脑脉泰胶囊对外周神经元损伤模型斑马鱼神经再生的影响

Effects of Naomaitai Capsule(脑脉泰胶囊)on Nerve Regeneration in Zebrafish Model with Peripheral Neuronal Injury

目的探讨脑脉泰胶囊治疗脑梗死的可能作用机制。方法脑脉泰胶囊用超纯水溶解成50、100、250、500、1000、1500μg/ml 6个浓度,均分别处理30尾野生型AB系斑马鱼24h后,统计各组斑马鱼死亡数量,绘制浓度效应曲线,并计算最大非致死浓度,筛选实验药物浓度。取受精后1天运动神经元转基因荧光斑马鱼,分为空白对照组、模型组、阳性对照组和脑脉泰胶囊高、中、低剂量组,每组30尾。除空白对照组外,其余各组斑马鱼用1.5%乙醇诱导处理斑马鱼48h建立斑马鱼外周运动神经元损伤模型。造模结束后,阳性对照组给予L-甲状腺素钠0.08μmol/L,空白对照组和模型组给予养鱼用水,脑脉泰胶囊低、中、高剂量组分别给予筛选浓度的脑脉泰胶囊处理24h。药物处理结束后,利用图像处理软件测量外周运动神经元的长度,计算促神经再生率。结果脑脉泰胶囊浓度在50、100μg/ml时,斑马鱼无死亡;浓度为250μg/ml时斑马鱼开始出现死亡,根据浓度效应曲线得出脑脉泰胶囊最大非致死浓度为170μg/ml,故本研究选取17、57、170μg/ml为脑脉泰胶囊低、中、高剂量组干预浓度。与空白对照组比较,模型组斑马鱼外周运动神经元长度显著缩短(P<0.01);与模型组比较,阳性对照组和脑脉泰胶囊低、中、高剂量组斑马鱼外周运动神经元长度增加(P<0.01);与阳性对照组比较,脑脉泰胶囊中、高剂量组斑马鱼外周运动神经元长度差异无统计学意义(P>0.05),而脑脉泰胶囊低剂量组斑马鱼外周运动神经元长度降低(P<0.01)。阳性对照组和脑脉泰胶囊低、中、高组促神经再生率分别为31%、17%、29%、30%。结论脑脉泰胶囊对外周神经元具有促神经再生作用,可能为其治疗脑梗死作用机制之一。

Objective To explore the possible mechanism of Naomaitai Capsule(脑脉泰胶囊) for treatment of cerebral infarction. Methods Ultrapure water was used to dissolve Naomaitai Capsules;the solutions of Naomaitai Capsules at different concentrations of 50, 100, 250, 500, 1000, and 1500μg/ml were used to keep 30 wild-type AB zebrafish for 24 hours, respectively. The number of zebrafish deaths in each group was counted;the concentration-effect curve was drawn;the maximum non-lethal concentration was calculated;and the suitable concentration used for the experiment was screened. The transgenic zebrafish with fluorescent motor neurons one day after fertilization were grouped into blank control group, model group, positive control group, Naomaitai Capsule high, medium and low dose group, with 30 zebrafish in each group. Ethanol(1.5%) was used on zebrafish for 48 hours to establish the model of peripheral neuronal injury in all groups except for blank control group. After modelling, L-thyroxine sodium at concentration of 0.08μmol/L was given to positive control group, and fish-farming water was used in blank control group and model group;Naomaitai Capsules at the screened concentrations were given to zebrafish for 24 hours in Naomaitai Capsule low, medium and high dose groups, respectively. After the interventions, the image processing software was used to measure the neuronal length of peripheral motor neurons, and the nerve regeneration rate was calculated. Results There was no deaths of zebrafish when the concentration of Naomaitai Capsule solution was 50 or 100μg/ml, and zebrafish began to die when the concentration was 250μg/ml. According to the concentration-effect curve, the maximum non-lethal concentration was 170μg/ml, and therefore the concentrations used in the Naomaitai Capsule low, medium and high dose group were set as 17, 57 and 170μg/ml, respectively. The length of peripheral motor neurons of zebrafish in model group was significantly shorter than that of blank control group(P<0.01), and that in positive control group, Naomaitai Capsule low, medium and high dose groups was longer than that in the model group(P<0.01);There was no significant difference in length of peripheral motor neurons in comparison with positive control group, Naomaitai Capsule medium and high dose groups(P>0.05), while there was significantly decreased length of peripheral motor neurons in Naomaitai Capsule low dose group(P<0.01). The nerve regeneration rate of positive control group, Naomaitai Capsule low, medium and high dose groups were 31%, 17%, 29%, and 30% respectively. Conclusion The effect of Naomaitai Capsule on promoting nerve regeneration of peripheral neurons may be related to its mechanisms of treating cerebral infarction.