基于H4K16ac介导的细胞自噬探讨电针血清对脑缺血后神经元的保护机制

徐疏影; 沈燕; 彭拥军; 杨沙; 李文倩

doi:10.3969/j.issn.1006-2157.2021.04.012

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基于H4K16ac介导的细胞自噬探讨电针血清对脑缺血后神经元的保护机制

针灸 | 更新时间：2021-06-03

- 基于H4K16ac介导的细胞自噬探讨电针血清对脑缺血后神经元的保护机制
- Protective mechanism of electroacupuncture serum on neurons after cerebral ischemia based on H4K16AC-mediated autophagy
- 北京中医药大学学报 2021年44卷第4期页码：366-373
- 作者机构：
  
  1.南京中医药大学附属医院　江苏　 210029
  2.天津中医药大学第一附属医院国家中医针灸临床医学研究中心
- 作者简介：
  
  徐疏影，女，在读博士生
  #通信作者：彭拥军，男，博士，主任医师，博士生导师，主要研究方向：针灸治疗脑血管疾病及变态反应性疾病，E-mail：pengyongjun2004@126.com
- 基金信息：
  
  国家自然科学基金项目(81973932;81574060;81001556);天津市自然科学基金重点项目(18JCZDJC99200);长江学者和创新团队发展计划项目(IRT1167)
- DOI：10.3969/j.issn.1006-2157.2021.04.012
  中图分类号： R245
- 收稿：2020-05-18，
  
  纸质出版：2021-04-30
- 稿件说明：
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徐疏影, 沈燕, 彭拥军, 等. 基于H4K16ac介导的细胞自噬探讨电针血清对脑缺血后神经元的保护机制[J]. 北京中医药大学学报, 2021,44(4):366-373.

Shuying Xu, Yan Shen, Yongjun Peng, et al. Protective mechanism of electroacupuncture serum on neurons after cerebral ischemia based on H4K16AC-mediated autophagy[J]. Journal of Beijing University of traditional Chinese Medicine, 2021, 44(4): 366-373.
徐疏影, 沈燕, 彭拥军, 等. 基于H4K16ac介导的细胞自噬探讨电针血清对脑缺血后神经元的保护机制[J]. 北京中医药大学学报, 2021,44(4):366-373. DOI： 10.3969/j.issn.1006-2157.2021.04.012.

Shuying Xu, Yan Shen, Yongjun Peng, et al. Protective mechanism of electroacupuncture serum on neurons after cerebral ischemia based on H4K16AC-mediated autophagy[J]. Journal of Beijing University of traditional Chinese Medicine, 2021, 44(4): 366-373. DOI： 10.3969/j.issn.1006-2157.2021.04.012.

摘要

目的

从组蛋白H4第16位赖氨酸的乙酰化(H4K16ac)对细胞自噬调控的角度，研究电针血清对脑缺血后神经元的保护机制。

方法

电针血清的制备：建立改良的大鼠急性局灶性脑缺血再灌注模型，在造模成功后5 min和16 h进行电针干预，针刺人中、百会穴，并接电针治疗仪治疗30 min。第2次电针治疗后7 h取大鼠血清备用。细胞实验：将大鼠大脑皮质神经元细胞分为对照组、模型组、电针组、组蛋白乙酰转移酶MOF(hMOF)抑制剂组与细胞沉默调节蛋白1(Sirt1)抑制剂组，除对照组外，其余各组剥夺氧、糖3 h后再复氧、复糖24 h，并于复氧、复糖的同时给予2%电针血清、15 g/L的hMOF siRNA及8 g/L的Sirt1 siRNA处理。CCK-8检测细胞处理48 h后细胞增殖情况；流式细胞术检测细胞处理48 h后细胞凋亡情况；Western blot检测细胞中hMOF、Sirt1、组蛋白H4第16位赖氨酸的乙酰化(H4K16ac)、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、自噬相关蛋白Beclin1表达；qPCR检测hMOF、Sirt1、Beclin1 mRNA表达；染色质免疫沉淀技术(ChIP)检测各组神经元细胞的H4K16ac在自噬靶基因Beclin1启动子区的结合程度。

结果

与模型组相比，电针干预可使细胞增殖活性提高(

0.05)，细胞凋亡率下降(

0.05)。与模型组比较，电针组和hMOF抑制组剂均可降低hMOF和H4K16ac蛋白的表达(

0.05)，升高Sirt1、LC3-II和Beclin1的表达(

0.05)；而与Sirt1抑制剂组差异无统计学意义(

0.05)。qPCR结果显示，与模型组比较，电针组和hMOF抑制组剂均可抑制hMOF mRNA表达(

0.05)，而Sirt1和Beclin1 mRNA表达上调(

0.05)。且与模型组相比，电针组在自噬靶基因Beclin1启动子区域中H4K16ac的富集量增加(

0.05)。

结论

电针血清对糖－氧剥夺再灌注损伤神经元细胞具有保护作用，其抗脑缺血再灌注损伤的作用可能是电针通过调节组蛋白H4K16ac的表达进而调控细胞自噬，从而减轻脑缺血再灌注损伤，发挥对神经细胞的保护作用。

Abstract

Objective

To study the protective mechanism of electroacupuncture serum on neurons after cerebral ischemia from the perspective of autophagy regulation by acetylation of histone H4 16 lysine (H4K16ac).

Methods

1) Preparation of electroacupuncture serum: The improved rat model of acute focal cerebral ischemia reperfusion was established. Electroacupuncture intervention was performed 5 at min and 16 h after the model was successfully established. Acupuncture was performed at the points of Renzhong (GV 26) and Baihui (GV 20)

and electroacupuncture treatment was applied for 30 mins. The samples were collected 7 h after the second electroacupuncture treatment

and the serum of the rats was used for reserve. 2) Cell experiment: The rat cerebral cortex neuron cells were divided into control group

model group

electroacupuncture group

hMOF inhibitor group and Sirt1 inhibitor group. All cells were deprived of oxygen and sugar for 3 h and then re-oxygenated and re-glycated for 24 h except the control group. Meanwhile

all these cells were treated with 2% electroacupuncture serum

15 g/L hMOF siRNA and 8 g/L Sirt1 siRNA. Cell proliferation was measured by using CCK-8 after 48 h of cell treatment. Cell apoptosis was measured by using flow cytometry after 48 h treatment. hMOF

Sirt1

H4K16ac

LC3-Ⅱ

and Beclin1 protein expression in rat cerebral cortex neurons were measured with Western blot assay. The expression of hMOF

Sirt1 and Beclin1 mRNA was detected by using qPCR. ChIP was used to detect the binding degree of H4K16ac of neurons in each group in the Beclin1 promoter region of autophagy target gene.

Results

Compared with the model group

the cell proliferation activity was significantly increased (

0.05)

and the cell apoptosis rate was significantly decreased (

0.05). Compared with model group

both electroacupuncture and hMOF inhibitor decreased the expression of hMOF and H4K16ac proteins (

0.05)

and increased the expression of Sirt1

LC3-II and Beclin1 (

0.05). There was no significant difference between Sirt1 inhibitor group and model group (

0.05). According to qPCR results

compared with model group

both electroacupuncture and hMOF inhibitor could inhibit hMOF mRNA expression (

0.05)

while Sirt1 and Beclin1 mRNA expression were up-regulated (

0.05). Compared with the model group

the enrichment of H4K16ac in the Beclin1 promoter region of autophagy target gene was significantly increased in the electroacupuncture group (

0.05).

Conclusion

Electroacupuncture serum has a protective effect on OGD/R injured neurons

and its anti-cerebral ischemia reperfusion injury may be that electroacupuncture regulates autophagy by regulating the expression of hitin H4K16ac

thereby reducing cerebral ischemia reperfusion injury and playing a protective effect on nerve cells.

关键词

Keywords

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