基于JNK通路探讨调更汤对GT1-7下丘脑神经元细胞凋亡的保护作用

李盛楠; 刘洋; 刘小菲; 丛超; 刘慧聪; 归雯佳; 王月娇; 肖珊; 单鑫; 徐莲薇

doi:10.13422/j.cnki.syfjx.20192037

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基于JNK通路探讨调更汤对GT1-7下丘脑神经元细胞凋亡的保护作用

药理 | 更新时间：2021-02-09

- 基于JNK通路探讨调更汤对GT1-7下丘脑神经元细胞凋亡的保护作用
- Protective Effect of Tiaogeng Decoction in Improving Apoptosis of Hypothalamic Neurons Based on JNK Pathway
- 中国实验方剂学杂志 2019年25卷第20期页码：49-55
- 作者机构：
  
  上海中医药大学　附属龙华医院，上海　 200032
- 作者简介：
  
  李盛楠，在读博士，主治医师，从事中医药诊治生殖内分泌疾病研究，E-mail：lisncq@163.com
  徐莲薇，教授，主任医师，博士生导师，从事中医药诊治生殖内分泌疾病和不孕症研究，E-mail：doctorsherryxlw@163.com
- 基金信息：
  
  国家自然科学基金面上项目(81574010)
- DOI：10.13422/j.cnki.syfjx.20192037
  中图分类号： R2-0; R285.5; R289
- 收稿日期：2019-03-02，
  
  网络出版日期：2019-07-05，
  
  纸质出版日期：2019-10-20
- 稿件说明：
移动端阅览
李盛楠, 刘洋, 刘小菲, 等. 基于JNK通路探讨调更汤对GT1-7下丘脑神经元细胞凋亡的保护作用[J]. 中国实验方剂学杂志, 2019,25(20):49-55.

Sheng-nan LI, Yang LIU, Xiao-fei LIU, et al. Protective Effect of Tiaogeng Decoction in Improving Apoptosis of Hypothalamic Neurons Based on JNK Pathway[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(20): 49-55.
李盛楠, 刘洋, 刘小菲, 等. 基于JNK通路探讨调更汤对GT1-7下丘脑神经元细胞凋亡的保护作用[J]. 中国实验方剂学杂志, 2019,25(20):49-55. DOI： 10.13422/j.cnki.syfjx.20192037.

Sheng-nan LI, Yang LIU, Xiao-fei LIU, et al. Protective Effect of Tiaogeng Decoction in Improving Apoptosis of Hypothalamic Neurons Based on JNK Pathway[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(20): 49-55. DOI： 10.13422/j.cnki.syfjx.20192037.

摘要

目的：

研究调更汤通过c-Jun氨基末端激酶(JNK)通路改善下丘脑神经元细胞凋亡的作用机制。

方法：

采用下丘脑神经元细胞GT1-7细胞株，以三丁基氯化锡(TBTC)诱导模拟神经元细胞凋亡模型作为模型组(1 mg·L

－1

)，以17

-雌二醇(17

-E

)为阳性药物作为17

-E

组(100 nmol·L

－1

)，调更汤低、中、高剂量组(31.25，62.5，125 mg·L

－1

)进行干预。以倒置荧光显微镜观察细胞形态学变化；细胞增殖毒性(CCK-8)法检测细胞活力；Hoechst 33258染色观察凋亡细胞核的形态改变；Annexin V-FITC/碘化丙啶(PI)双染色法检测下丘脑神经元细胞凋亡率；蛋白免疫印迹法(Western blot)检测JNK通路凋亡相关蛋白凋亡信号调节激酶1(ASK1)，JNK，p53，活化半胱氨酸蛋白酶(cleaved Caspase-3)表达水平及JNK抑制剂SP600125干预后磷酸化JNK(p-JNK)，cleaved Caspase-3的蛋白表达水平。

结果：

与正常组比较，模型组细胞活力显著降低(

0.01)，细胞凋亡率显著升高(

0.01)，JNK通路相关蛋白磷酸化ASK1(p-ASK1)，p-JNK，磷酸化p53(p-p53)，cleaved Caspase-3的表达显著增加(

0.01)。与模型组比较，调更汤显著改善GT1-7细胞活力(

0.01)，降低细胞凋亡率(

0.01)，调节p-ASK1，p-JNK，p-p53，cleaved Caspase-3蛋白的表达；高剂量调更汤联合应用SP600125(10 μmol·L

－1

)处理后，更显著的抑制GT1-7细胞p-JNK，cleaved Caspase-3的蛋白表达(

0.01)。

结论：

调更汤通过JNK通路改善下丘脑神经元细胞凋亡，为调更汤治疗绝经综合征和中枢神经保护作用提供理论依据。

Abstract

Objective:

To study the mechanism of Tiaogeng decoction in improving apoptosis of hypothalamic neurons through c-Jun

-terminal kinase (JNK) pathway.

Method:

The GT1-7 cell line of hypothalamic neuron cells treated by tributyltin chloride (TBTC) was used to induce a model of neuronal apoptosis

with 17

-estradiol as a positive drug. They were divided into control group

model group (1 mg·L

-1

)

-E

group(100 nmol·L

-1

) and low

middle and high-dose Tiaogeng decoction groups (31.25

62.5

125 mg·L

-1

). The cell morphology was observed under a fluorescent inverted microscope

and cell counting kit-8 (CCK-8) was used to detect the cell viability. The cell nuclear morphology and the apoptosis rate of hypothalamic neurons were detected by Hoechst 33258 and Annexin V-FITC/propidium iodide(PI) double staining. Western blot was used to detect the expression levels of apoptosis signal-regulating kinase 1(ASK1)

JNK

p53 and cleaved Caspase-3 of JNK pathway in GT1-7 cell. Western blot was used to analyze apoptosis-associated protein apoptosis signal-regulated kinase 1 (ASK1)

JNK

p53

cleaved Caspase-3 expressions in JNK pathway and phosphorylation of JNK after intervention with JNK inhibitor SP600125

protein expression level of cleaved Caspase-3.

Result:

Compared with normal control group

the cell viability of the model group was significantly decreased (

0.01)

whereas the apoptotic rate was significantly increased (

0.01). The expressions of phosphorylated ASK1(p-ASK1)

p-JNK

phosphorylated p53 (p-p53) and cleaved Caspase-3 increased significantly (

0.01). After intervention with Tiaogeng decoction

compared with model group

the activity of GT1-7 cells was significantly improved(

0.01)

while the apoptotic rate was decreased (

0.01). The expressions of p-ASK1

p-JNK

p-p53

and cleaved Caspase-3 proteins were down-regulated. High-dose Tiaogeng decoction combined with SP600125(10 μmol·L

-1

)treatment significantly inhibited the expressions of p-JNK and cleaved Caspase-3 in GT1-7 cells (

0.01).

Conclusion:

Tiaogeng decoction can improve the apoptosis of hypothalamic neurons through JNK pathway

and provide a theoretical basis for the treatment of menopausal syndrome and central nervous system protection.

关键词

Keywords

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