Molecular Regulatory Mechanism of Modified Gandou Decoction on Cyt C/Caspase Signaling Pathway in Neurons of Wilson's Disease Model-TX Mice

XU Chen-chen; DONG Jian-jian; CHENG Nan; HAN Yong-zhu; WANG Xun

doi:10.13422/j.cnki.syfjx.2017060143

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Molecular Regulatory Mechanism of Modified Gandou Decoction on Cyt C/Caspase Signaling Pathway in Neurons of Wilson's Disease Model-TX Mice

更新时间：2024-01-04

- Molecular Regulatory Mechanism of Modified Gandou Decoction on Cyt C/Caspase Signaling Pathway in Neurons of Wilson's Disease Model-TX Mice
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 23, Issue 6, Pages: 143-148(2017)
- 作者机构：
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- DOI：10.13422/j.cnki.syfjx.2017060143
  CLC： R-332;R285.5
- Published：2017
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XU Chen-chen, DONG Jian-jian, CHENG Nan, et al. Molecular Regulatory Mechanism of Modified Gandou Decoction on Cyt C/Caspase Signaling Pathway in Neurons of Wilson's Disease Model-TX Mice[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(6): 143-148.
DOI：

XU Chen-chen, DONG Jian-jian, CHENG Nan, et al. Molecular Regulatory Mechanism of Modified Gandou Decoction on Cyt C/Caspase Signaling Pathway in Neurons of Wilson's Disease Model-TX Mice[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(6): 143-148. DOI： 10.13422/j.cnki.syfjx.2017060143.

摘要

目的：探讨肝豆汤改良方调控TX乳鼠神经元内细胞色素C（Cyt C）/半胱氨酸天冬氨酸蛋白酶（Caspase）信号通路的分子靶点并观察其相应的调控机制。方法：本实验乳鼠神经元通过原代方法分离培养所得，分为正常组、模型组、肝豆汤改良方组、丁苯酞组，正常组为正常DL乳鼠神经元，用完全培养基培养，模型组为TX乳鼠神经元，用10%空白兔血清培养，肝豆汤改良方组为TX乳鼠神经元，加入含体积浓度（5%，10%，15%，20%）肝豆汤改良方兔血清的培养基继续培养，丁苯酞组为TX乳鼠神经元，用10%含丁苯酞兔血清培养。采用原子吸收分光光度法检测不同浓度含肝豆汤改良方兔血清作用24 h后，对TX乳鼠神经元内微量元素的影响；流式细胞仪检测经肝豆汤改良方兔血清作用后活性氧（ROS）释放量的变化；蛋白质免疫印迹（Western blot）法检测经含肝豆汤改良方兔血清作用后Cyt C，Caspase-9，Caspase-3蛋白的表达。结果：与模型组比较，含肝豆汤改良方兔血清可显著降低TX乳鼠神经元内铜、铁含量，增加锌含量（P<0.01）。流式细胞仪检测发现，含肝豆汤改良方兔血清较模型组可显著降低TX乳鼠神经元内ROS的释放量（P<0.01）。Western blot检测结果显示与模型组比较，含肝豆汤改良方兔血清可显著降低TX乳鼠神经元内Cyt C，Caspase-9，Caspase-3蛋白表达（P<0.01）。结论：肝豆汤改良方可能是通过促进过量铜排出而抑制神经元内Cyt C，Caspase-9，Caspase-3表达，从而减轻高铜对神经元的损伤作用。肝豆汤改良方可通过减少脑内铜含量，进而调控Cyt C/Caspase信号通路达到减轻高铜诱导的神经元损伤的治疗效果。

Abstract

Objective: To investigate the molecular targets of Modified GanDou decoction (MGDD) treatment in Cyt C/Caspase signaling pathway in neurons of TX neonatal mice

and explore its corresponding mechanism on regulating Cyt C/Caspase signaling pathway. Method: neurons of TX neonatal mice were obtained by using primary culture method

and were divided into normal group (normal DL neonatal mice neurons

cultured by complete medium)

model group (TX neonatal mice neurons

cultured by 10% blank rabbit serum)

MGDD group (TX neonatal mice neurons

continuously cultured by rabbit serum containing 5%

10%

15% and 20% MGDD)

and Butylphthalide group (TX neonatal mice neurons

cultured by rabbit serum containing 10% Butylphthalide). Atomic absorption method was used to detect the effect of different concentrations of MGDD rabbit serum on microelement in the neurons of TX neonatal mice after 24 h treatment flow cytometry was used to analyze the expression of reactive oxygen (ROS) in the neurons of TX neonatal mice after treatment with rabbit serum containing MGDD; Western blot was used to detect the protein expression levels of Cyt C

Caspase-9 and Caspase-3 in the neurons of TX neonatal mice after treatment with rabbit serum containing MGDD. Result: As compared with the model group

MGDD could reduce the contents of copper and iron in the neurons of TX neonatal mice

and increase the content of zinc in a dose-dependent manner (P<0.01). Flow cytometry results showed that MGDD can significantly reduce the release of ROS as compared with model group (P<0.01). Western blot results indicated that MGDD significantly decreased the protein expression levels of Cyt C

Caspase-9

and Caspase-3 in the neurons of TX neonatal mice as compared with the model group (P<0.01). Conclusion: MGDD could relieve the high copper-induced neurons damage

probably by promoting the copper excretion and inhibiting the expression levels of Cyt C

Caspase-9 and Caspase-3 in TX neonatal mice neurons. MGDD achieves the treatment effect by reducing copper content in brain and regulating Cyt C/Caspase signaling pathway.

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