泊洛沙姆188对脑缺血再灌注损伤的保护作用及其机制研究
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摘要
目的：阐明泊洛沙姆188对脑缺血再灌注损伤的保护作用并探讨其保护机制
方法：
第一部分，P188对脑缺血再灌注损伤的保护作用
采用大脑中动脉栓塞法建立小鼠局灶性脑缺血再灌注损伤模型，再灌注前尾静脉单次注射不同剂量的P188，24小时后测定脑梗死体积（TTC染色法），脑含水量以及神经症状评分以评价短期保护作用。为进一步观察P188的长期效应，小鼠尾静脉注射P188后再每天单次腹腔注射P188，3周后评估小鼠的生存期，应用爬杆实验和悬挂实验测定肢体运动功能，尼氏染色法测定脑萎缩。此外，体外建立海马神经细胞系(HT22)的糖氧剥夺模型，检测P188对细胞活力和乳酸脱氢酶释放的影响。
第二部分，泊洛沙姆188对脑缺血保护作用的机制研究
小鼠缺血再灌注损伤后，碘化丙啶（PI，非膜穿透性染料）侧脑室注射应用荧光显微镜观察细胞膜完整性。体外应用低浓度Triton X-100轻度裂解细胞膜，观察P188处理前后，细胞对碘化丙啶（红色荧光）和SYTOX Green（非膜穿透性染料，绿色荧光）两种染料的通透性，探讨P188对细胞膜的直接修复作用。进一步，应用伊文思蓝漏出实验检测小鼠缺血再灌注损伤后的血脑屏障的完整性，同时应用酶谱法和免疫印迹分析P188对基质金属蛋白酶9的活性和表达的影响。此外，为了探讨P188对细胞内膜的影响，对P188进行铱配合物标记（自带黄绿色荧光），荧光显微镜观察P188在HT22细胞和小鼠缺血再灌注后神经细胞中的分布。在证实P188能穿透细胞膜后，分离出脑缺血再灌注组织的溶酶体或线粒体，western blot检测P188应用后对Cyt c或Cathepsin L从线粒体或溶酶体中漏出的情况。
第三部分，泊洛沙姆188与银杏内酯B合用对脑缺血再灌注损伤的保护作用
采用大脑中动脉栓塞法建立小鼠局灶性脑缺血再灌注损伤模型，比较P188和银杏内酯B联合应有与相同剂量的P188和银杏内酯B单用的差异，观察指标为脑梗死体积，脑含水量和神经症状评分。
结果：
第一部分，P188对脑缺血再灌注损伤的影响
中剂量和高剂量的P188（0.2g，0.4g/kg）尾静脉给药后能显著降低缺血再灌注损伤小鼠的脑梗死体积，脑含水量和神经症状评分。长期多次给药后能延长小鼠生存期，提高运动能力和减轻脑萎缩。体外OGD模型证实P188能显著提高细胞活力，降低LDH的释放。
第二部分，泊洛沙姆188对脑缺血保护作用的机制研究
脑缺血再灌注后小鼠海马和纹状体部位出现大量PI染色阳性的细胞，表明细胞膜的破损，P188干预后显著减少PI染色阳性细胞的数量。当低浓度Triton X-100处理细胞后，大量细胞对PI的通透性增加（细胞产生红色荧光），P188干预后，部分PI阳性细胞对SYTOX Green通透性降低（PI阳性/SYTOX Green阴性），表明P188对细胞膜的直接修复阻止SYTOX Green进入细胞内。伊文思蓝漏出实验发现P188能明显抑制缺血再灌注导致的血脑屏障的开放，这可能与P188直接修复血脑屏障或者抑制基质金属蛋白酶9的活性和表达有关。此外，铱配合物标记的P188能穿透正常细胞外膜以及脑缺血再灌注损伤部位的神经细胞，抑制缺血再灌注组织细胞色素C从线粒体释放入胞浆中，抑制caspase3的活化，此外，P188也能抑制溶酶体中的组织蛋白酶Cathepsin L的释放，这些结果提示P188进入细胞内后对细胞器膜产生一定的保护作用。
第三部分，泊洛沙姆188与银杏内酯B合用对脑缺血再灌注损伤的保护
单用银杏内酯B可以产生明显的保护作用，联合P188和银杏内酯B比单用P188和银杏内酯B有更为显著的保护作用。
结论：
P188对脑缺血再灌注损伤具有保护作用，其可能的机制有：直接修复受损的细胞质膜；降低血脑屏障的通透性；进入细胞内保护线粒体和溶酶体等细胞器膜。此外，P188与其它作用机制的神经保护药-银杏内酯B合用可以产生增强效应。
Aim: To explore whether P188has a protective effect against cerebralischemia/reperfusion injury and its underlying mechanisms.
Methods:
Part1: The effects of P188on the cerebral ischemia/reperfusion injury
Firstly we established the mouse middle cerebral artery occlusion (MCAO) modelby intraluminal occlusion using monofilament. P188was intravenously injected justbefore reperfuion, mice were sacrificed to determine the infarct volume using TTCstaining, brain water content and neurological symptoms24h after ischemia/reperfusion.To learn the long-term protective effects, P188were administered by IP injection once aday for three weeks, the survival rate was determined, motor function was evaluatedwith wire hanging and pole test and the loss of brain volume was calculated using cresylviolet staining. In addition, we also apply the oxygen and glucose deprivation cellmodel to evaluate the effects of P188on the cell viability and LDH leakage.
Part2: The underlying mechanisms of P188against the cerebralischemia/reperfusion injury.
In this part we firstly applied the Propidium Iodide (PI) to evaluate the membraneintegrity24h after ischemia/reperfusion. Furthermore, in vitro0.03%Triton X-100, anonionic detergent, was used to directly disrupt neuronal membranes. Two differentmembrane-impermeant fluorescence dyes (PI and SYTOX Green) were sequentiallyadded to cell medium to observe the changes in membrane integrity after P188treatment. Secondly, we applied Evans Blue Dye Extravasation to assess thepermeability of blood-brain barrier (BBB), and analyzed the activity and expressionlevels of MMP-9using zymogram and western blot. Thirdly, to explore whether P188can penetrate the cell membrane into the cytosol, P188was labeled with Ir complexeswhich can emit the fluencerence and observe its distribution in HT-22cell and in mousebrain suffered from ischemia-reperfusion injury. Furthermore, mitochondrial and lysosome were separated from ischemic hemisphere of MCAO mice to analyze theleakage of cytochrome c and Cathepsin L.
Part3: The effects of combined P188and Ginkgolide B on theischemia/reperfusion injury
Combined P188and Ginkgolide B were compared with same dosage of P188orGinkgolide B by the infarct volume, brain water content and neurological symptoms.
Results:
Part1: The effects of P188on the cerebral ischemia/reperfusion injury
The results from in vivo MCAO model showed that P188significantly reduced theinfarct volume, ameliorated the brain edema and neurological symptoms24h afterischemia/reperfusion. In the long-term outcome study, P188markedly alleviated brainatrophy and motor impairments and increased survival rate in3weeks of post strokeperiod. Additionally, P188protected cultured hippucampal HT22cells againstoxygen–glucose deprivation and reoxygenation (OGD/R) injury
Part2: The underlying mechanisms of P188against the cerebralischemia/reperfusion injury.
Firstly, P188treatment significantly reduced the PI-positive cells followingischemia/reperfusion injury and repaired the HT22cell membrane rupture induced byTriton X-100. Secondly, P188inhibited ischemia/reperfusion-induced activation ofmatrixmetalloproteinase (MMP)-9and leakage of Evans blue, which suggests that P188can preserve the integrity of blood-brain barrier. Thirdly, we found P188labeled with Ircomplexes can penetrate the cell membrane, thus continued to assess the effects of P188on organelles including mitochondrial and lysosomes and also discovered that P188caninhibit the leakage of Cyt c and Cathepsin L.
Part3: The effects of combined P188and Ginkgolide B on theischemia/reperfusion injury
Combined P188and Ginkgolide B had greater protective effects on theischemia/reperfusion than that of P188or Ginkgolide B alone.
Conclusions:
P188can protect against cerebral ischemia/reperfusion injury, and the protectionmechanisms involve:1) directly sealing the cell membranes;2) preserve the integrity ofblood-brain barrier and3) penetrate the membrane and render the protection on mitochondrial and lysosomes. In addition, the combination of P188and Ginkgolide Bcan produce greater protective effects on ischemia/reperfusion injury.
方法：
第一部分，P188对脑缺血再灌注损伤的保护作用
采用大脑中动脉栓塞法建立小鼠局灶性脑缺血再灌注损伤模型，再灌注前尾静脉单次注射不同剂量的P188，24小时后测定脑梗死体积（TTC染色法），脑含水量以及神经症状评分以评价短期保护作用。为进一步观察P188的长期效应，小鼠尾静脉注射P188后再每天单次腹腔注射P188，3周后评估小鼠的生存期，应用爬杆实验和悬挂实验测定肢体运动功能，尼氏染色法测定脑萎缩。此外，体外建立海马神经细胞系(HT22)的糖氧剥夺模型，检测P188对细胞活力和乳酸脱氢酶释放的影响。
第二部分，泊洛沙姆188对脑缺血保护作用的机制研究
小鼠缺血再灌注损伤后，碘化丙啶（PI，非膜穿透性染料）侧脑室注射应用荧光显微镜观察细胞膜完整性。体外应用低浓度Triton X-100轻度裂解细胞膜，观察P188处理前后，细胞对碘化丙啶（红色荧光）和SYTOX Green（非膜穿透性染料，绿色荧光）两种染料的通透性，探讨P188对细胞膜的直接修复作用。进一步，应用伊文思蓝漏出实验检测小鼠缺血再灌注损伤后的血脑屏障的完整性，同时应用酶谱法和免疫印迹分析P188对基质金属蛋白酶9的活性和表达的影响。此外，为了探讨P188对细胞内膜的影响，对P188进行铱配合物标记（自带黄绿色荧光），荧光显微镜观察P188在HT22细胞和小鼠缺血再灌注后神经细胞中的分布。在证实P188能穿透细胞膜后，分离出脑缺血再灌注组织的溶酶体或线粒体，western blot检测P188应用后对Cyt c或Cathepsin L从线粒体或溶酶体中漏出的情况。
第三部分，泊洛沙姆188与银杏内酯B合用对脑缺血再灌注损伤的保护作用
采用大脑中动脉栓塞法建立小鼠局灶性脑缺血再灌注损伤模型，比较P188和银杏内酯B联合应有与相同剂量的P188和银杏内酯B单用的差异，观察指标为脑梗死体积，脑含水量和神经症状评分。
结果：
第一部分，P188对脑缺血再灌注损伤的影响
中剂量和高剂量的P188（0.2g，0.4g/kg）尾静脉给药后能显著降低缺血再灌注损伤小鼠的脑梗死体积，脑含水量和神经症状评分。长期多次给药后能延长小鼠生存期，提高运动能力和减轻脑萎缩。体外OGD模型证实P188能显著提高细胞活力，降低LDH的释放。
第二部分，泊洛沙姆188对脑缺血保护作用的机制研究
脑缺血再灌注后小鼠海马和纹状体部位出现大量PI染色阳性的细胞，表明细胞膜的破损，P188干预后显著减少PI染色阳性细胞的数量。当低浓度Triton X-100处理细胞后，大量细胞对PI的通透性增加（细胞产生红色荧光），P188干预后，部分PI阳性细胞对SYTOX Green通透性降低（PI阳性/SYTOX Green阴性），表明P188对细胞膜的直接修复阻止SYTOX Green进入细胞内。伊文思蓝漏出实验发现P188能明显抑制缺血再灌注导致的血脑屏障的开放，这可能与P188直接修复血脑屏障或者抑制基质金属蛋白酶9的活性和表达有关。此外，铱配合物标记的P188能穿透正常细胞外膜以及脑缺血再灌注损伤部位的神经细胞，抑制缺血再灌注组织细胞色素C从线粒体释放入胞浆中，抑制caspase3的活化，此外，P188也能抑制溶酶体中的组织蛋白酶Cathepsin L的释放，这些结果提示P188进入细胞内后对细胞器膜产生一定的保护作用。
第三部分，泊洛沙姆188与银杏内酯B合用对脑缺血再灌注损伤的保护
单用银杏内酯B可以产生明显的保护作用，联合P188和银杏内酯B比单用P188和银杏内酯B有更为显著的保护作用。
结论：
P188对脑缺血再灌注损伤具有保护作用，其可能的机制有：直接修复受损的细胞质膜；降低血脑屏障的通透性；进入细胞内保护线粒体和溶酶体等细胞器膜。此外，P188与其它作用机制的神经保护药-银杏内酯B合用可以产生增强效应。
Aim: To explore whether P188has a protective effect against cerebralischemia/reperfusion injury and its underlying mechanisms.
Methods:
Part1: The effects of P188on the cerebral ischemia/reperfusion injury
Firstly we established the mouse middle cerebral artery occlusion (MCAO) modelby intraluminal occlusion using monofilament. P188was intravenously injected justbefore reperfuion, mice were sacrificed to determine the infarct volume using TTCstaining, brain water content and neurological symptoms24h after ischemia/reperfusion.To learn the long-term protective effects, P188were administered by IP injection once aday for three weeks, the survival rate was determined, motor function was evaluatedwith wire hanging and pole test and the loss of brain volume was calculated using cresylviolet staining. In addition, we also apply the oxygen and glucose deprivation cellmodel to evaluate the effects of P188on the cell viability and LDH leakage.
Part2: The underlying mechanisms of P188against the cerebralischemia/reperfusion injury.
In this part we firstly applied the Propidium Iodide (PI) to evaluate the membraneintegrity24h after ischemia/reperfusion. Furthermore, in vitro0.03%Triton X-100, anonionic detergent, was used to directly disrupt neuronal membranes. Two differentmembrane-impermeant fluorescence dyes (PI and SYTOX Green) were sequentiallyadded to cell medium to observe the changes in membrane integrity after P188treatment. Secondly, we applied Evans Blue Dye Extravasation to assess thepermeability of blood-brain barrier (BBB), and analyzed the activity and expressionlevels of MMP-9using zymogram and western blot. Thirdly, to explore whether P188can penetrate the cell membrane into the cytosol, P188was labeled with Ir complexeswhich can emit the fluencerence and observe its distribution in HT-22cell and in mousebrain suffered from ischemia-reperfusion injury. Furthermore, mitochondrial and lysosome were separated from ischemic hemisphere of MCAO mice to analyze theleakage of cytochrome c and Cathepsin L.
Part3: The effects of combined P188and Ginkgolide B on theischemia/reperfusion injury
Combined P188and Ginkgolide B were compared with same dosage of P188orGinkgolide B by the infarct volume, brain water content and neurological symptoms.
Results:
Part1: The effects of P188on the cerebral ischemia/reperfusion injury
The results from in vivo MCAO model showed that P188significantly reduced theinfarct volume, ameliorated the brain edema and neurological symptoms24h afterischemia/reperfusion. In the long-term outcome study, P188markedly alleviated brainatrophy and motor impairments and increased survival rate in3weeks of post strokeperiod. Additionally, P188protected cultured hippucampal HT22cells againstoxygen–glucose deprivation and reoxygenation (OGD/R) injury
Part2: The underlying mechanisms of P188against the cerebralischemia/reperfusion injury.
Firstly, P188treatment significantly reduced the PI-positive cells followingischemia/reperfusion injury and repaired the HT22cell membrane rupture induced byTriton X-100. Secondly, P188inhibited ischemia/reperfusion-induced activation ofmatrixmetalloproteinase (MMP)-9and leakage of Evans blue, which suggests that P188can preserve the integrity of blood-brain barrier. Thirdly, we found P188labeled with Ircomplexes can penetrate the cell membrane, thus continued to assess the effects of P188on organelles including mitochondrial and lysosomes and also discovered that P188caninhibit the leakage of Cyt c and Cathepsin L.
Part3: The effects of combined P188and Ginkgolide B on theischemia/reperfusion injury
Combined P188and Ginkgolide B had greater protective effects on theischemia/reperfusion than that of P188or Ginkgolide B alone.
Conclusions:
P188can protect against cerebral ischemia/reperfusion injury, and the protectionmechanisms involve:1) directly sealing the cell membranes;2) preserve the integrity ofblood-brain barrier and3) penetrate the membrane and render the protection on mitochondrial and lysosomes. In addition, the combination of P188and Ginkgolide Bcan produce greater protective effects on ischemia/reperfusion injury.
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