Although in recent,accumulating evidencesuggests that humane TK gene transfer or exogenous TK administration,mainly viaB2R,exerts a beneficial effect on cerebral ischemia/reperfusion （I/R） injury,however,little is known about its definitely neuroprotective mechanisms.Among the diversemechanisms involved in the pathophysiology of post-ischemic injuries,it has beingincreasingly accepted that intracellular Ca²⁺ overload is a crucial factor ofischemic/hypoxic brain damage.There are two main pathways underlyingintracellular Ca²⁺ overload during brain ischemia/ hypoxia:One is theglutamate-dependent pathway mediated by ionotropic glutamate receptors includingNMDA and AMPA receptors,and another is the glutamate-independent pathwaymediated by a class of plasma-membrane proteins including acid sensing ion channels（ASICs）.In order to explore the mechanism involved in the neuroprotection of TKagainst cerebral ischemia/hypoxia,in the present study we designed to investigate atlength the potential effects of exogenous human TK on glutamate-inducedneurotoxicity and ASICs-mediated neurotoxicity in vitro and its underlyingmechanisms.PartⅠEffects of tissue kallikrein on glutamate-inducedneurotoxicity and its underlying mechanisms1.Effects of tissue kallikrein on glutamate-induced neurotoxicityObjective:To investigate the potential role of TK in the high concentration ofglutamate-induced neurotoxicity on cultured cortical neurons. Methods:In the present study,we established the glutamate （Glu） neurotoxicitymodel by the exposure of cultured cortical neurons to modified Mg²⁺-free Earle’sbalanced salt solution （EBSS） containing 100μM Glu and 10μM glycine for 1 hfollowed by 0.5-24 h of restoration or reperfusion.Cell injury was determined bymeasuring the amount of LDH released into the medium during 1-24 h of restorationafter 1 h Glu exposure.Cell viability was assessed with a WST-8 assay using a CellCounting Kit-8 （CCK-8） after the Glu exposure and 24 h restoration.To compare theeffects of different agents on Glu-induced neuronal damage,LDH release and cellviable at 24 h restoration was measured after pretreatment of cells with TK （1.0nM-1.0μM）,B2R agonist or antagonist for 24 h,or glutamate receptor （GluR）inhibitors 30 min before and during the Glu insult.The immunofluoresence method tolabel a neuron marker MAP-2 was applied to observe the morphological changes ofneurons exposed to 1 h Glu and 3 h restoration with or without TK and GluRinhibitors pretreatment.Apoptosis were assessed by TUNEL staining DNA fragmentand Hoechst neuclear staining.Results:1.Cultured neurons exposed to 1 h of Glu （100μM） combined withglycine （10μM）,displayed a time-dependent increase in LDH release during thesubsequent 24 h of reperfusion.2.GluR antagonists induced a reduction in LDHrelease and an increase in neuronal survival.Pretreatment with TK （1.0 nM-l.0μM）elicited suppression of LDH release and enhancement of cell viability in aconcentration-dependent manner.Furthermore,the effects of TK （100 nM）adminstration was abolished by a specific B2R inhibitor HOE140 but simulated by aspecific B2R agonist BK.3.After 3 h of reperfusion following the Glu exposure,cortical neurons showed obvious morphological changes compared to the untreatednormal cells such as prominent somatic swelling,dendritic beading and disappearanceand a loss of cellular MAP-2 immunoreactivity.However,cells pretreated with TK orGluR antagonists had less such changes.Conclusion:A brief exposure to a high concentration of Glu followed byrestoration （reperfusion） induces obvious neuronal damage.TK administration,viaB2R,has comparable protective effects of GluR blockade on Glu-induced neuronalinjury. 2.Effects of tissue kallikrein on glutamate-induced oxidative/nitrosative stressObjective:To observe the pontential role of TK in Glu-induced oxidative andnitrosative stress.Methods:Cells on DIV 8-10 were randomly assigned to various pretreatmentconditions including normal control,untreated model control,TK,B2R agonist orB2R antagonist pretreatment 24 h,and GluR inhibitors pretreatment 30 min beforeand during the 100μM Glu exposure.1.Intracellular reactive oxygen species （ROS）were labeled by using fluorescent probe 2’,7’-dichlorofluorescin diacetate（DCFH-DA）.DCF fluorescent photomicrographs of cortical neurons on glass-bottomdishes were obtained with a fluorescent microscope and DCF fluroscent intensity ofcells on 96-well plates was read on a fluorescent microplate reader at an excitation of488 nm and an emission of 525 nm.2.NO was monitored by the NO-sensitive dye4-amino-5-methylamino-2’,7’-difluorofluorescein （DAF-FM/DA）,and the cellularDAF-FM fluorescence intensity was obtained by using a fluorescence microplatereader with excitation at 495 nm and emission at 519 nm.3.Under the condition ofplentiful L-Argine,intracellular NO production can represent the total NOS activity.The nNOS activity was represented by the difference between NO production in thepresence and absence of calcium.4.Mitochondrial membrane potential （MMP） wasmonitored with the fluorescent cationic dye Rhodamine 123 （Rh 123） and the cellularfluorescence intensity of Rh 123 was quantified using a fluorescence microplatereader with excitation at 488 nm and emission at 525 nm.5.Intracellular free Ca²⁺level was detected by using Ca²⁺-sensitive fluorescent probe Fluo-3/AM,and Fluo-3fluorescence intensity was obtained by using a fluorescence microplate reader withexcitation at 488 nm and emission at 525 nm.Intracellular ROS and NO production,MMP and free Ca²⁺ level was expresses the fluorescent intensity of treated groupsfold relative to the normal control after background fluorescence obtained frommedium alone was subtracted.The nNOS activity was expressed a ratio of thedifference between fluorescence DAF-AM intensity in the presence and absence ofCa²⁺,over that of the fluorescence intensity in the presence of Ca²⁺.Each group hadeight samples,and each experiment was repeated at least 3 times.Resutls:1.When compared to the normal control,cells exposed continuously toGlu for 0.5- 6 h had higher ROS and NO production,the increased Ca²⁺ but thereduced MMP level at the same time points.2.Pretreatment with TK,B2R agonist or GluR inhibitors 24 h belfore and during the Glu exposure significantly reducedgeneration of intracellular ROS and NO and the Ca²⁺ level,but elevated the MMPlevel at the same time points of Glu exposure,particularly at the time point of 2 h.3.Addition of B2R antagonist before TK pretreatment could partially block the TKeffects.4.According to the results from the assay of nNOS activity at 2 h of Gluexposure,when compared to cultures under normal circumstances,the Glu exposureelicited a significant increase in nNOS activity.TK or GluR inhibitors pretreatmentmarkedly reduced the elevated nNOS activity.Blockade of B2R prior to TKpretreatment could abrogate the suppressive effects of TK on nNOS activity.Conclusion:TK is capable to prevent Glu-induced oxidative/nitrosative stress,viaactivation of B2R,by inhibiting intracellular ROS and NO production,nNOS activityand Ca²⁺ overload,and preventing mitochondrial depolarization to maintain the MMPlevel.3.The protective mechanisms of tissue kailikrein against glutamate-inducedneurotoxicityObjective:To elucidate the underlying signaling mechanisms of TK againstGlu-induced neurotoxicity.Methods:Immunocytochemical staining was performed at 1 h of restorationafter 1 h of Glu exposure to examine the expression of phospho-ERKl/2 （p-ERK1/2）.At the end of Glu exposure immediately and at 1 h of reperfusion after the exposure,Western blot was performed to analyze the expression of phospho-Akt （p-Akt）,phospho-JNK （p-JNK）,phospho-P38 （p-P38）,p-ERK1/2 and phospho-IKKα/β（p-IKKα/β）.Results:1.Immunocytochemical staining showed that cortical neurons exposedto Glu alone showed stronger red fluorescence in their cytoplasm than those undernormal conditions,while neurons pretreated with TK prior to Glu insult exhibitedeven stronger fluorescence than those challenged by Glu alone.2.Western blotanalysis showed that one hour exposure to Glu immediately increased the level of p44-ERK1 by 153.7-fold and the p42-ERK2 level by 82.33-fold relative to normalcontrol levels.At 1 h of reperfusion,the increased p-ERK1/2 level that wassignificantly declined to 6.79- and 20.39-fold that of the normal control.Theexpression of p-ERK1/2 was almost totally blocked by GluR inhibitors or theERK-protein-kinase inhibitor PD98059.When compared to Glu exposure alone,a 24h pretreatment with TK before Glu insult induced a much greater increase in the levelof p44-ERK1,to 566-fold relative to the normal control.At 1 h of reperfusion,TKpretreatment elevated the p-ERK1 level to 51.05-fold,but not affect the expression ofp-ERK2.The B2R agonist BK pretreatment induced another 12.05-fold increase inthe level of p-ERK1 at 1 h of reperfusion after Glu exposure.The B2R antagonistHOE140 had a dramatic effect in preventing both the activation of ERK1 and 2induced by TK pretreatment.3.The expression of p-Akt was inhibited by Glu insultalone,and gradually increased during the subsequent reperfusion.Although TK andBK slightly increased the expression of p-Akt in neurons exposed to Glu,however theB2R blockade attenuated significantly the expression of p-Akt 1 h after Glu exposure.4.Neurons in normal conditions seldom expressed p-P38,but those insulted by Gluhad a significantly enhanced level of p-P38 expression,which decreased during thesubsequent reperfusion.Neither TK nor BK pretreatment reduced obviously theincreased expression of p-P38 induced by the Glu exposure.5.The exposure ofneurons to Glu produced an increase in the level of p-IKKα/β,TK pretreatmentenhanced the level of p-IKKα/βexpression in the normal cultured cells and thosechallenged by Glu.A specific ERK inhibitor PD98059 could reduce the level ofp-IKKα/βexpression 1 h after Glu exposure.6.The expression of p-JNK increasedsignificantly at the end of Glu exposure immediately and decreased to the basal levelat 1 h after Glu exposure.Either TK or B2R agonist pretreatment reduced theincreased level of p-JNK bythe exposure of Glu.The B2R inhibitor HOE140 couldabrogate the inhibition ofp-JNK expression induced by TK application.Conclusion:The exposure of cultured neurons to a high concentration of Glubriefly activates the three MAPK signal pathways,particularly ERK1/2,and NF-κBpathway but suppresses activation of PI3K/Akt pathway.TK plays a protective role inGlu-induced neurotoxicity via activation of B2R,correlating with differentiallyinfluencing activation of these signal pathways,strongly promotiong activation of theERK1/2,particularly by activation of the ERK1,and NF-κB pathways,slightlyenhancing activation of the PI3K/Akt pathway,moderately inhibiting the JNK activity, but does not influence the P38 activity.PartⅡEffects of TK on acid sensing ion channels-mediatedneurotoxicity and its involving mechanisms1.Effects of acid sensing ion channels activation on cultured neuronsObjective:To investigate the effects of acid-sensing-ion channels （ASICs）activation,especially activation of ASIC1a on cultured neurons under various acidpathological conditions.Methods:In the presence of blockers of major Ca²⁺ entry pathways includingiontrophic GluRs and voltage-gated Ca²⁺ and Na⁺ channels,we used primary culturesof cortical neurons to establish several acidosis （pH6.0） insult models includingacidosis/reperfusion （Ac）,continuous acidosis （C-Ac）,oxygen and glucosedeprivation （OGD） combined with acidosis （OGD-Ac） followed by reperfusion.Cytotoxicity was determined by assaying the amount of LDH released into themedium.Cell viability was measured with an assay kit of CCK-8.Theimmunofluoresent double label method was applied to observe the distribution ofASIC1a and the morphological changes of neurons exposed to 90 min of OGD-Acand 4 h ofreperfusion.Results:1.Cytotoxicity gradually increased and cell viability progressivelyreduced during reperfusion following 2 h acid incubation （pH6.0）.At 24 h ofreperfusion,the relative LDH release attained 45.6% and cell survival decreased to58.5%,when compared to the normal control.2.Continuous acid incubation （pH6.0）induced a more markedly increase in LDH release and decrease in cell viability thanthe Ac model.At the end of 24 h of C-Ac treatment,only about 10% neuronsremained alive.3.Neurons exposed to OGD-Ac 90 min followed by reperfusionexhibited more remarked injury,had a greater increase in LDH release and decreasein cell viability than those challenged by OGD 90 min with reperfusion.3.Blockadeof ASIC1a with a specific inhibitor PcTX1 or nonspecific inhibitor Amiloridealleviated neuronal damage induced by C-Ac and OGD-Ac.The immunofluorescent staining showed:at 4 h of reperfusion following OGD-Ac 90 min,cortical neuronsshowed obvious morphological changes relative to normal condition-treated cells,such as prominent somatic swelling,neurite disruption and loss,nuclear pyknosis（condensation） and a loss of cellular MAP-2 immunoreactivity.ASICla normallydistributed on cytoplaysm membrane and axons,but mainly on cytoplyasm membraneafter the OGD-Ac insult.Conclusion:In the presence of a cocktail of ionotropic Glu andvoltage-dependent Ca²⁺ and Na⁺ channels blockers,the Ac,C-Ac and OGD-Acmodels are effective to induce cultured neurons injury.Under various acidpathological conditions,ASICs,especially ASICla activation,inducesGlu-independent neuronal injury wherease ASICla blockage elicits a markedlyneuroprotective effect.2.Effects of TK on acidosis-induced neurotoxicity in vitroObjective:To observe the effects of TK on ASICs-mediated neurotoxicity,weintestigated the potential role of TK administration in acidosis- and OGD-combinedwith acidosis-induced neuronal injury.Methods:In the presence of a cocktail of glutamate and voltage-dependent Ca²⁺and Na⁺ channels blockers,neurons were treated with TK,B2R agonist and antagonistfor 24 h,or PcTX1 for 30 min before exposed to Ac,C-Ac,OGD alone or OGDcombined with acidosis,Cytotoxicity was determined by assaying LDH release.Neuronal viability was measured by CCK-8.Death cells were observed by PI/Hoechststaining.Apoptotic cells were analyzed by TUNEL staining at 24 h reperfusion afterOGD-Ac 90 min.Immunocytochemical staining was applied to observe thedistribution of ASICla and the effects of TK pretreatment on the morphological ofneurons exposed to OGD-Ac 90 min followed by 4 h reperfusion and C-Ac for 3 h.Results:1.Results from measurement of LDH release and cell viability showed:Primary cortical neurons were treated with various concentration of TK （0.01-1.0μM）for 24 h prior to C-Ac,2 h of acidosis with reperfusion or 90 min of OGD-Acfollowed by reperfusion for 1-24 h.TK elicited a concentration-dependent increase in cell viability and decrease in LDH release.2.PI/Hoechst staining revealed that TKpretreatement reduced the increased ratio of PI positive cells in various acidosismodels.3.PcTX1 and B2R agonist had comparable effects of TK wherease B2Rinhibitor HOE140 had an inverse effect.3.The immunofluoresent stainingdemonstrated that the addition of 100 nM TK 24 h before and during OGD-Ac or acidincubation remarkably inhibited the morphological changes of neurons exposed to 4 hreperfusion following OGD-Ac or 3 h of acidosis,and did not affect the expression ofASIC1a.5.TUNEL staining showed that at 24 h reperfusion after OGD-Ac 90 minTK and PcTX1 pretreated groups had lowered ratio of apoptotic cells than that ofuntreated OGD-Ac.Conclusion:These findings reveal that TK may have antioxidant properties.TKadministration produces a protective effect on acidisos- or OGD-Ac-induced neuronaldamage,similar to that seen with ASICs blockade,by alleviating neuronal injury,reducing cell death and apoptosis and promoting cell survival.3.Effects of TK on the expression of ASICs in cultured neuronsundergoing oxygen and glucose deprivationObjective:To investigate the effects of TK on the expression of ASICs mRNAand proteins.Methods:RT-PCR was used to examine the mRNA expression changes ofASIC1a,ASIC2a and ASIC2b in normal cultured neurons and those exposed to OGD90 min followed by reoxygenation l h,2 h,3 h,6 h and 12 h.Western blot was usedto investigate the changes of ASICla,ASIC2a and ASIC2b protein expression innormal control neurons and those challenged by 90 min of OGD with reoxygenation 1h,2 h,3 h,6 h,12 h and 24 h.And we further examine the effects ofTK pretreatmentfor 24 h on the expression ofASICla,ASIC2a and ASIC2b mRNA and proteins.Results:1.The expression of ASICla,ASIC2a and ASIC2b mRNA in culturedneurons was plentiful but had an obvious decline during 1-12 h of reoxygenationfollowing OGD 90 min.The expression ofASICla mRNA level had a slow decreaseduring the first 6 h of reoxygenation,but such a decrease became accelerated after that and reached its minimum at 12 h.The expression of ASIC2b mRNA level graduallyreduced during 1-12 h of reoxygenation.However,the ASIC2a mRNA expressionincreased slightly at the early stage of reoxygenation,reached it peak at 2 hreoxygenation and decreased after that.2.TK pretreatment significantly inhibited allthe three subunits of ASICs mRNA expression.3.Western blot results showed:ASIC la protein expression changed largely during the course of 1-24 h reoxygenation:during the first 6 h ofreoxyenation,its expression had no obvious change,during 6-12h of reoxyenation displayed a remarkable decrease and reached the minimal level at12 h reoxyenation;however,after that time point ASICla protein expressionexhibited a significant increase.TK treatment could reduce the expression ASIClaprotein level during 1-6 h and 12-24 h of reoxygenation.At the early stage ofreoxygenation,the level of ASIC2a protein expression had a significant increase,especially at the time point of 2 h,then experienced a slow increase from 3 h to 12 h.TK pretreatment did not reduce but enhanced slightly the ASIC2a protein expression.The ASIC2b protein expression exhibited a progressive reduction during the wholecourse of reoxygenation.TK treatment had no effect on the change of ASIC2b proteinexpression.Conclusion:Although during the course of reoxygenation following OGD 90min,ASICla,ASIC2a and ASIC2b expression experiences a reduction in the level ofmRNA,however their protein expression changes differently.TK has an inhibitoryeffect on the level of ASICla,ASIC2a and ASIC2b mRNA expression,butdifferential effects on their protein expression:significantly inhibits ASIClaexpression,slightly enhances the ASIC2a expression level but does not influenceASIC2b expression.TK application elicits a neuroprotective effect on acidosis or/andOGD-induced damage,in part by down-regulation of ASIC1a and up-regulation ofASIC2a protein expression.4.The intracellular signaling mechanisms of tissue kallikrein alleviatingneurotoxicity induced by the combination of OGD and acidosisObjective:To elucidate the underlying signaling mechanisms of TK alleviating neurotoxicity induced by the combination of OGD and acidosis.Methods:lmmunocytochemical staining was performed at 1 h of reperfusionfollowing the combination of OGD and acidosis for 90 min,to check the expressionof p-Akt,p-ERK1/2,p-JNK and p-P38.Western blot was used to quantitativelyanalyze the expression of p-Akt,p-ERK,p-JNK and p-P38 proteins at the end of 90min of OGD-acidosis insult and 1 h reperfusion after the insult.During theOGD-acidosis insult,a cocktail of ionotropic glutamate and voltage-dependent Ca²⁺and Na⁺ channels blockers was present to abolish activation of ionotropic glutamateand Ca²⁺ and Na⁺ channels.We further observed the effect of the ERK1/2 blockade onTK-promoting survival by assaying cell viability at 4 h of reperfusion following 90min of OGD-Ac.Results:1.Immunocytochemical staining revealed that neurons exposed toOGD-Ac insult with 1 h reperfusion exhibited stronger red fluorescence of p-Akt,p-ERK,p-JNK and p-P38 proteins than those in normal conditions.2.Western blotresults demonstrated that the p-Akt protein expression was inhibited by OGD-Acinsult alone,and significantly increased during subsequent reperfusion.TKpretreatment had no significant effect on the level of p-Akt expression.3.Culturedneurons in normal conditions could express p-ERK1/2,p-JNK and p-P38 proteins.At1 h of reperfusion following the 90 min of OGD-Ac,cells had significant increasedlevels ofp-ERKl/2 and p-JNK protein expression but no obvious change in the p-P38expression.4.TK treatment had differential effects on activation of MAPK signalpathways:At the end of OGD-Ac 90 min and subsequent reperfusion 1 h,cellspretreated with TK had a marked rise in the expression of p-ERK1/2,moderatereduction in that of p-JNK,however no significant effect on the p-P38 expression.PcTX1 pretreatment had effects consistent with those of TK adminstration.5.Treatemet with an inhibitor ERK1/2 PD98059 for 1 h,prior to TK pretreatment,significantly inhibited the survival-promoting effect of TK on cells exposed toOGD-Ac 90 min with reperfusion 4 h.Conclusion:The exposure of cultured neurons to OGD-Ac 90 min brieflyactivates the JNK and P38 but suppresses the PI3K/Akt pathways;however,1 h ofsubsequent reperfusion following the OGD-Ac induced activation of the PI3K/AktERK1/2 and JNK pathways.TK may play a protective role in OGD-Ac-inducedneurotoxicity on primary cultures of cortical neurons via potently promotingactivation of the ERK1/2,moderately inhibiting activation of the JNK pathway while the PI3K/Akt and the P38 signal pathways are not required for TK-mediatedneuroprotection.Conclusion1.TK,acting mainly through B2R,plays a protective role in glutamate-inducedneurotoxicity in cultured cortical neurons,by conferring resistance tooxidative/nitrosative stress,suppressing neuronal apoptosis and promotingneuronal survival through activation of the ERK1/2,in particular ERK1,theIKKα/β-NF-κB and the PI3K/Akt signal pathways,at the same time inhibition ofthe JNK signal pathway.2.TK adminstration,like ASICla blockade,protects cortical neurons againstdamage induced by ischemia-acidosis/reperfusion in vitro via allevation ofneuronal injury and apoptosis and enhancement of neuronal viability.And theneuroprotective mechansims of TK against neuronal injury produced by acidosisand/or hypoxia mainly involve down-regulation of the ASICla expression andup-regulation of the ASIC2a expression,probably through activation of theERK1/2 signal pathway and inhibition of the JNK signal pathway.3.In summary,based on our observations,we propose that TK,as a well-establishedneuroprotective agent,to a certain extent,may directly or indirectly interfere withglutamate-dependent and ASICs-mediated glutamate-independent Ca²⁺ neurotoxicmechanisms to ameliorate ischemic/hypoxic brain damage.更多还原