Analysis of Neuroprotective Effect of Danggui Shaoyaosan on APPswe/PS1ΔE9 Transgenic Mice Based on Circular RNA Sequencing

Chun-xiang HE; Zhen-yan SONG; Ze LI; Miao YANG; Wen-jing YU; Ping LI; Shao-wu CHENG

doi:10.13422/j.cnki.syfjx.20210104

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Analysis of Neuroprotective Effect of Danggui Shaoyaosan on APP_swe/PS1_Δ_E9 Transgenic Mice Based on Circular RNA Sequencing

Classic Prescriptions | 更新时间：2021-02-09

- Analysis of Neuroprotective Effect of Danggui Shaoyaosan on APP_swe/PS1_Δ_E9 Transgenic Mice Based on Circular RNA Sequencing
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 5, Pages: 16-24(2021)
- 作者机构：
  
  湖南中医药大学中西医结合心脑疾病防治湖南省重点实验室，长沙 410208
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20210104
  CLC： R2-0;R22;R285.5;R289
- Published：05 March 2021，
  
  Published Online：06 January 2021，
  
  Received：03 November 2020，
- 稿件说明：
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贺春香,宋祯彦,李泽等.基于环状RNA测序探讨当归芍药散对APP_swe/PS1_ΔE9转基因小鼠的神经保护作用[J].中国实验方剂学杂志,2021,27(05):16-24.

HE Chun-xiang,SONG Zhen-yan,LI Ze,et al.Analysis of Neuroprotective Effect of Danggui Shaoyaosan on APP_swe/PS1_Δ_E9 Transgenic Mice Based on Circular RNA Sequencing[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(05):16-24.
贺春香,宋祯彦,李泽等.基于环状RNA测序探讨当归芍药散对APP_swe/PS1_ΔE9转基因小鼠的神经保护作用[J].中国实验方剂学杂志,2021,27(05):16-24. DOI： 10.13422/j.cnki.syfjx.20210104.

HE Chun-xiang,SONG Zhen-yan,LI Ze,et al.Analysis of Neuroprotective Effect of Danggui Shaoyaosan on APP_swe/PS1_Δ_E9 Transgenic Mice Based on Circular RNA Sequencing[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(05):16-24. DOI： 10.13422/j.cnki.syfjx.20210104.

摘要

目的

研究环状RNA（circRNA）在当归芍药散对APP

swe

/PS1

ΔE9

双转基因（APP/PS1）小鼠神经保护作用及其机制。

方法

6月龄APP/PS1小鼠20只随机分为模型组和药物组，药物组予以当归芍药散水提液灌胃，10只野生C57BL/6小鼠为正常组，正常组和模型组给予等体积生理盐水，各组连续灌药8周。应用circRNA测序分析当归芍药散干预前后APP/PS1小鼠差异表达circRNA，构建circRNA-miRNA mRNA相互作用网络，实时荧光定量聚合酶链式反应（Real-time PCR）验证cricRNA测序结果，应用蛋白免疫印迹法（Western blot）检测海马区磷脂酰肌醇3-激酶（PI3K），磷酸化PI3K（p-PI3K），蛋白激酶B1（Akt1），p-Akt1，B淋巴细胞瘤-2和Bcl-2-相关X的蛋白质（Bax）的蛋白表达水平，应用免疫组化检测海马区的Caspase-3的蛋白表达水平，应用末端标记（TUNEL）法检测小鼠海马区神经元凋亡水平。

结果

与模型组比较，当归芍药散干预后有90个差异表达的circRNA，其中46个上调，44个下调。与正常组比较，模型组circRNA1398，circRNA1399表达水平下降，miR-103-3p，miR-153-3p，miR-143-3p及miR-143-5p表达水平上调（

0.01），与模型组比较，当归芍药散组circRNA1398，circRNA1399表达水平上调，miR-103-3p，miR-153-3p，miR-143-3p及miR-143-5p表达水平下降（

0.01）。通过circRNA-miRNA mRNA相互作用网络分析circRNA1398和circRNA1399调控Bcl-2和Akt1基因表达。与正常组比较，模型组的p-PI3K，Akt1，p-Akt1和Bcl-2表达量下降（

0.01），Bax和Caspase-3的蛋白表达量升高（

0.01）；与模型组比较，当归芍药散组APP/PS1小鼠海马区的p-PI3K，Akt1，p-Akt1和Bcl-2表达量增加（

0.01），Bax和Caspase-3的蛋白表达量下降（

0.01）。与正常组比较，模型组小鼠海马区神经元凋亡水平上升，细胞凋亡率为（43.76±2.92）%。与模型组比较，当归芍药散干预后细胞凋亡率为（24.64±3.39）%。

结论

当归芍药散可以激活PI3K/Akt通路和抑制APP/PS1小鼠海马区神经元凋亡，发挥神经保护作用，具体机制可能与其上调circRNA1398和circRNA1399表达及其相应miRNA的表达相关。

Abstract

Objective

To investigate the neuroprotective effects of Danggui Shaoyaosan （DSS） on APP

swe

/PS1

ΔE9

transgenic （APP/PS1） mice and its mechanism related to circular RNA （circRNA）.

Method

Totally twenty 6-month-old APP/PS1 mice were divided into model group and DSS group， and 10 C57BL/6 wild-type mice were set as the normal control group. The normal group and model group received the same volume of normal saline， and DSS group received drug by gavage administration， all for 8 weeks. The differentially expressed circRNA of APP/PS1 mice before and after DSS intervention was analyzed by circRNA sequencing to construct circRNA-miRNA mRNA interaction network. The results of cricRNA sequencing were then verified by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of phosphoinositide 3-kinase （PI3K）， p-PI3K， protein kinase B1 （Akt1）， p-Akt1， B lymphocytoma-2 （Bcl-2）， and Bcl-2-Associated X protein （Bax） in the hippocampus were detected by immunoblotting （Western blot）. The protein expression of Caspase-3 in the hippocampus was detected by immunohistochemistry and the level of apoptosis in the hippocampus was detected by the TUNEL method.

Result

Compared with the model group， there were 90 differentially expressed circRNA after intervention with DSS， of which 46 were up-regulated and 44 down-regulated. Compared with the normal group， the expression levels of circRNA1398 and circRNA1399 in the model group decreased， and the expression levels of miR-103-3p， miR-153-3p， miR-143-3p， and miR-143-5p increased. Compared with the model group， the expression levels of circRNA1398 and circRNA1399 in the DSS group were up-regulated， while the expression levels of miR-103-3p， miR-153-3p， miR-143-3p， and miR-143-5p were down-regulated. Compared with the normal group， the expression of p-PI3K， Akt1， p-Akt1， and Bcl-2 in the model group decreased （

0.05，

0.01）， and the expression of Bax and Caspase in the model group increased （

0.01）. Compared with the model group， the expression of p-PI3K， Akt1， p-Akt1， and Bcl-2 in the hippocampus of the DSS group increased （

0.01）， and the protein expression of Bax and Caspase decreased （

0.01）. Compared with the normal group， the apoptosis level in the hippocampus of the model group increased， with an apoptosis rate of （43.76±2.92）%. Compared with the model group， the apoptosis rate of DSS group was （24.64±3.39）%.

Conclusion

DSS can activate PI3K/Akt pathway and inhibit apoptosis in hippocampal neurons of APP / PS1 mice， and play a neuroprotective role. The specific mechanism may be related to the regulation of circRNA1398 and circRNA1399 expression and the corresponding miRNA expression.

关键词

当归芍药散阿尔兹海默病凋亡环状RNA（circRNA）microRNA（miRNA）磷脂酰肌醇3-激酶（PI3K）/蛋白激酶Bα（Akt1）通路

Keywords

Danggui ShaoyaosanAlzheimer's diseaseapoptosiscircular RNA （circRNA）microRNA （miRNA）phosphoinositide 3-kinase （PI3K）/protein kinase B α （Akt1） signaling pathway

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