Protective Effect of Gandou Decoction on Cer Signaling Pathway in HT-22 Induced by High Copper

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

doi:10.13422/j.cnki.syfjx.20190701

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Protective Effect of Gandou Decoction on Cer Signaling Pathway in HT-22 Induced by High Copper

Experimental Study on Treatment of Wilson's Disease with Gandou Decoction | 更新时间：2021-02-09

- Protective Effect of Gandou Decoction on Cer Signaling Pathway in HT-22 Induced by High Copper
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 7, Pages: 56-60(2019)
- 作者机构：
  
  1.安徽中医药大学　神经病学研究所　附属医院，合肥　 230061；
  2.安徽医科大学　第二附属医院，合肥　 230601
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20190701
  CLC： R2-0;R22;R285.5;R289
- Received：10 October 2018，
  
  Published Online：18 December 2018，
  
  Published：05 April 2019
- 稿件说明：
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Chen-chen XU, Jian-jian DONG, Xun WANG, et al. Protective Effect of Gandou Decoction on Cer Signaling Pathway in HT-22 Induced by High Copper[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(7): 56-60.
DOI：

Chen-chen XU, Jian-jian DONG, Xun WANG, et al. Protective Effect of Gandou Decoction on Cer Signaling Pathway in HT-22 Induced by High Copper[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(7): 56-60. DOI： 10.13422/j.cnki.syfjx.20190701.

摘要

目的：

探讨肝豆汤调控海马神经元内神经酰胺(Cer)信号通路的分子靶点并观察其相应的调控机制。

方法：

本实验采用海马神经元HT-22细胞，分为正常组，模型组，10%，15%及20%肝豆汤组5个组，模型组为硫酸铜(CuSO

)孵育HT-22细胞所得，正常组加入空白含药血清，10%，15%及20%肝豆汤组分别加含不同体积分数(10%，15%，20%)肝豆汤兔血清的培养基培养。噻唑蓝(MTT)比色法检测不同剂量CuSO

作用不同时间点对 HT-22细胞生长和增殖的影响；流式细胞仪检测细胞内活性氧(ROS)释放量的变化；蛋白免疫印迹法(Western blot)检测Cer信号通路相关蛋白表达情况。

结果：

MTT结果显示CuSO

在一定时间和浓度范围内对HT-22细胞生长和增殖有明显抑制作用；流式细胞仪检测发现，与正常组比较，模型组ROS荧光强度显著增加（

0.01），肝豆汤组较模型组可显著降低细胞内ROS的释放量（

0.01）。Western blot检测结果显示与模型组比较，肝豆汤组可明显降低细胞内酸性鞘磷脂水解酶（ASM），Cer，p38丝裂原活化蛋白激酶（p38 MAPK），细胞色素C（Cyt C），半胱氨酸天冬氨酸蛋白酶-9（Caspase-9），半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）蛋白表达（

0.05，

0.01）。

结论：

高铜可诱导HT-22细胞内线粒体氧化应激并引起 Cer信号通路失活，从而诱导海马神经元损伤的发生。肝豆汤可能通过促进过量铜排出而抑制细胞内ASM

Cer

p38 MAPK

Cyt C

Caspase-9

Caspase-3蛋白的表达，从而减轻高铜对海马神经元的损伤作用。肝豆汤可通过减少脑内铜含量，进而调控Cer信号通路达到减轻高铜诱导海马神经元损伤的治疗效果。

Abstract

Objective:

To detect ceramide(Cer) signaling pathway-related proteins expression levels in HT-22 with Gandou decoction (GDD)

in order to explore its molecular targets and mechanism in regulating Cer signaling pathway.

Method:

The experiment was divided into normal group (normal HT-22 cultured by 10%blank rabbit serum)

model group (HT-22 cells incubated with CuSO

)

and GDD group (HT-22 cells incubated with CuSO

continuously cultured by rabbit serum containing10%

15%

20%GDD). HT-22 cells were incubated with different concentrations of CuSO

.The cell growth and proliferation were assessed using methyl thiazolyl tetrazolium(MTT) method; flow cytometry was used to analyze the expression of reactive oxygen species (ROS); Western blot was used to detect relevant protein expression of Cer signaling pathway.

Result:

The results of MTT showed that CuSO

inhibited the growth and proliferation of HT-22 cells in a time and concentration-dependent manner; flow cytometry results showed that the model group increased the release of ROS compared with the normal group (

0.01); compared with the model group

GDD could significantly reduce the release of ROS (

0.01). Western blot indicated that GDD concentration-dependently decreased the expressions of acid sphingomyelinase(ASM)

Cer

p38 mitogen-activated protein kinase(p38 MAPK)

cytochrome C(Cyt C)

cysteinyl aspartate specific proteinase(Caspase)-9

Caspase-3 in HT-22 compared with the model group (

0.05

0.01).

Conclusion:

High copper can induce oxidative stress and deactivate Cer signaling pathway

which led to hippocampal neuron injury. These findings suggest that GDD reduces neurotoxicity induced by copper overload by increasing the copper excretion that inhibits the expressions of ASM

Cer

p38 MAPK

Cyt C

Caspase-9

Caspase-3.GDD reduces neurotoxicity induced by copper overload by decreasing copper levels in brain and then regulating Cer signaling pathway.

关键词

Keywords

references

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