基于NMDAR调控线粒体自噬探讨滋肾活血方对血管性痴呆大鼠海马氧化应激损伤的保护作用

赵梓婷; 张秀丽; 刘桐赫; 施佳仪; 向韵; 周梓洋; 谢乐; 刘芳; 曾珊珊; 杨瑾昱; 伍大华; 曾晶

doi:10.3969/j.issn.1006-2157.2023.08.011

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基于NMDAR调控线粒体自噬探讨滋肾活血方对血管性痴呆大鼠海马氧化应激损伤的保护作用

实验研究 | 更新时间：2023-09-08

- 基于NMDAR调控线粒体自噬探讨滋肾活血方对血管性痴呆大鼠海马氧化应激损伤的保护作用
- Exploring the protective effect of Zishen Huoxue Formula on oxidative stress injury in the hippocampus of vascular dementia rats based on NMDAR-regulated mitophagy
- 北京中医药大学学报 2023年46卷第8期页码：1128-1138
- 作者机构：
  
  1.湖南中医药大学　长沙　410208
  2.湖南中医药大学科技创新中心
  3.湖南省中医药研究院附属医院
  4.湖南中医药大学第一附属医院
- 作者简介：
  
  赵梓婷，女，在读硕士生
  # 伍大华，女，博士，教授，主任医师，博士生导师，主要研究方向：中医药防治脑病，E-mail：893049352@qq.com
- 基金信息：
  
  国家自然科学基金面上项目(81874462);湖南省重点研发计划项目(2020SK2123);湖南省教育厅优秀青年基金项目(19B426);长沙市自然科学基金项目(kq2202452);湖南中医药大学校级科研基金项目(2019XJJJ036)
- DOI：10.3969/j.issn.1006-2157.2023.08.011
  中图分类号： R285.5
- 收稿：2023-02-06，
  
  网络首发：2023-06-06，
  
  纸质出版：2023-08-30
- 稿件说明：
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赵梓婷, 张秀丽, 刘桐赫, 等. 基于NMDAR调控线粒体自噬探讨滋肾活血方对血管性痴呆大鼠海马氧化应激损伤的保护作用[J]. 北京中医药大学学报, 2023,46(8):1128-1138.

ZHAO Ziting, ZHANG Xiuli, LIU Tonghe, et al. Exploring the protective effect of Zishen Huoxue Formula on oxidative stress injury in the hippocampus of vascular dementia rats based on NMDAR-regulated mitophagy[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023, 46(8): 1128-1138. DOI: 10.3969/j.issn.1006-2157.2023.08.011.
赵梓婷, 张秀丽, 刘桐赫, 等. 基于NMDAR调控线粒体自噬探讨滋肾活血方对血管性痴呆大鼠海马氧化应激损伤的保护作用[J]. 北京中医药大学学报, 2023,46(8):1128-1138. DOI： 10.3969/j.issn.1006-2157.2023.08.011.

ZHAO Ziting, ZHANG Xiuli, LIU Tonghe, et al. Exploring the protective effect of Zishen Huoxue Formula on oxidative stress injury in the hippocampus of vascular dementia rats based on NMDAR-regulated mitophagy[J]. Journal of Beijing University of Traditional Chinese Medicine, 2023, 46(8): 1128-1138. DOI: 10.3969/j.issn.1006-2157.2023.08.011. DOI：

摘要

目的

探讨滋肾活血方通过调节N－甲基－D－天冬氨酸受体(NMDAR)促进线粒体自噬，从而减轻血管性痴呆(VD)大鼠海马氧化应激损伤的分子机制。

方法

采用双侧颈总动脉结扎法建立大鼠VD模型。60只雄性SD大鼠根据随机数字表法分为假手术组、模型组、盐酸多奈哌齐组(0.45 mg/kg)、滋肾活血方低剂量组(8.9 g/kg)、滋肾活血方中剂量组(17.8 g/kg)、滋肾活血方高剂量组(35.6 g/kg)，每组10只。灌胃给药14 d。通过水迷宫实验对各组大鼠的学习记忆能力进行评估。50只雄性SD大鼠根据随机数字表法分为假手术组、模型组、马来酸氢盐(MK－801)组(1 mg/kg)、滋肾活血方高剂量组(35.6 g/kg)、滋肾活血方高剂量联合MK－801组(35.6 g/kg＋1 mg/kg)，每组10只。MK－801组在造模后连续7 d腹腔注射MK－801，滋肾活血方高剂量联合MK－801组灌胃高剂量滋肾活血方联合腹腔注射MK－801。灌胃给药14 d。蛋白质印迹法检测PTEN诱导假定激酶1(PINK1)/帕金森病相关蛋白(Parkin)信号通路相关蛋白[PINK1、Parkin、抗增殖蛋白2(PHB2)、微管相关蛋白1轻链3B(LC3B)、泛素结合蛋白p62]表达，免疫荧光法检测海马PINK1、神经元特异性烯醇化酶(NSE)平均荧光强度，酶联免疫吸附测定检测海马丙二醛(MDA)、超氧化物歧化酶(SOD)含量，实时荧光PCR及蛋白质印迹法检测大鼠海马NMDAR2A(NR2A)、NMDAR2B(NR2B)mRNA及NR2B蛋白表达。

结果

水迷宫实验结果表明，与假手术组相比，模型组逃避潜伏期延长(

0.05)，穿越平台次数减少(

0.05)；与模型组相比，盐酸多奈哌齐组与滋肾活血方低、中、高剂量组大鼠逃避潜伏期缩短(

0.05，

0.01)，盐酸多奈哌齐组与滋肾活血方中、高剂量组穿越平台次数增加(

0.05，

0.01)。进一步研究表明，与模型组相比，滋肾活血方高剂量组大鼠海马Parkin、PINK1、PHB2、LC3Ⅱ/Ⅰ蛋白表达增加(

0.01)，p62蛋白表达减少(

0.01)，PINK1、NSE平均荧光强度增强(

0.05，

0.01)，SOD含量增加(

0.01)，MDA含量减少(

0.01)；MK－801组大鼠海马Parkin、PINK1、PHB2及LC3Ⅱ/Ⅰ蛋白表达减少(

0.01)，p62蛋白表达增加(

0.01)，PINK1、NSE平均荧光强度减弱(

0.05，

0.01)，SOD含量减少(

0.05)，MDA含量增加(

0.01)。与MK－801组相比，滋肾活血方高剂量联合MK－801组大鼠海马Parkin、PINK1、PHB2、LC3Ⅱ/Ⅰ蛋白表达增加(

0.01)，p62蛋白表达减少(

0.01)，PINK1、NSE平均荧光强度增强(

0.05，

0.01)，SOD含量增加(

0.01)，MDA含量减少(

0.01)，NR2B mRNA及蛋白表达增加(

0.01)。

结论

滋肾活血方可能通过NR2B介导的PINK1/Parkin信号通路调节线粒体自噬，从而改善VD大鼠海马神经元氧化应激损伤。

Abstract

Objective

To investigate the molecular mechanism of

Zishen Huoxue

Formula alleviating hippocampal oxidative stress injury in rats with vascular dementia (VD) by regulating N-methyl-D-aspartate receptor (NMDAR) to promote mitophagy.

Methods

The VD rat model was established by two-vessel occlusion. According to the random number table method

60 male SD rats were randomly divided into the sham group

the model group

the donepezil group (0.45 mg/kg)

the

Zishen Huoxue

Formula low-dose group (8.9 g/kg)

the

Zishen Huoxue

Formula medium-dose group (17.8 g/kg)

and the

Zishen Huoxue

Formula high-dose group (35.6 g/kg) (

= 10 rats per group). The drug was given by intragastric administration for 14 days. The learning and memory ability of rats in each group was evaluated by the water maze test. According to the random number table method

50 male SD rats were randomly divided into the sham group

the model group

the

Zishen Huoxue

Formula high-dose group (35.6 g/kg)

the MK-801 (an NMDAR inhibitor)group (1 mg/kg)

and the

Zishen Huoxue

Formula high-dose combined with MK-801 group (35.6 g/kg + 1 mg/kg) (

=10 rats per group). Rats in the MK-801 group were injected intraperitoneally for 7 consecutive days after modeling

while rats in the

Zishen Huoxue

Formula high-dose combined with MK-801 group were treated with

Zishen Huoxue

Formula by gavage combined with intraperitoneal injection of MK-801. The drug was given by intragastric administration for 14 days. Western blotting was used to determine the expression levels of proteins involved in the PTEN-induced kinase 1 (PINK1)/Parkinson protein (Parkin) signaling pathway[PINK1

Parkin

prohibitin 2 (PHB2)

LC3B

and ubiquitin-binding protein p62]. The mean fluorescence intensity of PINK1 and neuron-specific enolase (NSE) in the hippocampus was measured by immunofluorescence. The contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in the hippocampus were measured by enzyme-linked immunosorbent assay. The mRNA expression levels of NR2A and NR2B and the protein level of NR2B in the hippocampus were detected by real-time PCR and Western blotting.

Results

The result of the water maze test showed that compared with the sham group

the escaping incubation period was prolonged (

0.05) and the times of crossing platform were reduced (

0.05) in the model group. Compared with the model group

the escaping incubation period was shortened in the donepezil and

Zishen Huoxue

Formula low-

medium-

and high-dose groups (

0.05

0.01)

and the times of crossing platform were increased in the donepezil and

Zishen Huoxue

Formula medium- and high-dose groups (

0.05

0.01). Further research showed that: compared with the model group

the protein expression levels of Parkin

PINK1

PHB2

and LC3Ⅱ/Ⅰ in the hippocampus of rats in the

Zishen Huoxue

Formula high-dose group were increased (

0.01)

the protein expression of p62 was decreased (

0.01)

the mean fluorescence intensity of PINK1 and NSE was increased (

0.05

0.01)

the content of SOD was increased (

0.01)

and the content of MDA was decreased (

0.01); the protein expression levels of Parkin

PINK1

PHB2

and LC3Ⅱ/Ⅰ in the hippocampus of the MK-801 group were decreased (

0.01)

the protein expression of p62 was increased (

0.01)

and the mean fluorescence intensity of PINK1 and NSE was decreased (

0.05

0.01)

the content of SOD was decreased (

0.05)

and the content of MDA was increased (

0.01). Compared with the MK-801 group

the protein expression levels of Parkin

PINK1

PHB2

and LC3Ⅱ/Ⅰ in the hippocampus of the

Zishen Huoxue

Formula high-dose combined with MK-801 group were increased (

0.01)

the protein expression of p62 was decreased (

0.01)

the mean fluorescence intensity of PINK1 and NSE was increased (

0.05

0.01)

the content of SOD was increased (

0.01)

the content of MDA was decreased (

0.01)

and the mRNA and protein expression levels of NR2B were increased (

0.01).

Conclusion

Zishen Huoxue

Formula can improve oxidative stress injury of hippocampal neurons in VD rats by regulating mitophagy through the NR2B-mediated PINK1/Parkin signaling pathway.

关键词

Keywords

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