丹红注射液对体外培养胎鼠神经元NMDA损伤后的神经保护作用
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摘要
背景神经保护治疗是急性脑缺血等神经系统疾病的研究热点之一。中药治疗是国内急性脑缺血常用治疗手段,具有巨大潜力。近年来,多项临床研究及脑缺血动物模型体内实验表明,丹红注射液对急性脑缺血治疗有效。体外实验主要集中在丹红注射液对血管内皮细胞的作用方面,尚少见丹红注射液对于体外培养神经元模拟缺血损伤后是否具有神经保护作用的报道。由N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体介导的损伤途径是兴奋性氨基酸神经毒性作用的主要通路之一,可导致细胞凋亡。
目的以NMDA诱导体外培养胎鼠皮层神经元的兴奋性毒性损伤,研究丹红注射液对于神经元NMDA损伤后是否具有保护作用。为进一步的研究包括分析该药有效成分并阐明其具体的保护机制等打下基础。方法采用孕17-18天的KM孕小鼠,以神经元完全培养液进行胎鼠原代皮层神经元培养。通过神经元微管相关蛋白(Microtubule-associated protein-2,MAP-2)染色进行神经元鉴定。神经元培养至10d时,随机分为3组,分别加入浓度为50umol/l、150umol/l及300umol/l的NMDA,根据噻唑兰(methyl-thiazole-tetrazolium,MTT)法细胞生存率检测及乳酸脱氢酶(lactatedehydrogenase,LDH)释放率测定结果,选取3组中神经元损伤最明显的一组,以该组NMDA浓度建立NMDA损伤模型。将培养的神经元随机分为3组:正常对照组、NMDA损伤组、丹红给药组。正常对照组按原神经元培养条件继续培养。NMDA损伤组按上述方法建立NMDA损伤模型。丹红给药组分别在加入NMDA后即刻给予0.005μl/ml及0.01μl/ml的丹红注射液治疗。以MTT法检测细胞生存率,LDH释放率测定细胞损伤,通过Hoechst及TUNEL染色检测细胞凋亡情况。
结果NMDA损伤后神经元肿胀、轴突断裂。随着NMDA浓度的增高,细胞生存率依次下降,LDH释放率依次增加,以NMDA 300umol/l组损伤最明显。丹红给药组细胞损伤减轻,胞体肿胀程度减轻,细胞形态尚保持完整,细胞生存率提高,LDH释放率下降,两种剂量丹红注射液均可减轻NMDA引起的神经元损伤,两组问细胞生存率及LDH释放率无明显统计学差异。小剂量丹红给药组Hoechst染色及TUNEL细胞凋亡检测示丹红给药组凋亡细胞百分比下降。
结论丹红注射液可减轻NMDA诱导的体外培养胎鼠皮层神经元的损伤,抑制NMDA引起的神经元凋亡。
Backgrounds The neuroprotection after acute cerebral ischemia is a hot topic in stroke research. As a common therapy for cerebral ischemia, traditional Chinese medicine has tremendous potential. Clinical trials and researches on animal models have shown that Danhong injection is effective in treating acute cerebral ischemia. Up till now, most researches in vitro have studied effects of Danhong injection on vascular endothelial cells, it remains to be explored whether Danhong injection can protect neurons from damage induced by NMDA, which mediates excitotoxicity as one of the main sources of damage after cerebral ischemia and can result in cell apoptosis.
Objective To provide cultured embryonic mice neurons with exogenous NMDA in vitro. To study whether Danhong injection can protect neurons from excitotoxicity induced by NMDA. To make the basis for further analysis of effective components of Danhong injection and its protective mechanisms.
Methods Primary cortical cultures were prepared from 17 or 18-day-old embryos obtained from pregnant KM mice. Neuronal cell populations are determined by immunocytochemistry against microtubule-associated protein (MAP-2). 10 days later, cultures were exposed to exogenous NMDA (50,150 and 300umol/l) randomly. Cell survival was evaluated by MTT assay and LDH release. The concentration of NMDA leading to the most marked cell damage was chosen to establish the model of NMDA induced neuronal injury. Cultured neurons were divided into 3 groups: normal control group, NMDA injury group and Danhong treatment group. Danhong injection(0.005μl/ml and 0.01μl/ml) was added to the culture medium of the treatment group simultaneously with NMDA. Cell survival was evaluated by MTT assay. Cell injury was determined by LDH release. Apoptosis was estimated by Hoechst staining and TUNEL.
Results A dose-response curve of NMDA neurotoxicity after 24 h of exposure to cultured neurons was performed and revealed the most significant reduction in MTT activity and the highest LDH release at a concentration of 300umol/l. Therefore, this concentration was chosen to perform all the following experiments. Under microscopy, after NMDA incubation, cultured neurons were damaged as evidenced by the presence of swelling and fragmented neuritis. When cortical cells were coincubated with NMDA and Danhong injection, cultures were better preserved and more intact neurons were visible. Danhong treatment group had both increased MTT activity and decreased LDH release and no statistical difference was observed between the 2 treatment groups(0.005μl/ml and 0.01μl/ml). The percentage of apoptotic neurons decreased as was labeled by Hoechst staining and TUNEL.
Conclusions Danhong injection can relieve NMDA induced neuronal injury in cultured cortical neurons of embryonic mice and inhibit NMDA induced neuronal apotosis.
目的以NMDA诱导体外培养胎鼠皮层神经元的兴奋性毒性损伤,研究丹红注射液对于神经元NMDA损伤后是否具有保护作用。为进一步的研究包括分析该药有效成分并阐明其具体的保护机制等打下基础。方法采用孕17-18天的KM孕小鼠,以神经元完全培养液进行胎鼠原代皮层神经元培养。通过神经元微管相关蛋白(Microtubule-associated protein-2,MAP-2)染色进行神经元鉴定。神经元培养至10d时,随机分为3组,分别加入浓度为50umol/l、150umol/l及300umol/l的NMDA,根据噻唑兰(methyl-thiazole-tetrazolium,MTT)法细胞生存率检测及乳酸脱氢酶(lactatedehydrogenase,LDH)释放率测定结果,选取3组中神经元损伤最明显的一组,以该组NMDA浓度建立NMDA损伤模型。将培养的神经元随机分为3组:正常对照组、NMDA损伤组、丹红给药组。正常对照组按原神经元培养条件继续培养。NMDA损伤组按上述方法建立NMDA损伤模型。丹红给药组分别在加入NMDA后即刻给予0.005μl/ml及0.01μl/ml的丹红注射液治疗。以MTT法检测细胞生存率,LDH释放率测定细胞损伤,通过Hoechst及TUNEL染色检测细胞凋亡情况。
结果NMDA损伤后神经元肿胀、轴突断裂。随着NMDA浓度的增高,细胞生存率依次下降,LDH释放率依次增加,以NMDA 300umol/l组损伤最明显。丹红给药组细胞损伤减轻,胞体肿胀程度减轻,细胞形态尚保持完整,细胞生存率提高,LDH释放率下降,两种剂量丹红注射液均可减轻NMDA引起的神经元损伤,两组问细胞生存率及LDH释放率无明显统计学差异。小剂量丹红给药组Hoechst染色及TUNEL细胞凋亡检测示丹红给药组凋亡细胞百分比下降。
结论丹红注射液可减轻NMDA诱导的体外培养胎鼠皮层神经元的损伤,抑制NMDA引起的神经元凋亡。
Backgrounds The neuroprotection after acute cerebral ischemia is a hot topic in stroke research. As a common therapy for cerebral ischemia, traditional Chinese medicine has tremendous potential. Clinical trials and researches on animal models have shown that Danhong injection is effective in treating acute cerebral ischemia. Up till now, most researches in vitro have studied effects of Danhong injection on vascular endothelial cells, it remains to be explored whether Danhong injection can protect neurons from damage induced by NMDA, which mediates excitotoxicity as one of the main sources of damage after cerebral ischemia and can result in cell apoptosis.
Objective To provide cultured embryonic mice neurons with exogenous NMDA in vitro. To study whether Danhong injection can protect neurons from excitotoxicity induced by NMDA. To make the basis for further analysis of effective components of Danhong injection and its protective mechanisms.
Methods Primary cortical cultures were prepared from 17 or 18-day-old embryos obtained from pregnant KM mice. Neuronal cell populations are determined by immunocytochemistry against microtubule-associated protein (MAP-2). 10 days later, cultures were exposed to exogenous NMDA (50,150 and 300umol/l) randomly. Cell survival was evaluated by MTT assay and LDH release. The concentration of NMDA leading to the most marked cell damage was chosen to establish the model of NMDA induced neuronal injury. Cultured neurons were divided into 3 groups: normal control group, NMDA injury group and Danhong treatment group. Danhong injection(0.005μl/ml and 0.01μl/ml) was added to the culture medium of the treatment group simultaneously with NMDA. Cell survival was evaluated by MTT assay. Cell injury was determined by LDH release. Apoptosis was estimated by Hoechst staining and TUNEL.
Results A dose-response curve of NMDA neurotoxicity after 24 h of exposure to cultured neurons was performed and revealed the most significant reduction in MTT activity and the highest LDH release at a concentration of 300umol/l. Therefore, this concentration was chosen to perform all the following experiments. Under microscopy, after NMDA incubation, cultured neurons were damaged as evidenced by the presence of swelling and fragmented neuritis. When cortical cells were coincubated with NMDA and Danhong injection, cultures were better preserved and more intact neurons were visible. Danhong treatment group had both increased MTT activity and decreased LDH release and no statistical difference was observed between the 2 treatment groups(0.005μl/ml and 0.01μl/ml). The percentage of apoptotic neurons decreased as was labeled by Hoechst staining and TUNEL.
Conclusions Danhong injection can relieve NMDA induced neuronal injury in cultured cortical neurons of embryonic mice and inhibit NMDA induced neuronal apotosis.
引文
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[1]谭延国,陈瑞.兴奋性氨基酸类神经递质与缺血性脑疾患[J].国外医学临床生物化学与检脸学分册,2003,24(1):36-37.
[2]张忱,孟书聪,徐亚萍,程兰英.局灶性脑缺血大鼠脑内NMDA受体活性变化的研究[J].中华神经外科杂志,2001,17(5):321-323.
[3]张忱,张文,徐亚萍,李刚,程兰英.应用NMDA受体拮抗剂MK-801治疗局灶性脑缺血的实验研究[J].中日友好医院学报,2002,16(56):293-297.
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