蝎毒神经元营养活性肽抗癫痫作用的研究
摘要
目的：
癫痫症(Epilepsy, EP)是常见多发的慢性脑部疾患之一,其发病机制目前并不十分清楚。但其发作的电生理本质都是脑部神经元过度同步化放电,从而引起大脑短暂的功能障碍。本病长期反复发作,不仅使患者体躯遭受痛苦,而且在一定程度上导致精神及社会心理障碍,使其在智能及人格方面均受到损害。正是由于癫痫病的复杂性、难治性及用药的多样性,因此抗癫痫药物的开发急需等待突破。
在药物筛选中,我们发现东亚钳蝎(Buthus martensi Karsch, BmK)对难治性癫痫有明显药效。全蝎入药治疗癫痫已有几千年的历史,其药效在于蝎尾中的毒液组分蝎毒(scorpion venom, SV)。SV具有明显的抗癫痫作用,但成分复杂,毒性很强,使得其临床应用受到很大限制。我室长期从事蝎毒药物开发研究,从蝎毒毒素中提取出具有抗癫痫作用的组分通过特殊工艺,得到分子量为3177Da的蝎毒神经营养活性肽(scorpion venom neuro-nourishing peptide, svNNP)(国家发明专利：ZL011061669)。与SV粗毒相比,svNNP不仅毒性明显降低,且抗癫痫作用增强。
因此本研究的主要目的是从整体和细胞水平进一步探讨svNNP抗癫痫的神经保护机制,为蝎毒药物的开发提供新的理论依据。
方法：
在体大鼠及体外培养神经元制备癫痫模型,应用全细胞膜片钳技术、免疫组织化学、免疫细胞化学、MTT法及Western-Blot等方法多层次,多水平观察蝎毒神经营养活性肽抗癫痫作用的机制。
结果：
一、体内试验：
氯化锂(LiCl)-匹罗卡品(PILO)诱导大鼠制备癫痫模型,给予动物svNNP,观察行为学改变、免疫组织化学影响,结果显示：
1.svNNP给药组25min内癫痫发作次数较对照组减少,但发作级别尚无统计学意义(n=5,P>0.05),而各实验组动物隐藏平台获得实验及空间搜索实验逃避潜伏期和穿越平台的次数比较无显著意义(n=5,P>0.05)。
2.GFAP免疫组织化学实验显示svNNP用药后GFAP阳性反应细胞突起不规则,分支多而短曲,表面不光滑,细胞类型由模型组的纤维型星形胶质细胞转化为用药组的原浆型星形胶质细胞。
3.MAP2免疫组织化学显示svNNP给药组与模型组相比树突断裂数明显减轻,神经元脱失及树突形状明显改善。
二、体外实验：
1.免疫细胞化学：体外培养海马神经元及胶质细胞,预孵育SV粗毒和svNNP24h后脑源神经生长因子(brain derived neutrophic factor, BDNF)免疫染色：①胶质细胞BDNF免疫细胞化学结果：svNNP给药组与SV粗毒给药组及正常对照组相比较BDNF免疫反应染色加深,阳性细胞数目明显增加(P<0.01),染色变深,推测svNNP可增加胶质细胞BDNF的表达。②海马神经元BNDF免疫细胞化学结果：svNNP预处理组较对照组比较神经元形态完好,细胞之间连接紧密,BDNF免疫反应阳性染色及免疫反应阳性细胞明显增强(P<0.01)。
2.细胞存活能力：MTT法观察蝎毒提出物对原代培养海马神经元存活的影响,发现SV粗毒组与正常组相比较OD值明显降低(P<0.05)；svNNP组同正常组相比较OD值明显增高(P<0.05)。说明svNNP可提高细胞存活力,而粗毒却具有毒性作用,降低原代培养海马神经元的细胞存活力。
3.受体门控通道：体外培养海马神经元,急性加入NMDA诱导NMDA受体激活电流(ⅠNMDA),观察不同浓度svNNP(10-3μg/ml、10-4μg/ml、10-5μg/ml、10-6μg/ml、10-7μg/m、10-8μg/ml、)对ⅠNMDA的影响,结果显示：五种不同浓度的svNNP均能降低海马神经元ⅠNMDA(n=8,P<0.01),而且具有浓度依赖性。
4.电压门控钠通道：采用红藻氨酸(Kainic acid, KA)制备原代培养海马神经元的兴奋毒性损伤模型,孵育svNNP 24h,结果显示：①svNNP可抑制KA预处理的海马神经元电压门控钠通道峰电流。对照组(n=26)与svNNP组(n=24)相比差异具有显著意义(P<0.05)。②svNNP药物处理组海马神经元电压门控钠通道稳态激活曲线发生变化,10μmol/LKA处理后钠通道稳态激活曲线的半数激活电压V1/2为-40.1±1.80(n=20),2μg／ml svNNP+10μmol／L KA处理后V1/2变为-36.26±1.44(n=19),KA处理组和KA+svNNP处理组V1/2相比较具有统计学意义(P<0.05)。③药物不同处理组的海马神经元钠通道稳态失活曲线亦发生变化,正常钠通道半数失活电压为-59.03±0.29(n=16)；10μmol／LKA处理后V1/2-61.07+0.43(n=18)；给予2μg／ml svNNP+10μmol／L KA处理后V1/2变为-64.24±0.97(n=16)。表明2μg／ml svNNP+10μmol／L KA处理后钠通道半数失活电压左移,与正常对照组、KA模型组相比差异均具有显著意义(P<0.05)。④svNNP用药后钠通道从失活中恢复减慢,与对照组相比差异具有显著意义(n=22,P<0.05)。
结论：
1.svNNP用药后癫痫大鼠星形胶质细胞由纤维型变为原浆型。
2.svNNP可改善癫痫发作所致树突断裂及神经元脱失。
3.svNNP可增加原代培养海马神经元存活能力,而SV粗毒却减低神经元存活能力。
4.svNNP可增加神经元和胶质细胞BDNF的分泌,具有营养和保护神经元作用。
5.svNNP可浓度依赖性的抑制海马神经元NMDA受体激活电流,减低癫痫过程中NMDA受体过度激活而引起的神经损伤作用。
6.svNNP可抑制体外KA致痫的海马神经元钠通道电流,减慢钠通道的激活,加快钠通道的失活,减缓钠通道的恢复,从而降低癫痫过程中的神经元的过度兴奋。
Objective:The pathogenesis of epilepsy, one of the common multiple chronic brain diseases, is not very clear now. However, the electrophysiological nature of the epilepsy onsets is that brain neurons synchronized over-discharge, causing temporary dysfunction of the brain. It's a long-term recurrent disease. Patients suffer not only physically, but to some extent, also have mental and psychosocial obstacles, with their intelligence and personality aspects damaged. It is because of the complexity and refractoriness of epilepsy, the discovery of antiepileptic drugs needs a breakthrough urgently. In the screening of drug, we found Scorpion (BmK) show a significant efficacy on refractory epilepsy. Scorpion has been used for medicine treatment of epilepsy for many years. Its efficacy lies in the venom component of scorpion, scorpion venom (SV). SV has a significant antiepileptic effect. However, the elements are complex, highly toxic, so that its clinical application is limited. Our laboratory has been engaged in the research for discovering scorpion poison for a long time and has extracted active peptide which has the anti-epileptic effect from scorpion venom. Through a special process, we have obtained the scorpion venom neuro-nourishing peptide (svNNP), which has the anti-epileptic effect. Compared with SV venom, not only the toxicity of svNNP is significantly reduced, but aslo the anti-epileptic efficacy is enhanced. The purpose of this study is to further explore the neuroprotective antiepileptic mechanism of svNNP on the general and whole cell level, in order to provide new experimental evidences for the discovery of Scorpion Poison.
Method:Using whole cell patch clamp, immunohistochemistry, immunocytochemistry, MTT and Western-Blot technique, the mechanism of antiepileptic effect of svNNP was approached by epilepsy model in vivo and in vitro.
Results:
Section 1 (In vivo experiment)
Using lithium chloride(LiCl)-pilocarpine (PILO) epilepsy rat model, the effects of svNNP were observed by behavior, immunohistochemistry. The results show that
1. The times of seizures within 25min after treatment of svNPP were decreased than the control group, but the grade of seizures has no significance (n=5, P>0.05). The escape latencies and times to pass through the platform for each group animals were not significant among each other (n=5,P> 0.05)
2. GFAP immunohistochemistry slices results show that svNNP treatment group could increase GFAP immunoreactive cells, The morphology of astrocytes showed irregular processes, many short curved branches and not smooth surface which demonstrated the type of most astrocytes belong to protoplasmic astrocyte.
3. Map2 immunohistochemistry staining show that the number of fractured dendrites and lossing of neuron for post-treatment svNNP group were significantly reduced compared with the model control group.
Section 2 (In vitro experiment)
1. Brain derived neurotrophic factor (BDNF) immunostaining for cultured hippocampal neurons and glial cells which were pre-incubated with svNNP for 24hrs shows that①the number of BDNF immunoreactive glial cells of post-treatment svNNP group increases significantly and the staining becomes deeper compared with those of the post-treatment SV venom group, suggesting that svNNP could increase the expression of BDNF of glial cells.②hippocampal neuronal morphology of post-treatment svNNP group is intact with tight connections among cells, and BDNF immunoreactive staining and immunoreactive cells increased significantly (P<0.01) compared with the control group.
2. The effects of svNNP on the survival of the primary cultured hippocampal neurons were observed by MTT assay. It was found that in SV venom group the OD value decreased significantly (P<0.05) compared with the normal control group; in svNNP group the OD value increased significantly (P<0.05) compared with the normal control group, suggesting that svNNP could increase neuron survival-rate, and protecte them from damage, while the SV venom has toxic effects and decreases the survival of the primary cultured hippocampal neurons
3. NMDA-activated current (ⅠNMDA) were induced from cultured hippocampal neurons. The effects of different concentrations of svNNP (10-3μg/ml,10-4μg/ml,10-5μg/ml,10-6μg/ml,10-7μg/m,10-8μg/ml) onⅠNMDA were observed. The results show that the five different concentrations of svNNP all could reduceⅠNMDA of hippocampal neurons (P<0.01, n=8), and appered dose-dependent manner.
4. Voltage-gated sodium channel currents from primary cultured hippocampal neurons were recorded using patch clamp technique in kainic acid (KA) excitotoxic injury model which incubated with svNNP for 24hrs. The results show that①svNNP could inhibit voltage-gated sodium channel peak currents from KA-induced hippocampal epilepsy model neurons (P<0.05).②svNNP could shift toward depolarzied potentials in steady-state activation curve of voltage-gated sodium channel on hippocampal neurons compared with those of normal control group. After treatment of 10μmol/L KA, the voltage of half-activation (V1/2) was-40.1±1.80 (n=20), post-treatment 2μg/ml svNNP+10μmol/L KA the V1/2 became-36.26±1.44 (n=19). The V1/2 was significant (P<0.05) between post-treatment KA group with post-treatment KA+svNNP group.③The curve for steady-state inactivation of sodium channel on hippocampal KA-induced epilepsy model neurons was shifted toward negative potential after treament of svNNP. The half inactivation voltage (V1/2) of the normal sodium channel was-59.03±0.29 (n= 16);post-treatment 10μmol/L KA the V1/2 became-61.59±0.58 (n=28); post-treatment 10μmol/L KA+2μg/ml svNNP the V1/2 became-64.24±0.97 (n=16). It is suggested that post-treatment 2μg/ml svNNP+10μmol/L KA the sodium channel half inactivation voltage shifted to the left, compared with the control group and KA model group. The differences were significant (P<0.05).④After svNNP treatment, the sodium channels recovered slowly from inactivation. The difference was significant (P<0.05) compared with the control group.
Conclusion:
1. svNNP could make epileptic rats'astrocytes change from the fibrous type into the protoplasmic type after the treatment.
2. svNNP could reduce the dendritic fracture and the neuron-loss in the epileptic attack process.
3. svNNP could improve the survival of primary cultured hippocampal neurons,while SV venom toxicity has a contrary effect.
4. svNNP could increase the secretion of BDNF of neurons and glial cells, playing the role of nutrition and protection of neurons.
5. svNNP could inhibit the NMDA receptor (NMDAR)-activated currents of the hippocampal neurons in a dose-dependent manner and reduce the nerve injury caused by excessive activation of NMDAR during seizures.
6. svNNP could inhibit sodium channel current in vitro KA-induced epileptic hippocampal neuronal, slow the activation of sodium channels, speed up the inactivation of sodium channels and slow the recovery of sodium channels, thereby reducing the excessive activation of neurons in the epileptic process.
癫痫症(Epilepsy, EP)是常见多发的慢性脑部疾患之一,其发病机制目前并不十分清楚。但其发作的电生理本质都是脑部神经元过度同步化放电,从而引起大脑短暂的功能障碍。本病长期反复发作,不仅使患者体躯遭受痛苦,而且在一定程度上导致精神及社会心理障碍,使其在智能及人格方面均受到损害。正是由于癫痫病的复杂性、难治性及用药的多样性,因此抗癫痫药物的开发急需等待突破。
在药物筛选中,我们发现东亚钳蝎(Buthus martensi Karsch, BmK)对难治性癫痫有明显药效。全蝎入药治疗癫痫已有几千年的历史,其药效在于蝎尾中的毒液组分蝎毒(scorpion venom, SV)。SV具有明显的抗癫痫作用,但成分复杂,毒性很强,使得其临床应用受到很大限制。我室长期从事蝎毒药物开发研究,从蝎毒毒素中提取出具有抗癫痫作用的组分通过特殊工艺,得到分子量为3177Da的蝎毒神经营养活性肽(scorpion venom neuro-nourishing peptide, svNNP)(国家发明专利：ZL011061669)。与SV粗毒相比,svNNP不仅毒性明显降低,且抗癫痫作用增强。
因此本研究的主要目的是从整体和细胞水平进一步探讨svNNP抗癫痫的神经保护机制,为蝎毒药物的开发提供新的理论依据。
方法：
在体大鼠及体外培养神经元制备癫痫模型,应用全细胞膜片钳技术、免疫组织化学、免疫细胞化学、MTT法及Western-Blot等方法多层次,多水平观察蝎毒神经营养活性肽抗癫痫作用的机制。
结果：
一、体内试验：
氯化锂(LiCl)-匹罗卡品(PILO)诱导大鼠制备癫痫模型,给予动物svNNP,观察行为学改变、免疫组织化学影响,结果显示：
1.svNNP给药组25min内癫痫发作次数较对照组减少,但发作级别尚无统计学意义(n=5,P>0.05),而各实验组动物隐藏平台获得实验及空间搜索实验逃避潜伏期和穿越平台的次数比较无显著意义(n=5,P>0.05)。
2.GFAP免疫组织化学实验显示svNNP用药后GFAP阳性反应细胞突起不规则,分支多而短曲,表面不光滑,细胞类型由模型组的纤维型星形胶质细胞转化为用药组的原浆型星形胶质细胞。
3.MAP2免疫组织化学显示svNNP给药组与模型组相比树突断裂数明显减轻,神经元脱失及树突形状明显改善。
二、体外实验：
1.免疫细胞化学：体外培养海马神经元及胶质细胞,预孵育SV粗毒和svNNP24h后脑源神经生长因子(brain derived neutrophic factor, BDNF)免疫染色：①胶质细胞BDNF免疫细胞化学结果：svNNP给药组与SV粗毒给药组及正常对照组相比较BDNF免疫反应染色加深,阳性细胞数目明显增加(P<0.01),染色变深,推测svNNP可增加胶质细胞BDNF的表达。②海马神经元BNDF免疫细胞化学结果：svNNP预处理组较对照组比较神经元形态完好,细胞之间连接紧密,BDNF免疫反应阳性染色及免疫反应阳性细胞明显增强(P<0.01)。
2.细胞存活能力：MTT法观察蝎毒提出物对原代培养海马神经元存活的影响,发现SV粗毒组与正常组相比较OD值明显降低(P<0.05)；svNNP组同正常组相比较OD值明显增高(P<0.05)。说明svNNP可提高细胞存活力,而粗毒却具有毒性作用,降低原代培养海马神经元的细胞存活力。
3.受体门控通道：体外培养海马神经元,急性加入NMDA诱导NMDA受体激活电流(ⅠNMDA),观察不同浓度svNNP(10-3μg/ml、10-4μg/ml、10-5μg/ml、10-6μg/ml、10-7μg/m、10-8μg/ml、)对ⅠNMDA的影响,结果显示：五种不同浓度的svNNP均能降低海马神经元ⅠNMDA(n=8,P<0.01),而且具有浓度依赖性。
4.电压门控钠通道：采用红藻氨酸(Kainic acid, KA)制备原代培养海马神经元的兴奋毒性损伤模型,孵育svNNP 24h,结果显示：①svNNP可抑制KA预处理的海马神经元电压门控钠通道峰电流。对照组(n=26)与svNNP组(n=24)相比差异具有显著意义(P<0.05)。②svNNP药物处理组海马神经元电压门控钠通道稳态激活曲线发生变化,10μmol/LKA处理后钠通道稳态激活曲线的半数激活电压V1/2为-40.1±1.80(n=20),2μg／ml svNNP+10μmol／L KA处理后V1/2变为-36.26±1.44(n=19),KA处理组和KA+svNNP处理组V1/2相比较具有统计学意义(P<0.05)。③药物不同处理组的海马神经元钠通道稳态失活曲线亦发生变化,正常钠通道半数失活电压为-59.03±0.29(n=16)；10μmol／LKA处理后V1/2-61.07+0.43(n=18)；给予2μg／ml svNNP+10μmol／L KA处理后V1/2变为-64.24±0.97(n=16)。表明2μg／ml svNNP+10μmol／L KA处理后钠通道半数失活电压左移,与正常对照组、KA模型组相比差异均具有显著意义(P<0.05)。④svNNP用药后钠通道从失活中恢复减慢,与对照组相比差异具有显著意义(n=22,P<0.05)。
结论：
1.svNNP用药后癫痫大鼠星形胶质细胞由纤维型变为原浆型。
2.svNNP可改善癫痫发作所致树突断裂及神经元脱失。
3.svNNP可增加原代培养海马神经元存活能力,而SV粗毒却减低神经元存活能力。
4.svNNP可增加神经元和胶质细胞BDNF的分泌,具有营养和保护神经元作用。
5.svNNP可浓度依赖性的抑制海马神经元NMDA受体激活电流,减低癫痫过程中NMDA受体过度激活而引起的神经损伤作用。
6.svNNP可抑制体外KA致痫的海马神经元钠通道电流,减慢钠通道的激活,加快钠通道的失活,减缓钠通道的恢复,从而降低癫痫过程中的神经元的过度兴奋。
Objective:The pathogenesis of epilepsy, one of the common multiple chronic brain diseases, is not very clear now. However, the electrophysiological nature of the epilepsy onsets is that brain neurons synchronized over-discharge, causing temporary dysfunction of the brain. It's a long-term recurrent disease. Patients suffer not only physically, but to some extent, also have mental and psychosocial obstacles, with their intelligence and personality aspects damaged. It is because of the complexity and refractoriness of epilepsy, the discovery of antiepileptic drugs needs a breakthrough urgently. In the screening of drug, we found Scorpion (BmK) show a significant efficacy on refractory epilepsy. Scorpion has been used for medicine treatment of epilepsy for many years. Its efficacy lies in the venom component of scorpion, scorpion venom (SV). SV has a significant antiepileptic effect. However, the elements are complex, highly toxic, so that its clinical application is limited. Our laboratory has been engaged in the research for discovering scorpion poison for a long time and has extracted active peptide which has the anti-epileptic effect from scorpion venom. Through a special process, we have obtained the scorpion venom neuro-nourishing peptide (svNNP), which has the anti-epileptic effect. Compared with SV venom, not only the toxicity of svNNP is significantly reduced, but aslo the anti-epileptic efficacy is enhanced. The purpose of this study is to further explore the neuroprotective antiepileptic mechanism of svNNP on the general and whole cell level, in order to provide new experimental evidences for the discovery of Scorpion Poison.
Method:Using whole cell patch clamp, immunohistochemistry, immunocytochemistry, MTT and Western-Blot technique, the mechanism of antiepileptic effect of svNNP was approached by epilepsy model in vivo and in vitro.
Results:
Section 1 (In vivo experiment)
Using lithium chloride(LiCl)-pilocarpine (PILO) epilepsy rat model, the effects of svNNP were observed by behavior, immunohistochemistry. The results show that
1. The times of seizures within 25min after treatment of svNPP were decreased than the control group, but the grade of seizures has no significance (n=5, P>0.05). The escape latencies and times to pass through the platform for each group animals were not significant among each other (n=5,P> 0.05)
2. GFAP immunohistochemistry slices results show that svNNP treatment group could increase GFAP immunoreactive cells, The morphology of astrocytes showed irregular processes, many short curved branches and not smooth surface which demonstrated the type of most astrocytes belong to protoplasmic astrocyte.
3. Map2 immunohistochemistry staining show that the number of fractured dendrites and lossing of neuron for post-treatment svNNP group were significantly reduced compared with the model control group.
Section 2 (In vitro experiment)
1. Brain derived neurotrophic factor (BDNF) immunostaining for cultured hippocampal neurons and glial cells which were pre-incubated with svNNP for 24hrs shows that①the number of BDNF immunoreactive glial cells of post-treatment svNNP group increases significantly and the staining becomes deeper compared with those of the post-treatment SV venom group, suggesting that svNNP could increase the expression of BDNF of glial cells.②hippocampal neuronal morphology of post-treatment svNNP group is intact with tight connections among cells, and BDNF immunoreactive staining and immunoreactive cells increased significantly (P<0.01) compared with the control group.
2. The effects of svNNP on the survival of the primary cultured hippocampal neurons were observed by MTT assay. It was found that in SV venom group the OD value decreased significantly (P<0.05) compared with the normal control group; in svNNP group the OD value increased significantly (P<0.05) compared with the normal control group, suggesting that svNNP could increase neuron survival-rate, and protecte them from damage, while the SV venom has toxic effects and decreases the survival of the primary cultured hippocampal neurons
3. NMDA-activated current (ⅠNMDA) were induced from cultured hippocampal neurons. The effects of different concentrations of svNNP (10-3μg/ml,10-4μg/ml,10-5μg/ml,10-6μg/ml,10-7μg/m,10-8μg/ml) onⅠNMDA were observed. The results show that the five different concentrations of svNNP all could reduceⅠNMDA of hippocampal neurons (P<0.01, n=8), and appered dose-dependent manner.
4. Voltage-gated sodium channel currents from primary cultured hippocampal neurons were recorded using patch clamp technique in kainic acid (KA) excitotoxic injury model which incubated with svNNP for 24hrs. The results show that①svNNP could inhibit voltage-gated sodium channel peak currents from KA-induced hippocampal epilepsy model neurons (P<0.05).②svNNP could shift toward depolarzied potentials in steady-state activation curve of voltage-gated sodium channel on hippocampal neurons compared with those of normal control group. After treatment of 10μmol/L KA, the voltage of half-activation (V1/2) was-40.1±1.80 (n=20), post-treatment 2μg/ml svNNP+10μmol/L KA the V1/2 became-36.26±1.44 (n=19). The V1/2 was significant (P<0.05) between post-treatment KA group with post-treatment KA+svNNP group.③The curve for steady-state inactivation of sodium channel on hippocampal KA-induced epilepsy model neurons was shifted toward negative potential after treament of svNNP. The half inactivation voltage (V1/2) of the normal sodium channel was-59.03±0.29 (n= 16);post-treatment 10μmol/L KA the V1/2 became-61.59±0.58 (n=28); post-treatment 10μmol/L KA+2μg/ml svNNP the V1/2 became-64.24±0.97 (n=16). It is suggested that post-treatment 2μg/ml svNNP+10μmol/L KA the sodium channel half inactivation voltage shifted to the left, compared with the control group and KA model group. The differences were significant (P<0.05).④After svNNP treatment, the sodium channels recovered slowly from inactivation. The difference was significant (P<0.05) compared with the control group.
Conclusion:
1. svNNP could make epileptic rats'astrocytes change from the fibrous type into the protoplasmic type after the treatment.
2. svNNP could reduce the dendritic fracture and the neuron-loss in the epileptic attack process.
3. svNNP could improve the survival of primary cultured hippocampal neurons,while SV venom toxicity has a contrary effect.
4. svNNP could increase the secretion of BDNF of neurons and glial cells, playing the role of nutrition and protection of neurons.
5. svNNP could inhibit the NMDA receptor (NMDAR)-activated currents of the hippocampal neurons in a dose-dependent manner and reduce the nerve injury caused by excessive activation of NMDAR during seizures.
6. svNNP could inhibit sodium channel current in vitro KA-induced epileptic hippocampal neuronal, slow the activation of sodium channels, speed up the inactivation of sodium channels and slow the recovery of sodium channels, thereby reducing the excessive activation of neurons in the epileptic process.
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