体外缺血再灌注损伤中神经元凋亡发生机制及白藜芦醇苷保护作用的研究
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摘要
目的:
在新生大鼠大脑皮层神经元分离、纯化、培养和鉴定的基础上,建立体外大脑皮层神经元缺血再灌注(ischemia/reperfusion,I/R)损伤模型,阐明体外I/R损伤中神经元凋亡发生机制,探讨白藜芦醇苷(polydatin, PD)对体外I/R神经元损伤的作用。
方法:
在无菌条件下,将出生24h内的新生Sprague Dawley大鼠断头取脑,剔除脑膜及血管,分离皮层神经元,原代培养7d,进行纯度测定后,再随机分为3组:对照组、I/R损伤组和PD治疗组。对照组用DMEM/F12培养基处理15min后,用无血清培养基继续培养;I/R损伤组用连二亚硫酸钠(Na_2S_2O_4)溶液处理15min后,用无血清培养基继续培养;PD治疗组在Na_2S_2O_4溶液处理前、中、后均加入PD进行干预;在继续培养0h、6h、12h、24h、48h五个不同时间点获取标本待检。用AO/EB及DAPI染色,在荧光显微镜下观察神经元的形态学变化,用TUNEL检测神经元凋亡率,免疫组化检测神经元Bcl-2、Bax及NF-κBp65的表达。
结果:
(1)在倒置相差显微镜下观察,原代培养7d的大鼠大脑皮层神经元胞体饱满,立体感强,突起较长,相互连接,形成致密的网络。
(2)AO/EB及DAPI染色可见I/R损伤组有凋亡细胞的特征性形态变化。
(3)I/R损伤组神经元凋亡率在0h时无变化,6h增高,24h达峰值,48h降低;并在6h、12h、24h及48h对应时间点均高于对照组,有统计学差异(P<0.05)。PD治疗组神经元凋亡率在6h、12h、24h及48h对应时间点均低于I/R损伤组,有统计学差异(P<0.05)。
(4)I/R损伤组NF-κBp65的表达量随再灌注时间的延长而增加,24h达高峰, 24h～48h从胞浆向胞核移位明显,NF-κBp65与Bcl-2/Bax间存在较强的负相关性(P<0.05)。PD治疗组NF-κBp65表达量降低,在6h、12h、24h及48h对应时间点低于I/R损伤组,有统计学差异(P<0.05)。I/R损伤组Bax的表达量随再灌注时间的延长而明显增加,24h达高峰,之后降低;Bcl-2的表达量及Bcl-2/Bax比率均在6h达高峰,随后渐降低;且Bcl-2/Bax比率与凋亡间存在较强的负相关性(P<0.05)。PD治疗组Bcl-2增高而Bax降低,与I/R损伤组在6h、12h、24h及48h对应时间点的值相比有统计学差异(P<0.05)。
结论:
(1)在分离、纯化、培养和鉴定新生大鼠大脑皮层神经元的基础上,成功的建立了体外大脑皮层神经元I/R损伤模型。
(2)PD通过降低I/R损伤神经元凋亡的发生率而起保护作用,其减少神经元凋亡作用的可能机制是抑制NF-κBp65激活,上调Bcl-2/Bax比率。
Objectives
On the basis of successful rat cortical neuron isolation, purification, culture and identification, to establish the model of ischemia/reperfusion (I/R), to clarify the mechanism of neuronal apoptosis during I/R induced injury and to explore the effect of polydatin (PD)on I/R induced cortical neuron injury in vitro.
Methods
The 24-hours-old newborn Sprague Dawley rats were sacrified by disconnecting the neck under aseptic conditions and the brain was taken out. The meninges and blood vessels on the surface of the brain were removed. The cortical nerons were dissociated from the cerebra, cultured for 7 days and the purity of the cortical nerons was identified. Then were randomly divided into three groups, the control group, the I/R injury group and the PD treatment group. The cortical neurons were cultuered with DMEM/F12 medium in control group. The neurons were treated with Sodium dithionite for 15 minutes and then cultuered with DMEM/F12 medium in I/R injury group. The PD was added before, at the same time with and after Sodium dithionite treatment in PD treatment group. The morphological features of the neurons were observed under fluorescence microscope with AO/EB and DAPI immunofluorescence staining. Apoptosis were measured through TUNEL method. The neuronal expressions of Bcl-2, Bax and NF-κBp65 were determined by immunohistochemical method.
Results
(1)At the 7th day, the primary cortical neurons grown well, there are plump and solid bodies, nervous processus contacted with eath other and a dense network formed under inverted phase contrast microscope.
(2)Morphological changes of neuronal apoptosis can be observed under fluorescence microscope (AO/EB and DAPI) in I/R injury group.
(3)The number of apoptotic cells in I/R injury group kept no change at 0h, increased at 6h, peaked at 24h, and decreased at 48h. The rate of neuronal apoptosis was increased in I/R injury group compared with that in control group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance(P<0.05).The rate of neuronal apoptosis was lower in PD treatment group than that in I/R injury group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance (P<0.05).
(4)The expressions of neuron NF-κBp65 increased with time after reperfusion, peaked at 24h and then decreased in I/R injury group. Marked translocation of NF-κBp65 from cytolymph into nucleus were found from 24h to 48h in I/R injury group. There exists a strong negative correlation between NF-κBp65 and Bcl-2/Bax, with statistical significance (P<0.05). The expressions of neuron NF-κBp65 were decreased in PD treatment group, and compared with those in control group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance (P<0.05). The expression of Bax increased significantly with time after reperfusion, peaked at 24h and then decreased in I/R injury group. The expressions of Bcl-2 and Bcl-2/Bax ratio increased with time after reperfusion, peak at 6h and then decreased. There was significantly negative correlation between Bcl-2/Bax and apoptosis (P<0.05). Increased expression of Bcl-2 but decreased Bax expresion were found in PD treatment group, and compared with those in I/R group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significances (P<0.05).
Conclusions
(1)On the basis of newborn rat primary cortical neuron isolation, purification and culture, we successfully established the model of I/R induced cortical neuron injury in vitro.
(2)The Polydatin may have protective effect by decreasing the rate of neuron apoptosis during I/R insult in vitro. The possible mechanism of decreased neuron apoptosis may be through the suppression of NF-κBp65 activiation and up-regulation the ratio between Bcl-2 and Bax.
在新生大鼠大脑皮层神经元分离、纯化、培养和鉴定的基础上,建立体外大脑皮层神经元缺血再灌注(ischemia/reperfusion,I/R)损伤模型,阐明体外I/R损伤中神经元凋亡发生机制,探讨白藜芦醇苷(polydatin, PD)对体外I/R神经元损伤的作用。
方法:
在无菌条件下,将出生24h内的新生Sprague Dawley大鼠断头取脑,剔除脑膜及血管,分离皮层神经元,原代培养7d,进行纯度测定后,再随机分为3组:对照组、I/R损伤组和PD治疗组。对照组用DMEM/F12培养基处理15min后,用无血清培养基继续培养;I/R损伤组用连二亚硫酸钠(Na_2S_2O_4)溶液处理15min后,用无血清培养基继续培养;PD治疗组在Na_2S_2O_4溶液处理前、中、后均加入PD进行干预;在继续培养0h、6h、12h、24h、48h五个不同时间点获取标本待检。用AO/EB及DAPI染色,在荧光显微镜下观察神经元的形态学变化,用TUNEL检测神经元凋亡率,免疫组化检测神经元Bcl-2、Bax及NF-κBp65的表达。
结果:
(1)在倒置相差显微镜下观察,原代培养7d的大鼠大脑皮层神经元胞体饱满,立体感强,突起较长,相互连接,形成致密的网络。
(2)AO/EB及DAPI染色可见I/R损伤组有凋亡细胞的特征性形态变化。
(3)I/R损伤组神经元凋亡率在0h时无变化,6h增高,24h达峰值,48h降低;并在6h、12h、24h及48h对应时间点均高于对照组,有统计学差异(P<0.05)。PD治疗组神经元凋亡率在6h、12h、24h及48h对应时间点均低于I/R损伤组,有统计学差异(P<0.05)。
(4)I/R损伤组NF-κBp65的表达量随再灌注时间的延长而增加,24h达高峰, 24h～48h从胞浆向胞核移位明显,NF-κBp65与Bcl-2/Bax间存在较强的负相关性(P<0.05)。PD治疗组NF-κBp65表达量降低,在6h、12h、24h及48h对应时间点低于I/R损伤组,有统计学差异(P<0.05)。I/R损伤组Bax的表达量随再灌注时间的延长而明显增加,24h达高峰,之后降低;Bcl-2的表达量及Bcl-2/Bax比率均在6h达高峰,随后渐降低;且Bcl-2/Bax比率与凋亡间存在较强的负相关性(P<0.05)。PD治疗组Bcl-2增高而Bax降低,与I/R损伤组在6h、12h、24h及48h对应时间点的值相比有统计学差异(P<0.05)。
结论:
(1)在分离、纯化、培养和鉴定新生大鼠大脑皮层神经元的基础上,成功的建立了体外大脑皮层神经元I/R损伤模型。
(2)PD通过降低I/R损伤神经元凋亡的发生率而起保护作用,其减少神经元凋亡作用的可能机制是抑制NF-κBp65激活,上调Bcl-2/Bax比率。
Objectives
On the basis of successful rat cortical neuron isolation, purification, culture and identification, to establish the model of ischemia/reperfusion (I/R), to clarify the mechanism of neuronal apoptosis during I/R induced injury and to explore the effect of polydatin (PD)on I/R induced cortical neuron injury in vitro.
Methods
The 24-hours-old newborn Sprague Dawley rats were sacrified by disconnecting the neck under aseptic conditions and the brain was taken out. The meninges and blood vessels on the surface of the brain were removed. The cortical nerons were dissociated from the cerebra, cultured for 7 days and the purity of the cortical nerons was identified. Then were randomly divided into three groups, the control group, the I/R injury group and the PD treatment group. The cortical neurons were cultuered with DMEM/F12 medium in control group. The neurons were treated with Sodium dithionite for 15 minutes and then cultuered with DMEM/F12 medium in I/R injury group. The PD was added before, at the same time with and after Sodium dithionite treatment in PD treatment group. The morphological features of the neurons were observed under fluorescence microscope with AO/EB and DAPI immunofluorescence staining. Apoptosis were measured through TUNEL method. The neuronal expressions of Bcl-2, Bax and NF-κBp65 were determined by immunohistochemical method.
Results
(1)At the 7th day, the primary cortical neurons grown well, there are plump and solid bodies, nervous processus contacted with eath other and a dense network formed under inverted phase contrast microscope.
(2)Morphological changes of neuronal apoptosis can be observed under fluorescence microscope (AO/EB and DAPI) in I/R injury group.
(3)The number of apoptotic cells in I/R injury group kept no change at 0h, increased at 6h, peaked at 24h, and decreased at 48h. The rate of neuronal apoptosis was increased in I/R injury group compared with that in control group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance(P<0.05).The rate of neuronal apoptosis was lower in PD treatment group than that in I/R injury group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance (P<0.05).
(4)The expressions of neuron NF-κBp65 increased with time after reperfusion, peaked at 24h and then decreased in I/R injury group. Marked translocation of NF-κBp65 from cytolymph into nucleus were found from 24h to 48h in I/R injury group. There exists a strong negative correlation between NF-κBp65 and Bcl-2/Bax, with statistical significance (P<0.05). The expressions of neuron NF-κBp65 were decreased in PD treatment group, and compared with those in control group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significance (P<0.05). The expression of Bax increased significantly with time after reperfusion, peaked at 24h and then decreased in I/R injury group. The expressions of Bcl-2 and Bcl-2/Bax ratio increased with time after reperfusion, peak at 6h and then decreased. There was significantly negative correlation between Bcl-2/Bax and apoptosis (P<0.05). Increased expression of Bcl-2 but decreased Bax expresion were found in PD treatment group, and compared with those in I/R group at any corresponding time point of 6h, 12h, 24h and 48h, with statistical significances (P<0.05).
Conclusions
(1)On the basis of newborn rat primary cortical neuron isolation, purification and culture, we successfully established the model of I/R induced cortical neuron injury in vitro.
(2)The Polydatin may have protective effect by decreasing the rate of neuron apoptosis during I/R insult in vitro. The possible mechanism of decreased neuron apoptosis may be through the suppression of NF-κBp65 activiation and up-regulation the ratio between Bcl-2 and Bax.
引文
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