Effect of Gandou Decoction on Autophagy of SH-SY5Y Cell Induced by High Copper and Its Mechanism

Ya-nan HUANG; Jian-jian DONG; Chen-chen XU; Nan CHENG; Yong-zhu HAN

doi:10.13422/j.cnki.syfjx.20190702

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Effect of Gandou Decoction on Autophagy of SH-SY5Y Cell Induced by High Copper and Its Mechanism

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

- Effect of Gandou Decoction on Autophagy of SH-SY5Y Cell Induced by High Copper and Its Mechanism
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 7, Pages: 61-67(2019)
- 作者机构：
  
  安徽中医药大学　神经病学研究所　附属医院，合肥　 230061
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20190702
  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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Ya-nan HUANG, Jian-jian DONG, Chen-chen XU, et al. Effect of Gandou Decoction on Autophagy of SH-SY5Y Cell Induced by High Copper and Its Mechanism[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(7): 61-67.
DOI：

Ya-nan HUANG, Jian-jian DONG, Chen-chen XU, et al. Effect of Gandou Decoction on Autophagy of SH-SY5Y Cell Induced by High Copper and Its Mechanism[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(7): 61-67. DOI： 10.13422/j.cnki.syfjx.20190702.

摘要

目的：

研究肝豆汤对高铜诱导的人神经母细胞瘤(SH-SY5Y)细胞自噬效应的影响及其作用机制，为中医药防治脑型Wilson病(Wilson disease

WD)提供新的治疗靶点和研究思路。

方法：

噻唑蓝(MTT)比色法筛选硫酸铜(CuSO

)造模浓度(0，100，200，400，800，1 600 μmol·L

－1

)及时间；MTT比色法筛选含药血清浓度(5%，10%，15%，20%)及时间；乳酸脱氢酶(LDH)释放实验检测细胞LDH漏出率；流式细胞法检测细胞内活性氧(ROS)的含量；荧光染料JC-1检测细胞线粒体膜电位；流式细胞仪对自噬进行定量分析。蛋白免疫印迹法(Western blot)检测肝激酶B

(LKB

)，腺苷酸活化蛋白激酶(AMPK)，自噬微管相关蛋白轻链3A/B(LC3A/B)，哺乳动物雷帕霉素靶蛋白(mTOR)，unc-51样激酶1(ULK1)，磷酸化ULK(p-ULK)，磷酸化AMPK(p-AMPK)蛋白的表达。

结果：

MTT结果显示，CuSO

对细胞的损伤呈现一定的量效和时效关系（

0.01），随着CuSO

作用浓度及时间的增加，细胞存活率呈现下降趋势；10%含肝豆汤兔血清可显著抑制CuSO

诱导的细胞死亡（

0.01）。LDH释放实验显示，与正常组比较，CuSO

作用细胞后LDH漏出率显著增加（

0.01），与模型组比较，含肝豆汤兔血清明显降低CuSO

损伤细胞的LDH漏出率（

0.05）。DCFH-DA荧光染色显示，与正常组比较，CuSO

可显著增加细胞内ROS生成（

0.01），与模型组比较，含肝豆汤兔血清可显著抑制CuSO

诱导的细胞内ROS产生（

0.01）。JC-1染色结果显示，与正常组比较，CuSO

诱导细胞线粒体膜电位

Δψ

显著降低（

0.01），与模型组比较，含肝豆汤兔血清明显抑制CuSO

诱导的线粒体膜电位降低

Δψ

（

0.05）。Western blot结果显示，与正常组比较，模型组细胞内LKB

，AMPK

LC3A/B

ULK1及p-AMPK蛋白的表达显著增加，mTOR及p-ULK蛋白的表达显著降低（

0.01）。与模型组比较，含肝豆汤兔血清组LKB

，AMPK，LC3A/B，ULK1及p-AMPK蛋白表达显著降低，mTOR及p-ULK蛋白表达显著增加（

0.01）。

结论：

高铜可通过诱导细胞内线粒体氧化应激，上调自噬相关蛋白LKB

，p-AMPK

AMPK

LC3A/B及ULK1的表达，下调自噬相关蛋白mTOR及p-ULK的表达，导致细胞发生自噬性死亡，而肝豆汤可通过调控LKB

/AMPK信号通路，下调自噬相关蛋白LKB

，p-AMPK

LC3A/B

ULK及AMPK的表达，上调自噬相关蛋白及基因mTOR及p-ULK的表达，抑制自噬的发生，阻断高铜诱导的神经元损伤，从而发挥神经保护作用。

Abstract

Objective:

To explore the effect of Gandou decoction on autophagy of SH-SY5Y cells induced by high copper and its mechanism

in order to provide new therapeutic targets and research ideas for the prevention and treatment of brain-type Wilson disease (WD) with traditional Chinese medicine.

Method:

CuSO

model showed a certain dose-effect and time-effect relationship according to methyl thiazolyl tetrazolium(MTT); lactate dehydrogenase(LDH) leakage rate was detected by LDH release assay; flow cytometry method was used to detect intracellular reactive oxygen species (ROS) content. The fluorescent dye JC-1 was used to detect the mitochondrial membrane potential of the cells. Flow cytometry was used to quantify autophagy. The expressions of liver kinase B

(LKB

)

AMP-activated protein kinase (AMPK)

microtubule-associated protein 1 light chain 3 (LC3A/B)

mammalian target of rapamycin (mTOR) and UNC-51-like kinase-1 (ULK1)

phosphorylation-ULK (p-ULK)

phosphorylation-AMPK (p-AMPK) were detected by Western blot.

Result:

According to MTT results

CuSO

showed a dose-effect and time-effect relationship with cells (

0.01). With the increase of the dosage and time of CuSO

the survival rate of cells showed a downward trend (

0.01). MTT results showed that 10%of rabbit serum containing Gandou decoction significantly inhibited CuSO

-induced cell death (

0.01). The results of MTT showed that the leakage rate of LDH increased significantly after treatment with CuSO

compared with the normal group (

0.01)

and the rabbit serum of Gandou decoction significantly decreased the LDH leakage rate of CuSO

-injured cells (

0.05). DCFH-DA fluorescence staining showed that CuSO

significantly increased the production of ROS in cells (

0.01). The rabbit serum containing Gandou decoction significantly inhibited CuSO

-induced intracellular ROS production (

0.01). JC-1 staining showed that CuSO

induced a significant decrease in mitochondrial membrane potential in cells (

0.01)

while serum containing Gandou decoction inhibited CuSO

-induced mitochondrial membrane potential in a dose-dependent manner (

0.05). The western blot results showed that compared with the normal group

the protein expressions of LKB

AMPK

LC3A/B

ULK

p-AMPK in the model group were significantly increased

while the protein expressions of mTOR and p-ULK were significantly decreased (

0.01). Compared with the model group

the expressions of LKB

AMPK

LC3A/B

p-AMPK and ULK were significantly decreased

whereas the protein expressions of mTOR and p-ULK were significantly increased in the rabbit serum group containing Gandou decoction (

0.01).

Conclusion:

High copper can induce autophagic apoptosis in SH-SY5Y cells by inducing intracellular mitochondrial oxidative stress

up-regulating the expressions of autophagy-related proteins LKB

AMPK

LC3A/B

ULK

p-AMPK and down-regulating the expressions of mTOR and p-ULK. However

Gandou decoction can inhibit the occurrence of autophagy

and cut off high copper-induced neuronal damage by down-regulating the expressions of autophagy-related proteins LKB

AMPK

LC3A/B

ULK

p-AMPK

and up-regulating the expression of mTOR and p-ULK

so as to exert a neuroprotective effect.

关键词

Keywords

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