白香丹胶囊治疗经前期综合征肝气逆证模型大鼠的药理机制研究——含药血清对原代培养皮层神经元细胞相活力及GABAAR介导氯离子通道的影响
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摘要
目的:探讨白香丹胶囊(BXD)治疗经前期综合征(premenstrual syndrome,PMS)肝气逆证模型大鼠的中枢作用机理。
方法:阴道涂片镜检法和旷场实验筛选合适大鼠,以电刺激法复制PMS肝气逆证模型大鼠,同时采用宏观行为学观察和旷场实验来评价模型;结合血清药理学和细胞培养技术,MTT法和全细胞膜片钳技术分别测定大鼠含药血清对离体皮层神经元细胞相对活力和GABA_AR介导氯离子通道的影响。
结果:旷场实验中,与正常组相比,肝逆组大鼠水平得分、垂直得分及旷场总分均明显增加(P＜0.05);而与肝逆组相比,肝逆给药组大鼠得分又明显减少(P＜0.05)。MTT实验中,给药24h后,与正常组相比,正常给药组和肝逆给药组神经元MTT值显著增加(P＜0.05),肝逆组神经元MTT值无统计学差异;给药48h后,与正常组相比,正常给药组神经元MTT值显著增加(P＜0.05),空白组和肝逆组神经元MTT值显著降低(P＜0.05),肝逆给药组神经元MTT值无统计学差异。电生理实验中,离体大鼠皮层神经元细胞经PMS肝逆模型大鼠血清和PMS肝逆给药大鼠血清处理后,曲线拟合参数EC_(50)和n_H分别为63.5±8.2μM,1.04±0.10和29.0±4.4μM,1.07±0.16;其中EC_(50(Model))＞EC_(50(Model+BXD))(U检验,P=0.0000＜＜0.05),n_(H(Model+BXD))＞n_(H(Model))(t检验,P=0.8756＞0.05)。
结论:BXD可有效提高大鼠皮层神经元细胞活力和GABA_AR活性;PaeonimetabolinsⅠ、Paeonol可能就是BXD治疗PMS有效物质成分,而PaeonimetabolinsⅠ、Paeonol很可能通过作用于GABA_AR这一靶点来发挥药效作用。
Objectives: The aim of this study is to explore the effects of BDX capsule, a Chinese Medicinal Formula, on the PMS model rats with Liver-qi Invasion and the possible underlying micro-mechanisms.
Methods: After vaginal smear examination and open-field test, PMS model rats with Liver-qi Invasion were treated with electrical stimulating mothod, and evaluated by macro-behavior observation and open-field test. Then MTT assay and the whole-cell patch clamp recording were performed respectively to evaluate the effects of serum from BDX capsule-treated rats on the viability and GABA-induced currents of cortical neurons in vitro.
Results: In the open-field test, the crossing score, rearing score and total score of Model rats increased significantly (P<0.05), compared with the Normal rats, and that of Model+BXD group decreased (P<0.05), compared with model rats. Compared with cells exposed to serum of normal rats, the viability values of those incubated with serum of Normal+BXD group and Model+BXD group for 24h and of Normal+BXD group for 48h significantly increased (P<0.05), and that of Control and Model group for 48h significantly decreased (P<0.05), measured with MTT assay; that of others had no obvious difference. The results of whole-cell patch clamp recording showed that concentration-response relationship curves revealed an EC_(50) value of 29.0±4.4μM and a Hill coefficient of 1.07 for Model+BXD-exposed cultures, 63.5±8.2μM, 1.04 for Model-exposed cultures after incubation for 24h. Furthermore, the difference in EC_(50) values was statistically significant (t test, P <0.01), that in the Hill coefficient was not obvious (U test, P>0.05).
Conclusions: BDX capsule could effectively enhance the neuron viability and GABA_A receptor activity in rat cortex; Paeonimetabolins I and Paeonol may play a significant role in treating PMS model rats with Liver-qi Invasion by BDX capsule, and they may target at GABAA receptor, especially.
方法:阴道涂片镜检法和旷场实验筛选合适大鼠,以电刺激法复制PMS肝气逆证模型大鼠,同时采用宏观行为学观察和旷场实验来评价模型;结合血清药理学和细胞培养技术,MTT法和全细胞膜片钳技术分别测定大鼠含药血清对离体皮层神经元细胞相对活力和GABA_AR介导氯离子通道的影响。
结果:旷场实验中,与正常组相比,肝逆组大鼠水平得分、垂直得分及旷场总分均明显增加(P＜0.05);而与肝逆组相比,肝逆给药组大鼠得分又明显减少(P＜0.05)。MTT实验中,给药24h后,与正常组相比,正常给药组和肝逆给药组神经元MTT值显著增加(P＜0.05),肝逆组神经元MTT值无统计学差异;给药48h后,与正常组相比,正常给药组神经元MTT值显著增加(P＜0.05),空白组和肝逆组神经元MTT值显著降低(P＜0.05),肝逆给药组神经元MTT值无统计学差异。电生理实验中,离体大鼠皮层神经元细胞经PMS肝逆模型大鼠血清和PMS肝逆给药大鼠血清处理后,曲线拟合参数EC_(50)和n_H分别为63.5±8.2μM,1.04±0.10和29.0±4.4μM,1.07±0.16;其中EC_(50(Model))＞EC_(50(Model+BXD))(U检验,P=0.0000＜＜0.05),n_(H(Model+BXD))＞n_(H(Model))(t检验,P=0.8756＞0.05)。
结论:BXD可有效提高大鼠皮层神经元细胞活力和GABA_AR活性;PaeonimetabolinsⅠ、Paeonol可能就是BXD治疗PMS有效物质成分,而PaeonimetabolinsⅠ、Paeonol很可能通过作用于GABA_AR这一靶点来发挥药效作用。
Objectives: The aim of this study is to explore the effects of BDX capsule, a Chinese Medicinal Formula, on the PMS model rats with Liver-qi Invasion and the possible underlying micro-mechanisms.
Methods: After vaginal smear examination and open-field test, PMS model rats with Liver-qi Invasion were treated with electrical stimulating mothod, and evaluated by macro-behavior observation and open-field test. Then MTT assay and the whole-cell patch clamp recording were performed respectively to evaluate the effects of serum from BDX capsule-treated rats on the viability and GABA-induced currents of cortical neurons in vitro.
Results: In the open-field test, the crossing score, rearing score and total score of Model rats increased significantly (P<0.05), compared with the Normal rats, and that of Model+BXD group decreased (P<0.05), compared with model rats. Compared with cells exposed to serum of normal rats, the viability values of those incubated with serum of Normal+BXD group and Model+BXD group for 24h and of Normal+BXD group for 48h significantly increased (P<0.05), and that of Control and Model group for 48h significantly decreased (P<0.05), measured with MTT assay; that of others had no obvious difference. The results of whole-cell patch clamp recording showed that concentration-response relationship curves revealed an EC_(50) value of 29.0±4.4μM and a Hill coefficient of 1.07 for Model+BXD-exposed cultures, 63.5±8.2μM, 1.04 for Model-exposed cultures after incubation for 24h. Furthermore, the difference in EC_(50) values was statistically significant (t test, P <0.01), that in the Hill coefficient was not obvious (U test, P>0.05).
Conclusions: BDX capsule could effectively enhance the neuron viability and GABA_A receptor activity in rat cortex; Paeonimetabolins I and Paeonol may play a significant role in treating PMS model rats with Liver-qi Invasion by BDX capsule, and they may target at GABAA receptor, especially.
引文
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