牛膝多肽神经保护作用的实验研究
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摘要
目的:
观察牛膝多肽(A. bidentata polypeptides, ABPP)的神经保护作用及其相关作用机制。
方法:
(1)采用大脑中动脉线栓法建立大鼠局灶性脑缺血再灌注模型,通过测定神经功能缺陷评分、脑梗死百分比(TTC法),在体观察牛膝多肽的神经保护作用。
(2)以体外原代培养胎鼠海马神经元为研究对象,建立N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)损伤模型。通过MTT检测,离体观察牛膝多肽的神经保护作用。
(3)Hoechst33258/PI双染色、PI流式细胞仪检测、LDH检测、DNA ladder检测等观察牛膝多肽对NMDA诱导的海马神经元调亡的影响。
(4)利用钙离子荧光探针Fluo-3/AM标记海马神经元,通过激光共聚焦显微镜检测,观察牛膝多肽对NMDA引起的海马神经元胞内钙离子浓度的影响。
(5)通过Western blot,观察牛膝多肽对NMDA引起的海马神经元Bax蛋白过表达的影响。
(6)通过Caspase-3活性检测,观察牛膝多肽对NMDA引起的海马神经元Caspase-3蛋白活性增高的影响。
(7)采用亲脂性阳离子荧光染料Rhodamine123、分子探针DCFH-DA标记海马神经元,通过多功能酶标仪检测NMDA引起的海马神经元线粒体跨膜电位、活性氧自由基含量的变化。同时观察牛膝多肽对NMDA引起的海马神经元线粒体跨膜电位、活性氧自由基含量的影响。
(8)采用全细胞膜片钳技术,观察牛膝多肽对NMDA电流峰值、平台期电流值、失敏系数的影响。
(9)采用了含NR2A、NR2B两种不同亚单位类型NMDA受体的选择性抑制剂:Ro-256981、NVP-AAM007,及两种不同培养时间的海马神经元:体外培养8天、13-14天,通过MTT检测,观察两种不同的选择性抑制剂对NMDA诱导神经元损伤的影响,同时观察牛膝多肽对NMDA诱导神经元损伤的影响。
(10)应用钙离子荧光探针Fluo-3/AM标记通过激光共聚焦显微镜检测,观察含NR2A、NR2B两种不同亚单位类型NMDA受体的选择性抑制剂:Ro-256981、NVP-AAM007,对NMDA和Bicuculline引起的海马神经元胞内钙离子浓度的影响,并观察牛膝多肽对含NR2A、NR2B两种不同亚单位类型NMDA受体介导的胞内钙离子浓度的影响。
(11)采用全细胞膜片钳技术,观察含NR2A、NR2B两种不同亚单位类型NMDA受体的选择性抑制剂:Ro-256981、NVP-AAM007,对NMDA电流峰值、平台期电流值、失敏系数的影响,并观察牛膝多肽对含NR2A、NR2B两种不同亚单位类型NMDA受体介导的NMDA电流峰值、平台期电流值、失敏系数的影响。
结果:
(1)局灶性脑缺血再灌注损伤能引起大鼠死亡,死亡率达到50%,对存活的大鼠,其神经功能缺陷评分明显增高(p<0.01)和脑梗死百分比增加(p<0.01),尾静脉给予牛膝多肽(0.2mg/kg)治疗,可以降低神经功能缺陷评分,降低脑梗死百分比(p<0.01)。
(2)通过MTT检测结果显示,NMDA能降低培养的海马神经元的活力,而且随着NMDA浓度的增加、作用时间的延长,神经元的活力逐渐下降。牛膝多肽能抑制NMDA引起的海马神经元活力下降,并与其剂量相关。
(3)Hoechst33258/PI双染色、PI流式细胞仪检测、LDH检测、DNA ladder检测结果显示,NMDA能诱导海马神经元调亡,牛膝多肽(1μg/ml)能拮抗NMDA诱导的海马神经元调亡。
(4)钙实时成像结果显示,NMDA能引起海马神经元胞内钙荧光强度增加,并随着NMDA浓度增加,荧光强度增强。AP-V、MK801能抑制胞内钙荧光强度增高。预先、同时、后加入牛膝多肽都能减弱NMDA引起海马神经元胞内钙荧光强度增加,并随着浓度增高,抑制程度增加。
(5)通过MTT检测结果显示,Bax通道阻断剂能拮抗NMDA引起的海马神经元活力的降低。RT-PCR和Western blot结果提示,海马神经元Bax mRNA及其蛋白表达随着NMDA作用时间的变化而变化,其中作用30min Bax蛋白表达明显增加(p<0.05)。牛膝多肽能拮抗NMDA损伤引起的海马神经元Bax蛋白表达增高。
(6)多功能酶标仪实时测定结果显示,分子探针DCFH-DA氧化后形成的荧光产物DCF的荧光强度,随着NMDA浓度的增加而逐渐增高,而牛膝多肽能抑制NMDA引起的荧光强度增强,并随着其浓度的增加,其抑制程度逐渐增强。
(7)多功能酶标仪实时测定线粒体跨膜电位,结果显示NMDA能引起线粒体跨膜电位明显下降,而牛膝多肽能拮抗线粒体跨膜电位的下降。
(8)Caspase-3活性检测结果显示,NMDA能引起海马神经元Caspase-3蛋白活性增强,牛膝多肽能拮抗NMDA引起海马神经元Caspase-3活性增强(p<0.05)。
(9)通过MTT检测结果显示,体外培养8天、13-14天的海马神经元,NMDA都能引起细胞活力急性下降,Ro-256981、NVP-AAM007能拮抗NMDA引起的细胞活力下降(p<0.05),然而牛膝多肽不能抑制NMDA引起的急性细胞活力下降。体外培养8天、13-14天的海马神经元,Ro-256981能拮抗NMDA引起的迟发性细胞活力下降(p<0.05)。体外培养8天的海马神经元,NVP-AAM007能拮抗NMDA引起的迟发性细胞活力下降(p<0.05),牛膝多肽(0.1μg/ml)能拮抗NMDA引起的迟发性细胞活力下降(p<0.01),而牛膝多肽(10μg/ml)不能拮抗NMDA引起的细胞活力迟发性下降,但NVP-AAM007能恢复牛膝多肽拮抗NMDA引起的迟发性细胞活力下降作用。体外培养13-14天的海马神经元,NVP-AAM007能加剧NMDA引起的细胞活力迟发性下降(p<0.01)。牛膝多肽(10μg/ml)能明显抑制NMDA引起的迟发性细胞活力下降(p<0.01)。
(10)钙实时成像结果显示,NVP-AAM007、Ro-256981能部分抑制NMDA引起的海马神经元胞内钙荧光强度增加。当NVP-AAM007存在的时候,牛膝多肽能抑制NMDA引起的海马神经元胞内钙荧光强度增加。当Ro-256981存在的时候,牛膝多肽能增强NMDA引起的海马神经元胞内钙荧光强度增加。NVP-AAM007、Ro-256 981能部分抑制Bicuculline引起的海马神经元胞内钙荧光强度增加。当NVP-AAM007存在的时候,牛膝多肽能抑制Bicuculline引起的海马神经元胞内钙荧光强度增加。当Ro-256981存在的时候,牛膝多肽能增强Bicuculline引起的海马神经元胞内钙荧光强度增加。
(11)全细胞模式记录NMDA电流,结果显示:体外培养8天的海马神经元,牛膝多肽能降低NMDA平台期电流值,增加其失敏系数;培养13-14天的海马神经元,牛膝多肽能使NMDA电流峰值、平台期电流值增高,但降低其失敏系数。Ro-256981、NVP-AAM007能降低NMDA电流峰值、平台期电流值。当NVP-AAM007存在的时候,牛膝多肽能减低NMDA电流峰值、平台期电流值、失敏系数;当Ro-256981存在的时候,牛膝多肽能增强NMDA电流峰值、平台期电流值,降低其失敏系数。
结论:
(1)牛膝多肽在离体和在体具有神经保护作用。
(2)牛膝多肽通过抗神经元调亡实现神经保护作用。
(3)牛膝多肽对NMDA受体过度激活引起的钙超载、ROS形成、Bax过表达、Caspase-3激活具有抑制作用。
(4)牛膝多肽对含NR2A、NR2B亚单位的NMDA受体具有不同的调节作用,对含NR2A亚单位的NMDA受体具有增强作用,而对NR2B亚单位NMDA受体具有抑制作用。
Objective:
To observe the neuroprotective effect of Achyranthes Bidentata ploypeptides (ABPP) and its related mechanisms.
Methods:
(1) Middle cerebral artery occlusion (MCAO) was employed to establish focal cerebral ischemia-reperfusion model in rats, then the neurological behavior score and the percentage of cerebral infarction (with 2, 3, 5-triphenyltetrazolium chloride staining) were measured to observe the neuroprotective effect of ABPP in vivo.
(2) N-methyl-D-aspartate (NMDA)-induced injury model was established in primary cultured rat embryonic hippocampal neurons, and methyl-thiazole-tetrazolium (MTT) assay was used to observe the neuroprotective effect of ABPP in vitro.
(3) Hoechst33258 and propidium iodide (PI) double fluorescent staining, flow cytometry detection of PI, detection of lactate dehydrogenase and DNA ladder were applied to observe the effect of ABPP against NMDA-induced apoptotic cell death in primary cultured hippocampal neurons.
(4) The primary cultured hippocampal neurons were loaded with fluo-3/AM (a fluorescent probe of calcium ion), and the dyed cells were used to observe the effect of ABPP on NMDA-induced rise in intracellular calcium ions with a confocal laser scanning microscope.
(5) Western Blotting detection was performed to examine the effect of ABPP on the overexpression of Bax protein, which was induced by NMDA in cultured hippocampal neurons.
(6) Caspase-3 activity assay was performed to examine the effect of ABPP on the protein activity levels of Caspase-3, which was enhanced by NMDA in cultured hippocampal neurons.
(7) The primary cultured hippocampal neurons were loaded with Rhodamine 123 (a lipophilic cation fluorescent dye), and the dyed cells were to observe the effect of ABPP on NMDA-induced changes in the cellular mitochondrial transmembrane potential with a microplate fluorometer. In addition, the primary cultured hippocampal neurons were loaded with 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), and the dyed cells were to observe the effect of ABPP on NMDA-induced changes in the intracellular radical oxygen species (ROS) with a microplate fluorometer.
(8) With conventioanal whole-cell patch-clamp recording method, NMDA response was measured by amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization. The influences of ABPP on these parameters were investigated in the same time.
(9) By MTT assay, the effect of the two subunit-specific NMDA receptor antagonists, Ro-256981 and NVP-AAM007 on NMDA-induced cellular injury was observed in primary cultured hippocampal neurons in DIV8 and DIV13-14 respectively. Furthermore, the influence of ABPP on NMDA-induced neuronal injury was also investigated at the same time.
(10) The primary cultured hippocampal neurons were loaded with fluo-3/AM, and the dyed neurons were used to observe the effect of Ro-256981 and NVP-AAM007 on NMDA or Bicuculline induced rise in intracellular calcium ions with a confocal laser scanning microscope. Furthermore, the influence of ABPP on the rise of intracellular calcium ion via NR2A-containing NMDA receptors or NR2B-containing NMDA receptors was also investigated respectively.
(11) With whole-cell patch-clamp recording technique, the effect of Ro-256981 and NVP-AAM007 on NMDA induced current was measured by amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization. Furthermore, the influence of ABPP on the NMDA induced current via NR2A-containing NMDA receptors or NR2B-containing NMDA receptors was also investigated by these parameters respectively.
Results:
(1) Focal cerebral ischemia-reperfusion, induced by MCAO, resulted in the mortality of 50%, and promoted the neurology deficit score of the survival rats and the percentage of cerebral infarction (p<0.01). ABPP (0.2mg/kg, iv) decreased the neurology deficit score and the percentage of cerebral infarction (p<0.01). (2) The results of MTT assay showed that NMDA could lower the activity of the primary cultured hippocampal neurons. Furthermore, with the increase of the concentration of NMDA or the incubation time the neuronal activity was fall-off. On the other hand, ABPP could inhibit the decrease of the neuonal activity in a concentration-dependent manner.
(3) Hoechst33258 and propidium iodide (PI) double fluorescent staining, flow cytometry detection of PI, detection of lactate dehydrogenase and DNA ladder showed that NMDA could induce the apoptotic cell death in primary cultured hippocampal neurons, and ABPP (1μg/ml) could inhibit the apoptotic neuronal cell death.
(4) Imaging of intracellular calcium ([Ca~(2+)]_i) displayed that NMDA leaded to enhancement of intracellular fluorescence intensity in a concentration-dependent manner. Both AP-V and MK801 can inhibit the rise of the fluorescence intensity. Pre-, concurrent and post-treatment with ABPP could attenuate the enhanced fluorescence intensity induced by NMDA in concentration-dependent manner.
(5) The Bax channel blocker can inhibit the NMDA-induced cell viability decrease by MTT assay. The results of RT-PCR and Western blot showed that NMDA could augment the expression of Bax in time dependence, and when the primary cultured hippocampal neurons incubated for 30min, the expression of Bax was significantly increased. Furthermore, ABPP could antagonize the up-regulation of Bax protein induced by NMDA (p<0.05).
(6) Molecular probes DCFH-DA was used to monitor alterations of intracellular ROS levels. Exposure of the primary cultured hippocampal neurons to NMDA caused an elevation of ROS production in a concentration-dependent manner, and ABPP inhibited the elevation of ROS levels induced by NMDA, also showing a concentration-dependent pattern.
(7) After exposure to NMDA, the mitochondrial membrane potential of cultured hippocampal neurons was lower than that in normal extracellular solution. ABPP inhibited the lowering of mitochondrial membrane potential induced by NMDA.
(8) A three fold increase in caspase-3 activity was found after exposure of cultured hippocampal neurons to NMDA, but pretreatment with ABPP (1μg/ml) or MK-801 (10μM) protected neurons against the NMDA-induced increase in caspase-3 activity (p<0.05).
(9) The results of MTT assay showed that either Ro-256981 or NVP-AAM007 could inhibit the acute lowering of cell viability induced by NMDA in primary cultured hippocampal neurons at DIV8 and DIV13-14 (p<0.05). However, ABPP could not inhibit the acute lowering of cell viability induced by NMDA. In primary cultured hippocampal neurons at DIV8, Ro-256981 and NVP-AAM007 could inhibit the delayed lowering of cell viability induced by NMDA (p<0.05). In addition, ABPP (0.1μg/ml) could inhibit the delayed lowering of cell viability induced by NMDA (p<0.01), but ABPP (10μg/ml) could not inhibit the delayed lowering of cell viability induced by NMDA, which was recovered by NVP-AAM007 (p<0.05). On the contrary, in primary cultured hippocampal neurons at DIV13-14, NVP-AAM007 could intensify the delayed lowering of cell viability induced by NMDA (p<0.01) and ABPP (10μg/ml) could inhibit the delayed lowering of cell viability induced by NMDA (p<0.01).
(10) During the sustained exposure of hippocampal neurons to NMDA, the addition of NVP-AAM077 alone lowered the [Ca~(2+)]_i, and the simultaneous addition of NVP-AAM077 and ABPP further lowered the [Ca~(2+)]_i. On the contrary, during the sustained exposure of hippocampal neurons to NMDA, the addition of Ro25-6981 alone lowered the [Ca~(2+)]_i, but the simultaneous addition of Ro25-6981 and ABPP inversely raised the [Ca~(2+)]_i. In addition, during the sustained exposure of hippocampal neurons to bicuculline, the addition of NVP-AAM077 alone lowered the [Ca~(2+)]_i, and the simultaneous addition of NVP-AAM077 and ABPP further lowered the [Ca~(2+)]_i. On the contrary, during the sustained exposure of hippocampal neurons to bicuculline, the addition of Ro25-6981 alone lowered the [Ca~(2+)]_i, and the simultaneous addition of Ro25-6981 and ABPP inversely raised the [Ca~(2+)]_i, which can be blocked by NVP-AAM077.
(11) ABPP could decrease the amplitude of NMDA-induced steady-state current and increase its coefficient of desensitization in cultured hippocampal neurons in DIV8. On the contrary, ABPP could enhance amplitude of NMDA-induced peak current and steady-state current and decrease its coefficient of desensitization in cultured hippocampal neurons in DIV13-14. In addition, treatment of Ro25-6981 alone can decrease amplitude of NMDA-induced peak current and steady-state current, and the simultaneous treatment of Ro25-6981 and ABPP could enhance amplitude of NMDA-induced peak current and steady-state current and decrease coefficient of desensitization of NMDA current. Furthermore, treatment of NVP-AAM007 alone can decrease amplitude of NMDA-induced peak current and steady-state current, and the simultaneous treatment of NVP-AAM007 and ABPP could reduce amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization.
Conclusions:
(1) Achyranthes Bidentata ploypeptides (ABPP) have the neuroprotective effect in vivo and in vitro.
(2) The protective effect of ABPP is probably related to its anti-apoptosis in primary cultured hippocampal neurons.
(3) ABPP can inhibit the excess Ca~(2+) influx, intracellular ROS production, Bax protein over-expression, the activity of Caspase-3 and mitochondrial dysfunction, which were induced by over-stimulation of NMDA receptors.
(4) ABPP can modulate NR2A- and NR2B-containing NMDA receptors differentially.
观察牛膝多肽(A. bidentata polypeptides, ABPP)的神经保护作用及其相关作用机制。
方法:
(1)采用大脑中动脉线栓法建立大鼠局灶性脑缺血再灌注模型,通过测定神经功能缺陷评分、脑梗死百分比(TTC法),在体观察牛膝多肽的神经保护作用。
(2)以体外原代培养胎鼠海马神经元为研究对象,建立N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)损伤模型。通过MTT检测,离体观察牛膝多肽的神经保护作用。
(3)Hoechst33258/PI双染色、PI流式细胞仪检测、LDH检测、DNA ladder检测等观察牛膝多肽对NMDA诱导的海马神经元调亡的影响。
(4)利用钙离子荧光探针Fluo-3/AM标记海马神经元,通过激光共聚焦显微镜检测,观察牛膝多肽对NMDA引起的海马神经元胞内钙离子浓度的影响。
(5)通过Western blot,观察牛膝多肽对NMDA引起的海马神经元Bax蛋白过表达的影响。
(6)通过Caspase-3活性检测,观察牛膝多肽对NMDA引起的海马神经元Caspase-3蛋白活性增高的影响。
(7)采用亲脂性阳离子荧光染料Rhodamine123、分子探针DCFH-DA标记海马神经元,通过多功能酶标仪检测NMDA引起的海马神经元线粒体跨膜电位、活性氧自由基含量的变化。同时观察牛膝多肽对NMDA引起的海马神经元线粒体跨膜电位、活性氧自由基含量的影响。
(8)采用全细胞膜片钳技术,观察牛膝多肽对NMDA电流峰值、平台期电流值、失敏系数的影响。
(9)采用了含NR2A、NR2B两种不同亚单位类型NMDA受体的选择性抑制剂:Ro-256981、NVP-AAM007,及两种不同培养时间的海马神经元:体外培养8天、13-14天,通过MTT检测,观察两种不同的选择性抑制剂对NMDA诱导神经元损伤的影响,同时观察牛膝多肽对NMDA诱导神经元损伤的影响。
(10)应用钙离子荧光探针Fluo-3/AM标记通过激光共聚焦显微镜检测,观察含NR2A、NR2B两种不同亚单位类型NMDA受体的选择性抑制剂:Ro-256981、NVP-AAM007,对NMDA和Bicuculline引起的海马神经元胞内钙离子浓度的影响,并观察牛膝多肽对含NR2A、NR2B两种不同亚单位类型NMDA受体介导的胞内钙离子浓度的影响。
(11)采用全细胞膜片钳技术,观察含NR2A、NR2B两种不同亚单位类型NMDA受体的选择性抑制剂:Ro-256981、NVP-AAM007,对NMDA电流峰值、平台期电流值、失敏系数的影响,并观察牛膝多肽对含NR2A、NR2B两种不同亚单位类型NMDA受体介导的NMDA电流峰值、平台期电流值、失敏系数的影响。
结果:
(1)局灶性脑缺血再灌注损伤能引起大鼠死亡,死亡率达到50%,对存活的大鼠,其神经功能缺陷评分明显增高(p<0.01)和脑梗死百分比增加(p<0.01),尾静脉给予牛膝多肽(0.2mg/kg)治疗,可以降低神经功能缺陷评分,降低脑梗死百分比(p<0.01)。
(2)通过MTT检测结果显示,NMDA能降低培养的海马神经元的活力,而且随着NMDA浓度的增加、作用时间的延长,神经元的活力逐渐下降。牛膝多肽能抑制NMDA引起的海马神经元活力下降,并与其剂量相关。
(3)Hoechst33258/PI双染色、PI流式细胞仪检测、LDH检测、DNA ladder检测结果显示,NMDA能诱导海马神经元调亡,牛膝多肽(1μg/ml)能拮抗NMDA诱导的海马神经元调亡。
(4)钙实时成像结果显示,NMDA能引起海马神经元胞内钙荧光强度增加,并随着NMDA浓度增加,荧光强度增强。AP-V、MK801能抑制胞内钙荧光强度增高。预先、同时、后加入牛膝多肽都能减弱NMDA引起海马神经元胞内钙荧光强度增加,并随着浓度增高,抑制程度增加。
(5)通过MTT检测结果显示,Bax通道阻断剂能拮抗NMDA引起的海马神经元活力的降低。RT-PCR和Western blot结果提示,海马神经元Bax mRNA及其蛋白表达随着NMDA作用时间的变化而变化,其中作用30min Bax蛋白表达明显增加(p<0.05)。牛膝多肽能拮抗NMDA损伤引起的海马神经元Bax蛋白表达增高。
(6)多功能酶标仪实时测定结果显示,分子探针DCFH-DA氧化后形成的荧光产物DCF的荧光强度,随着NMDA浓度的增加而逐渐增高,而牛膝多肽能抑制NMDA引起的荧光强度增强,并随着其浓度的增加,其抑制程度逐渐增强。
(7)多功能酶标仪实时测定线粒体跨膜电位,结果显示NMDA能引起线粒体跨膜电位明显下降,而牛膝多肽能拮抗线粒体跨膜电位的下降。
(8)Caspase-3活性检测结果显示,NMDA能引起海马神经元Caspase-3蛋白活性增强,牛膝多肽能拮抗NMDA引起海马神经元Caspase-3活性增强(p<0.05)。
(9)通过MTT检测结果显示,体外培养8天、13-14天的海马神经元,NMDA都能引起细胞活力急性下降,Ro-256981、NVP-AAM007能拮抗NMDA引起的细胞活力下降(p<0.05),然而牛膝多肽不能抑制NMDA引起的急性细胞活力下降。体外培养8天、13-14天的海马神经元,Ro-256981能拮抗NMDA引起的迟发性细胞活力下降(p<0.05)。体外培养8天的海马神经元,NVP-AAM007能拮抗NMDA引起的迟发性细胞活力下降(p<0.05),牛膝多肽(0.1μg/ml)能拮抗NMDA引起的迟发性细胞活力下降(p<0.01),而牛膝多肽(10μg/ml)不能拮抗NMDA引起的细胞活力迟发性下降,但NVP-AAM007能恢复牛膝多肽拮抗NMDA引起的迟发性细胞活力下降作用。体外培养13-14天的海马神经元,NVP-AAM007能加剧NMDA引起的细胞活力迟发性下降(p<0.01)。牛膝多肽(10μg/ml)能明显抑制NMDA引起的迟发性细胞活力下降(p<0.01)。
(10)钙实时成像结果显示,NVP-AAM007、Ro-256981能部分抑制NMDA引起的海马神经元胞内钙荧光强度增加。当NVP-AAM007存在的时候,牛膝多肽能抑制NMDA引起的海马神经元胞内钙荧光强度增加。当Ro-256981存在的时候,牛膝多肽能增强NMDA引起的海马神经元胞内钙荧光强度增加。NVP-AAM007、Ro-256 981能部分抑制Bicuculline引起的海马神经元胞内钙荧光强度增加。当NVP-AAM007存在的时候,牛膝多肽能抑制Bicuculline引起的海马神经元胞内钙荧光强度增加。当Ro-256981存在的时候,牛膝多肽能增强Bicuculline引起的海马神经元胞内钙荧光强度增加。
(11)全细胞模式记录NMDA电流,结果显示:体外培养8天的海马神经元,牛膝多肽能降低NMDA平台期电流值,增加其失敏系数;培养13-14天的海马神经元,牛膝多肽能使NMDA电流峰值、平台期电流值增高,但降低其失敏系数。Ro-256981、NVP-AAM007能降低NMDA电流峰值、平台期电流值。当NVP-AAM007存在的时候,牛膝多肽能减低NMDA电流峰值、平台期电流值、失敏系数;当Ro-256981存在的时候,牛膝多肽能增强NMDA电流峰值、平台期电流值,降低其失敏系数。
结论:
(1)牛膝多肽在离体和在体具有神经保护作用。
(2)牛膝多肽通过抗神经元调亡实现神经保护作用。
(3)牛膝多肽对NMDA受体过度激活引起的钙超载、ROS形成、Bax过表达、Caspase-3激活具有抑制作用。
(4)牛膝多肽对含NR2A、NR2B亚单位的NMDA受体具有不同的调节作用,对含NR2A亚单位的NMDA受体具有增强作用,而对NR2B亚单位NMDA受体具有抑制作用。
Objective:
To observe the neuroprotective effect of Achyranthes Bidentata ploypeptides (ABPP) and its related mechanisms.
Methods:
(1) Middle cerebral artery occlusion (MCAO) was employed to establish focal cerebral ischemia-reperfusion model in rats, then the neurological behavior score and the percentage of cerebral infarction (with 2, 3, 5-triphenyltetrazolium chloride staining) were measured to observe the neuroprotective effect of ABPP in vivo.
(2) N-methyl-D-aspartate (NMDA)-induced injury model was established in primary cultured rat embryonic hippocampal neurons, and methyl-thiazole-tetrazolium (MTT) assay was used to observe the neuroprotective effect of ABPP in vitro.
(3) Hoechst33258 and propidium iodide (PI) double fluorescent staining, flow cytometry detection of PI, detection of lactate dehydrogenase and DNA ladder were applied to observe the effect of ABPP against NMDA-induced apoptotic cell death in primary cultured hippocampal neurons.
(4) The primary cultured hippocampal neurons were loaded with fluo-3/AM (a fluorescent probe of calcium ion), and the dyed cells were used to observe the effect of ABPP on NMDA-induced rise in intracellular calcium ions with a confocal laser scanning microscope.
(5) Western Blotting detection was performed to examine the effect of ABPP on the overexpression of Bax protein, which was induced by NMDA in cultured hippocampal neurons.
(6) Caspase-3 activity assay was performed to examine the effect of ABPP on the protein activity levels of Caspase-3, which was enhanced by NMDA in cultured hippocampal neurons.
(7) The primary cultured hippocampal neurons were loaded with Rhodamine 123 (a lipophilic cation fluorescent dye), and the dyed cells were to observe the effect of ABPP on NMDA-induced changes in the cellular mitochondrial transmembrane potential with a microplate fluorometer. In addition, the primary cultured hippocampal neurons were loaded with 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), and the dyed cells were to observe the effect of ABPP on NMDA-induced changes in the intracellular radical oxygen species (ROS) with a microplate fluorometer.
(8) With conventioanal whole-cell patch-clamp recording method, NMDA response was measured by amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization. The influences of ABPP on these parameters were investigated in the same time.
(9) By MTT assay, the effect of the two subunit-specific NMDA receptor antagonists, Ro-256981 and NVP-AAM007 on NMDA-induced cellular injury was observed in primary cultured hippocampal neurons in DIV8 and DIV13-14 respectively. Furthermore, the influence of ABPP on NMDA-induced neuronal injury was also investigated at the same time.
(10) The primary cultured hippocampal neurons were loaded with fluo-3/AM, and the dyed neurons were used to observe the effect of Ro-256981 and NVP-AAM007 on NMDA or Bicuculline induced rise in intracellular calcium ions with a confocal laser scanning microscope. Furthermore, the influence of ABPP on the rise of intracellular calcium ion via NR2A-containing NMDA receptors or NR2B-containing NMDA receptors was also investigated respectively.
(11) With whole-cell patch-clamp recording technique, the effect of Ro-256981 and NVP-AAM007 on NMDA induced current was measured by amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization. Furthermore, the influence of ABPP on the NMDA induced current via NR2A-containing NMDA receptors or NR2B-containing NMDA receptors was also investigated by these parameters respectively.
Results:
(1) Focal cerebral ischemia-reperfusion, induced by MCAO, resulted in the mortality of 50%, and promoted the neurology deficit score of the survival rats and the percentage of cerebral infarction (p<0.01). ABPP (0.2mg/kg, iv) decreased the neurology deficit score and the percentage of cerebral infarction (p<0.01). (2) The results of MTT assay showed that NMDA could lower the activity of the primary cultured hippocampal neurons. Furthermore, with the increase of the concentration of NMDA or the incubation time the neuronal activity was fall-off. On the other hand, ABPP could inhibit the decrease of the neuonal activity in a concentration-dependent manner.
(3) Hoechst33258 and propidium iodide (PI) double fluorescent staining, flow cytometry detection of PI, detection of lactate dehydrogenase and DNA ladder showed that NMDA could induce the apoptotic cell death in primary cultured hippocampal neurons, and ABPP (1μg/ml) could inhibit the apoptotic neuronal cell death.
(4) Imaging of intracellular calcium ([Ca~(2+)]_i) displayed that NMDA leaded to enhancement of intracellular fluorescence intensity in a concentration-dependent manner. Both AP-V and MK801 can inhibit the rise of the fluorescence intensity. Pre-, concurrent and post-treatment with ABPP could attenuate the enhanced fluorescence intensity induced by NMDA in concentration-dependent manner.
(5) The Bax channel blocker can inhibit the NMDA-induced cell viability decrease by MTT assay. The results of RT-PCR and Western blot showed that NMDA could augment the expression of Bax in time dependence, and when the primary cultured hippocampal neurons incubated for 30min, the expression of Bax was significantly increased. Furthermore, ABPP could antagonize the up-regulation of Bax protein induced by NMDA (p<0.05).
(6) Molecular probes DCFH-DA was used to monitor alterations of intracellular ROS levels. Exposure of the primary cultured hippocampal neurons to NMDA caused an elevation of ROS production in a concentration-dependent manner, and ABPP inhibited the elevation of ROS levels induced by NMDA, also showing a concentration-dependent pattern.
(7) After exposure to NMDA, the mitochondrial membrane potential of cultured hippocampal neurons was lower than that in normal extracellular solution. ABPP inhibited the lowering of mitochondrial membrane potential induced by NMDA.
(8) A three fold increase in caspase-3 activity was found after exposure of cultured hippocampal neurons to NMDA, but pretreatment with ABPP (1μg/ml) or MK-801 (10μM) protected neurons against the NMDA-induced increase in caspase-3 activity (p<0.05).
(9) The results of MTT assay showed that either Ro-256981 or NVP-AAM007 could inhibit the acute lowering of cell viability induced by NMDA in primary cultured hippocampal neurons at DIV8 and DIV13-14 (p<0.05). However, ABPP could not inhibit the acute lowering of cell viability induced by NMDA. In primary cultured hippocampal neurons at DIV8, Ro-256981 and NVP-AAM007 could inhibit the delayed lowering of cell viability induced by NMDA (p<0.05). In addition, ABPP (0.1μg/ml) could inhibit the delayed lowering of cell viability induced by NMDA (p<0.01), but ABPP (10μg/ml) could not inhibit the delayed lowering of cell viability induced by NMDA, which was recovered by NVP-AAM007 (p<0.05). On the contrary, in primary cultured hippocampal neurons at DIV13-14, NVP-AAM007 could intensify the delayed lowering of cell viability induced by NMDA (p<0.01) and ABPP (10μg/ml) could inhibit the delayed lowering of cell viability induced by NMDA (p<0.01).
(10) During the sustained exposure of hippocampal neurons to NMDA, the addition of NVP-AAM077 alone lowered the [Ca~(2+)]_i, and the simultaneous addition of NVP-AAM077 and ABPP further lowered the [Ca~(2+)]_i. On the contrary, during the sustained exposure of hippocampal neurons to NMDA, the addition of Ro25-6981 alone lowered the [Ca~(2+)]_i, but the simultaneous addition of Ro25-6981 and ABPP inversely raised the [Ca~(2+)]_i. In addition, during the sustained exposure of hippocampal neurons to bicuculline, the addition of NVP-AAM077 alone lowered the [Ca~(2+)]_i, and the simultaneous addition of NVP-AAM077 and ABPP further lowered the [Ca~(2+)]_i. On the contrary, during the sustained exposure of hippocampal neurons to bicuculline, the addition of Ro25-6981 alone lowered the [Ca~(2+)]_i, and the simultaneous addition of Ro25-6981 and ABPP inversely raised the [Ca~(2+)]_i, which can be blocked by NVP-AAM077.
(11) ABPP could decrease the amplitude of NMDA-induced steady-state current and increase its coefficient of desensitization in cultured hippocampal neurons in DIV8. On the contrary, ABPP could enhance amplitude of NMDA-induced peak current and steady-state current and decrease its coefficient of desensitization in cultured hippocampal neurons in DIV13-14. In addition, treatment of Ro25-6981 alone can decrease amplitude of NMDA-induced peak current and steady-state current, and the simultaneous treatment of Ro25-6981 and ABPP could enhance amplitude of NMDA-induced peak current and steady-state current and decrease coefficient of desensitization of NMDA current. Furthermore, treatment of NVP-AAM007 alone can decrease amplitude of NMDA-induced peak current and steady-state current, and the simultaneous treatment of NVP-AAM007 and ABPP could reduce amplitude of NMDA-induced peak current and steady-state current and its coefficient of desensitization.
Conclusions:
(1) Achyranthes Bidentata ploypeptides (ABPP) have the neuroprotective effect in vivo and in vitro.
(2) The protective effect of ABPP is probably related to its anti-apoptosis in primary cultured hippocampal neurons.
(3) ABPP can inhibit the excess Ca~(2+) influx, intracellular ROS production, Bax protein over-expression, the activity of Caspase-3 and mitochondrial dysfunction, which were induced by over-stimulation of NMDA receptors.
(4) ABPP can modulate NR2A- and NR2B-containing NMDA receptors differentially.
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