1.Guattegaumerine对H_2O_2合并血清剥夺诱导的培养大鼠皮质神经元损伤的保护作用研究 2.Betulinic acid对ApoE Knock Out小鼠脑缺血再灌注损伤的保护作用及作用机制研究
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摘要
第一部分Guattegaumerine对H_2O_2合并血清剥夺诱导的培养大鼠皮质神经元损伤的保护作用研究
目的：建立方便有效的培养的大鼠皮质神经元拟缺血性损伤模型，观察Guattegaumerine的神经细胞保护作用并初步探讨其作用机制。方法：原代Wistar大鼠乳鼠皮质神经元培养，不同浓度的H_2O_2和谷氨酸在无血清低糖培养基中与细胞共孵育24h后检测细胞活力(MTT法)和乳酸脱氢酶(lactate dehydrogenase，LDH)释放量。1．25μmol／L与2．5μmol／L Guattegaumerine与皮质神经元预孵育30min，随后加入含6．25μmol／L H_2O_2的无血清低糖培养基，继续培养24h后检测细胞活力及LDH释放量，细胞总抗氧化能力(total antioxidative capatity，TAC)及脂氧化产物MDA生成量；膜连蛋白(Annexin v)-FITC／碘化丙啶(propidium iodide，PI)双染流式细胞仪检测神经元的凋亡和坏死，及免疫组化检测细胞Bax与Bcl-2蛋白表达；钙荧光探针Fura-2AM检测H_2O_2在含钙及无钙细胞外液中诱发的胞内钙浓度变化及KCl诱导胞内钙浓度变化，并观察不同浓度Guattegaumerine对H_2O_2和KCl诱发的胞内钙浓度升高的影响。结果：6．25μmol／L～200μmol／L H_2O_2及12．5μmol／L～50μmol／L谷氨酸合并血清剥夺与神经元孵育24h后，使神经元的生长能力降低，LDH漏出量增加。Guattegaumerine能抑制6．25μmol／L H_2O_2合并血清剥夺诱导的皮质神经元损伤，表现为提高细胞活力，减少LDH漏出量，提高细胞TAC的活力，减少MDA生成量，并抑制细胞凋亡，抑制Bax及促进Bcl-2蛋白表达；抑制含钙细胞外液试验中H_2O_2及KCl诱导的胞内钙浓度升高，高浓度guattegaumerine还能抑制无钙外液试验中H_2O_2诱导的胞内钙浓度升高。结论：6．25μmol／L～200μmol／L H_2O_2及12．5μmol／L～50μmol／L谷氨酸合并血清剥夺与神经元孵育24h是两种方便有效的诱导神经元拟缺血性损伤模型的方法。Guattegaumerine对6．25μmol／L H_2O_2合并血清剥夺诱导的皮质神经元损伤有保护作用，其作用机制可能与其通过提高抗氧化酶活性，抑制脂质过氧化反应，调节凋亡相关基因及拮抗胞内钙超载有关。Guattegaumerine的钙拮抗作用可能与其阻断膜上电压依赖性钙通道及受体门控型钙通道，抑制钙库钙释放有关。
第二部分Betulinic acid对ApoE knock ont小鼠脑缺血再灌注损伤的保护作用及作用机制研究
目的：大脑中动脉栓塞法建立ApoE基因敲除(ApoE KO)小鼠的局灶性脑缺血再灌注损伤模型，并观察betulinic acid对脑缺血再灌注损伤的保护作用并初步探讨其作用机制。方法：50mg／kg betulinic acid灌胃给药，1次／天，共7天。灌胃结束后，左侧大脑中动脉栓塞2h复灌22h诱导ApoE KO小鼠的脑缺血再灌注损伤模型，脑组织切片TTC染色测定脑梗死体积；缺血侧及缺血对侧脑组织mRNA提取qRT-PCR检测NAPDH氧化酶亚型NOX1、NOX2、NOX4、p22phox，及NO合成酶eNOS、iNOS和nNOS的mRNA的表达；缺血侧及缺血对侧脑组织蛋白质提取western-blot检测NO合成酶eNOS和nNOS，及蛋白质的氧化产物酪氨酸硝基化蛋白的表达。结果：左侧大脑中动脉栓塞2h复灌22h能稳定地诱导ApoE KO小鼠脑缺血再灌注损伤，缺血侧脑组织NOX2、NOX4，nNOS及iNOS基因表达升高，蛋白质氧化产物生成量增多。50mg／kg betulinic acid灌胃给药能降低脑梗死体积，增加脑组织eNOS mRNA及蛋白质的表达，降低缺血侧脑组织nNOS mRNA及蛋白质的表达，同时降低缺血侧脑组织iNOS，NOX2的mRNA的表达。50mg／kg betulinic acid灌胃给药亦降低缺血侧脑组织酪氨酸硝基化蛋白质水平。结论：50mg／kg betulinic acid对ApoE KO小鼠脑缺血再灌注损伤有保护作用，其作用机制可能与其抑制NOX2、nNOS及iNOS基因表达从而抑制活性氧和活性氮的产生，抑制脑缺血再灌注后氧化应激损伤有关。
First Part Protective Effects of Guattegaumerine Against HydrogenPeroxide Concomitant with Serum Deprivation Incuced Injury inPrimary Cultured Cortical Neurons
Aims: To establish the convenient and effective model of ischemia induced injury inprimary cultured neurons, and evaluate the neuroprotective effects of guattegaumerine onrat primary cultured cortical neurons. Methods: Research on primary cultured corticalneurons of Wistar rats, different concentrations of H_2O_2 and Glutamate (Glu) expoured tocells in serum free and low glucose medium for 24h and cell viability determined by3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and lactatedehydrogenase (LDH) leakage were detected. 1.25μmol/L and 2.5μmol/L guattegaumerinewere added 30 min before cells exposure to 6.25μmol/L H_2O_2 in serum free and lowglucose medium for 24h, then cell viability、LDH leakage、malondialhehyde (MDA) andtotal antioxidative capacity (TAC) were observred. Cells apoptosis and necrosis weremeasured by Annexin V/Propidium iodide double staining assay using flow cytometry, andBax, Bcl-2 protein expression were detected by immunohistochemistry. The calciumfluorescent label fura-2 AM was used to measure the intracellular calcium concentrationinduced by H_2O_2 and KC1, and the effects of guattegaumerine on the increased intracellularcalcium stimulated by H_2O_2 and KC1. Results: 6.25μmol/L～200μmol/L H_2O_2 and12.5μmol/L～50μmol/L Glu could decreased cell viability and increased leakage of LDH.Preincubation of guattegaumerine dramatically improved the cell viability and inhibitedLDH release. Preincubation of guattegaumerine also dramatically inhibited MDAproduction and elevated TAC in cells. Results of flow cytometry andimmunohistochemistry showed that preaddition of guattegaumerine interrupted theapoptosis of the neurons, reversed the up regulation of the pro-apoptotic gene (Bax) and thedown regulation of the anti-apoptotic gene (Bcl-2). Furthermore, guattegaumerinesuppressed the increase of intracellular calcium concentration ([Ca~(2+)]_i) stimulated by eitherH_2O_2 or KC1 in Ca~(2+)-containing extracellular solution, and high concentration of guattegaumerine also suppressed the increase of [Ca~(2+)]_i induced by H_2O_2 in Ca~(2+)-freesolution. Conclusions: 6.25μmol/L～200μmol/L H_2O_2 and 12.5μmol/L～50μmol/L Glu aretwo convenient and effective models to induce ischemic injury in neurons.Guattegaumerine protects cultured cortical neurons against 6.25μmol/L H_2O_2 concomitantwith serum deprivation incuced injury, which may relate to its inhibition of lipidperoxidation, regulation of apoptotic related gene expression and prevention of intracellularcalcium elevation by blocking the selective and non-selective calcium channels on the cellmembrane, and the release of calcium from ER.
Second Part Betulinic Acid Protects against CerebralIschemia/Reperfusion Injury in ApoE Knock Out icem
Aims: To establish focal cerebral ischemia-reperfusion injury by middle cerebral arteryocclusion in ApoE knock out (KO) mice, and investigate the neuroprotective potential ofbetulinic acid against brain ischemia-reperfusion. Methods: ApoE KO mice were treatedwith 50mg/kg betulinic acid via gavage per day for 7days, then subjected to 2h of the leftmiddle cerebral artery occlusion (MCAO) followed by 22h of reperfusion. Brain infarctionwas indicated by TTC staining. NADPH oxidase (NOX) subunits (NOX1、NOX2、NOX4and p22phox) and nitric oxide synthase (NOS) subunits (nNOS、iNOS and eNOS) mRNAexpression were measured by qRT-PCR, and NOS subunits (nNOS and eNOS) andnitrotyrosine protein expression were measured by western-blot. Results: Inatherosclerosis-prone apolipoprotein E knockout (ApoE-KO) mice, 2h of middle cerebralartery occlusion (MCAO) followed by 22h of reperfusion led to an enhanced expression ofseveral NADPH oxidase subunits (NOX2, NOX4 and p22phox) and an up-regulation ofNOS isoforms (nNOS and iNOS) in the ischemic hemisphere. This was associated withelevated levels of 3-nitrotyrosine, an indicator of peroxynitrite-mediated oxidative proteinmodification. Pretreatment of the mice with betulinic acid (50 mg/kg/day for 7 days via gavage) prior to MCAO reduced infarct volume. In addition, treatment of betulinic acidresulted in a reduction of the ischemia/reperfusion-induced up-regulation of NOX2, nNOSand iNOS. Moreover, treatment with betulinic acid enhanced the expression of endothelialNOS (eNOS), both in the ischemic and non-ischemic hemispheres. In parallel, betulinicacid reduced the levels of 3-nitrotyrosine. Conclusions: these results suggest theneuroprotective potential of BA in cerebral ischemia-reperfusion injury, the mechanismsmay involve inhibiting of both superoxide and NO·production via down-regulated NOX2,iNOS and nNOS gene expression, leading to decreased oxidative stress injury duringcerebral ischemia-reperfusion.
目的：建立方便有效的培养的大鼠皮质神经元拟缺血性损伤模型，观察Guattegaumerine的神经细胞保护作用并初步探讨其作用机制。方法：原代Wistar大鼠乳鼠皮质神经元培养，不同浓度的H_2O_2和谷氨酸在无血清低糖培养基中与细胞共孵育24h后检测细胞活力(MTT法)和乳酸脱氢酶(lactate dehydrogenase，LDH)释放量。1．25μmol／L与2．5μmol／L Guattegaumerine与皮质神经元预孵育30min，随后加入含6．25μmol／L H_2O_2的无血清低糖培养基，继续培养24h后检测细胞活力及LDH释放量，细胞总抗氧化能力(total antioxidative capatity，TAC)及脂氧化产物MDA生成量；膜连蛋白(Annexin v)-FITC／碘化丙啶(propidium iodide，PI)双染流式细胞仪检测神经元的凋亡和坏死，及免疫组化检测细胞Bax与Bcl-2蛋白表达；钙荧光探针Fura-2AM检测H_2O_2在含钙及无钙细胞外液中诱发的胞内钙浓度变化及KCl诱导胞内钙浓度变化，并观察不同浓度Guattegaumerine对H_2O_2和KCl诱发的胞内钙浓度升高的影响。结果：6．25μmol／L～200μmol／L H_2O_2及12．5μmol／L～50μmol／L谷氨酸合并血清剥夺与神经元孵育24h后，使神经元的生长能力降低，LDH漏出量增加。Guattegaumerine能抑制6．25μmol／L H_2O_2合并血清剥夺诱导的皮质神经元损伤，表现为提高细胞活力，减少LDH漏出量，提高细胞TAC的活力，减少MDA生成量，并抑制细胞凋亡，抑制Bax及促进Bcl-2蛋白表达；抑制含钙细胞外液试验中H_2O_2及KCl诱导的胞内钙浓度升高，高浓度guattegaumerine还能抑制无钙外液试验中H_2O_2诱导的胞内钙浓度升高。结论：6．25μmol／L～200μmol／L H_2O_2及12．5μmol／L～50μmol／L谷氨酸合并血清剥夺与神经元孵育24h是两种方便有效的诱导神经元拟缺血性损伤模型的方法。Guattegaumerine对6．25μmol／L H_2O_2合并血清剥夺诱导的皮质神经元损伤有保护作用，其作用机制可能与其通过提高抗氧化酶活性，抑制脂质过氧化反应，调节凋亡相关基因及拮抗胞内钙超载有关。Guattegaumerine的钙拮抗作用可能与其阻断膜上电压依赖性钙通道及受体门控型钙通道，抑制钙库钙释放有关。
第二部分Betulinic acid对ApoE knock ont小鼠脑缺血再灌注损伤的保护作用及作用机制研究
目的：大脑中动脉栓塞法建立ApoE基因敲除(ApoE KO)小鼠的局灶性脑缺血再灌注损伤模型，并观察betulinic acid对脑缺血再灌注损伤的保护作用并初步探讨其作用机制。方法：50mg／kg betulinic acid灌胃给药，1次／天，共7天。灌胃结束后，左侧大脑中动脉栓塞2h复灌22h诱导ApoE KO小鼠的脑缺血再灌注损伤模型，脑组织切片TTC染色测定脑梗死体积；缺血侧及缺血对侧脑组织mRNA提取qRT-PCR检测NAPDH氧化酶亚型NOX1、NOX2、NOX4、p22phox，及NO合成酶eNOS、iNOS和nNOS的mRNA的表达；缺血侧及缺血对侧脑组织蛋白质提取western-blot检测NO合成酶eNOS和nNOS，及蛋白质的氧化产物酪氨酸硝基化蛋白的表达。结果：左侧大脑中动脉栓塞2h复灌22h能稳定地诱导ApoE KO小鼠脑缺血再灌注损伤，缺血侧脑组织NOX2、NOX4，nNOS及iNOS基因表达升高，蛋白质氧化产物生成量增多。50mg／kg betulinic acid灌胃给药能降低脑梗死体积，增加脑组织eNOS mRNA及蛋白质的表达，降低缺血侧脑组织nNOS mRNA及蛋白质的表达，同时降低缺血侧脑组织iNOS，NOX2的mRNA的表达。50mg／kg betulinic acid灌胃给药亦降低缺血侧脑组织酪氨酸硝基化蛋白质水平。结论：50mg／kg betulinic acid对ApoE KO小鼠脑缺血再灌注损伤有保护作用，其作用机制可能与其抑制NOX2、nNOS及iNOS基因表达从而抑制活性氧和活性氮的产生，抑制脑缺血再灌注后氧化应激损伤有关。
First Part Protective Effects of Guattegaumerine Against HydrogenPeroxide Concomitant with Serum Deprivation Incuced Injury inPrimary Cultured Cortical Neurons
Aims: To establish the convenient and effective model of ischemia induced injury inprimary cultured neurons, and evaluate the neuroprotective effects of guattegaumerine onrat primary cultured cortical neurons. Methods: Research on primary cultured corticalneurons of Wistar rats, different concentrations of H_2O_2 and Glutamate (Glu) expoured tocells in serum free and low glucose medium for 24h and cell viability determined by3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and lactatedehydrogenase (LDH) leakage were detected. 1.25μmol/L and 2.5μmol/L guattegaumerinewere added 30 min before cells exposure to 6.25μmol/L H_2O_2 in serum free and lowglucose medium for 24h, then cell viability、LDH leakage、malondialhehyde (MDA) andtotal antioxidative capacity (TAC) were observred. Cells apoptosis and necrosis weremeasured by Annexin V/Propidium iodide double staining assay using flow cytometry, andBax, Bcl-2 protein expression were detected by immunohistochemistry. The calciumfluorescent label fura-2 AM was used to measure the intracellular calcium concentrationinduced by H_2O_2 and KC1, and the effects of guattegaumerine on the increased intracellularcalcium stimulated by H_2O_2 and KC1. Results: 6.25μmol/L～200μmol/L H_2O_2 and12.5μmol/L～50μmol/L Glu could decreased cell viability and increased leakage of LDH.Preincubation of guattegaumerine dramatically improved the cell viability and inhibitedLDH release. Preincubation of guattegaumerine also dramatically inhibited MDAproduction and elevated TAC in cells. Results of flow cytometry andimmunohistochemistry showed that preaddition of guattegaumerine interrupted theapoptosis of the neurons, reversed the up regulation of the pro-apoptotic gene (Bax) and thedown regulation of the anti-apoptotic gene (Bcl-2). Furthermore, guattegaumerinesuppressed the increase of intracellular calcium concentration ([Ca~(2+)]_i) stimulated by eitherH_2O_2 or KC1 in Ca~(2+)-containing extracellular solution, and high concentration of guattegaumerine also suppressed the increase of [Ca~(2+)]_i induced by H_2O_2 in Ca~(2+)-freesolution. Conclusions: 6.25μmol/L～200μmol/L H_2O_2 and 12.5μmol/L～50μmol/L Glu aretwo convenient and effective models to induce ischemic injury in neurons.Guattegaumerine protects cultured cortical neurons against 6.25μmol/L H_2O_2 concomitantwith serum deprivation incuced injury, which may relate to its inhibition of lipidperoxidation, regulation of apoptotic related gene expression and prevention of intracellularcalcium elevation by blocking the selective and non-selective calcium channels on the cellmembrane, and the release of calcium from ER.
Second Part Betulinic Acid Protects against CerebralIschemia/Reperfusion Injury in ApoE Knock Out icem
Aims: To establish focal cerebral ischemia-reperfusion injury by middle cerebral arteryocclusion in ApoE knock out (KO) mice, and investigate the neuroprotective potential ofbetulinic acid against brain ischemia-reperfusion. Methods: ApoE KO mice were treatedwith 50mg/kg betulinic acid via gavage per day for 7days, then subjected to 2h of the leftmiddle cerebral artery occlusion (MCAO) followed by 22h of reperfusion. Brain infarctionwas indicated by TTC staining. NADPH oxidase (NOX) subunits (NOX1、NOX2、NOX4and p22phox) and nitric oxide synthase (NOS) subunits (nNOS、iNOS and eNOS) mRNAexpression were measured by qRT-PCR, and NOS subunits (nNOS and eNOS) andnitrotyrosine protein expression were measured by western-blot. Results: Inatherosclerosis-prone apolipoprotein E knockout (ApoE-KO) mice, 2h of middle cerebralartery occlusion (MCAO) followed by 22h of reperfusion led to an enhanced expression ofseveral NADPH oxidase subunits (NOX2, NOX4 and p22phox) and an up-regulation ofNOS isoforms (nNOS and iNOS) in the ischemic hemisphere. This was associated withelevated levels of 3-nitrotyrosine, an indicator of peroxynitrite-mediated oxidative proteinmodification. Pretreatment of the mice with betulinic acid (50 mg/kg/day for 7 days via gavage) prior to MCAO reduced infarct volume. In addition, treatment of betulinic acidresulted in a reduction of the ischemia/reperfusion-induced up-regulation of NOX2, nNOSand iNOS. Moreover, treatment with betulinic acid enhanced the expression of endothelialNOS (eNOS), both in the ischemic and non-ischemic hemispheres. In parallel, betulinicacid reduced the levels of 3-nitrotyrosine. Conclusions: these results suggest theneuroprotective potential of BA in cerebral ischemia-reperfusion injury, the mechanismsmay involve inhibiting of both superoxide and NO·production via down-regulated NOX2,iNOS and nNOS gene expression, leading to decreased oxidative stress injury duringcerebral ischemia-reperfusion.
引文
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