UQCRC1的高甲基化参与发育期小鼠七氟烷暴露所致的认知功能障碍

LIU Yan; CHEN Yanjuan; ZHANG Min; LONG Zonghong; LI Yu; PEI Jie; WANG Qiuyue; LI Hong

doi:10.16016/j.2097-0927.202411044

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UQCRC1的高甲基化参与发育期小鼠七氟烷暴露所致的认知功能障碍

Neuroscience | 更新时间：2025-04-25

- UQCRC1的高甲基化参与发育期小鼠七氟烷暴露所致的认知功能障碍
- Journal of Army Medical University Vol. 47, Issue 8, Pages: 775-783(2025)
- 作者机构：
  
  陆军军医大学（第三军医大学）第二附属医院麻醉科，重庆
- 作者简介：
  
  LI Hong， E-mail： lh78553@163.com
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(82171265)
- DOI：10.16016/j.2097-0927.202411044
  CLC： R363.21;R749.61;R971.2
- Received：09 November 2024，
  
  Revised：12 February 2025，
  
  Published：30 April 2025
- 稿件说明：
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刘燕,陈彦娟,张敏,等.UQCRC1的高甲基化参与发育期小鼠七氟烷暴露所致的认知功能障碍[J].陆军军医大学学报,2025,47(08):775-783.

LIU Yan,CHEN Yanjuan,ZHANG Min,et al.Hypermethylation of UQCRC1 is involved in cognitive impairment after neonatal sevoflurane exposure[J].J Army Med Univ,2025,47(08):775-783.
刘燕,陈彦娟,张敏,等.UQCRC1的高甲基化参与发育期小鼠七氟烷暴露所致的认知功能障碍[J].陆军军医大学学报,2025,47(08):775-783. DOI：10.16016/j.2097-0927.202411044.

LIU Yan,CHEN Yanjuan,ZHANG Min,et al.Hypermethylation of UQCRC1 is involved in cognitive impairment after neonatal sevoflurane exposure[J].J Army Med Univ,2025,47(08):775-783. DOI：10.16016/j.2097-0927.202411044.

摘要

目的

探究泛醇-细胞色素C还原酶核心蛋白1（ubiquinol-cytochrome C reductase core protein 1，UQCRC1）的甲基化异常是否与发育期小鼠七氟烷暴露所致的认知功能障碍相关。

方法

SPF级健康C57小鼠94只，雌雄不限，6日龄，体质量4~6 g。采用随机数字表法分为7组：对照组（Con组，

=6），七氟烷-6 h组（Sev-6 h组，

=6），七氟烷-24 h组（Sev-24 h组，

=6），对照+DMSO组（Con+DMSO组，

=19），对照+甲基化抑制剂5-氮杂脱氧胞苷（5-aza-2'-deoxycytidine，5-AZA）组（Con+5-AZA组，

=19），七氟烷+DMSO组（Sev+DMSO组，

=19），七氟烷+5-A

ZA组（Sev+5-AZA组，

=19）。出生后第6~8天Sev-6 h组和Sev-24 h组每天进行3%七氟烷暴露（吸入氧浓度100%，流量2 L/min，2 h/d）；Con组进行100%的氧气暴露（流量2 L/min，2 h/d）。Con+5-AZA、Con+DMSO组每天行100%氧气暴露前30 min腹腔分别注射1 mg/kg 5-AZA或等体积DMSO；Sev+5-AZA、Sev+DMSO组每天行3%七氟烷暴露前30 min腹腔分别注射1 mg/kg 5-AZA或等体积DMSO。在最后1次七氟烷暴露后6 h和24 h时，Con、Sev-6 h、Sev-24 h组处死6只小鼠进行生化分析；在最后一次七氟烷暴露后24 h时，Con+DMSO、Con+5-AZA、Sev+DMSO、Sev+5-AZA组采用随机数字表法抽取6只小鼠处死进行生化分析，抽取3只小鼠处死进行形态学分析，于小鼠出生后第30~33天对每组剩余的10只小鼠进行行为学检测，在最后一次行为学测试后24 h处死小鼠。采用RT-qPCR、Western blot检测小鼠海马组织UQCRC1、DNA甲基转移酶（DNA methyltransferases， Dnmts）和甲基CpG结合蛋白2（methyl CpG binding protein 2， Mecp2）表达水平，采用免疫荧光染色观察UQCRC1在小鼠海马的分布及表达，采用亚硫酸氢盐转化后测序（bisulfite sequencing PCR，BSP）检测UQCRC1启动子区甲基化水平，采用旷场、新物体和Y迷宫实验于小鼠出生后第30~33天评估小鼠认知功能。

结果

与Con组相比，Sev-6 h组和Sev-24 h组UQCRC1 mRNA和蛋白表达水平均显著下调（

0.05），Dnmts mRNA表达显著上调（

0.05）；且Sev-24 h组UQCRC1启动子区甲基化水平显著增加（

0.05）。与Sev+DMSO组相比，Sev+5-AZA组UQCRC1 mRNA和蛋白表达水平显著回升（

0.05），小鼠的认知功能障碍明显好转（

0.05）。

结论

增加UQCRC1启动子区的甲基化水平而下调UQCRC1 mRNA和蛋白的表达，可能是新生小鼠发育期反复暴露于七氟烷后引起小鼠认知功能障碍的重要机制之一。

Abstract

‍Objective‍　‍To investigate whether aberrant DNA methylation of ubiquinol-cytochrome C reductase core protein 1 (UQCRC1) is related to cognitive impairment caused by neonatal sevoflurane exposure. Methods‍　‍A total of 94 SPF C57 mice of either sex

aged 6 d

and weighing 4~6 g

were randomly divided into 7 groups: control group (Con

=6)

sevoflurane-6 and -24 h exposure groups (Sev-6 and -24 h

=6)

control+DMSO group (Con+DMSO

=19)

control+5-aza-2'-deoxycytidine (5-AZA

methylation inhibitor) group (Con+5-AZA

=19)

sevoflurane+DMSO group (Sev+DMSO group

=19)

and sevoflurane+5-AZA group (Sev+5-AZA group

=19). From 6 to 8 d after birth

the mice of the Sev-6 and -24 h exposure group

s were exposed to 3% sevoflurane daily (with 97% oxygen

2 L/min

2 h per day)

while those from the Con groups were given exposure of 100% oxygen (2 L/min

2 h per day). For the mice of the 5-AZA and DMSO groups

1 mg/kg of 5-AZA or an equal volume of DMSO was injected intraperitoneally 30 min before daily exposure. In 6 and 24 h after the last exposure to sevoflurane

6 mice from the Con

Sev-6 h

and Sev-24 h groups were euthanized for biochemical analysis

and in 24 h post-exposure

6 mice from the Con+DMSO

Con+5-AZA

Sev+DMSO

and Sev+5-AZA groups were randomly selected for biochemical analysis

while another 3 mice from above each group were also randomly selected for morphological analysis. The remaining 10 mice in these groups underwent behavioral testing (open field test

novel object test

and Y-maze test) at 30~33 d after birth to assess cognitive function

and were euthanized in 24 h after the final behavioral test. RT-qPCR and Western blotting were used to detect the hippocampal expression of UQCRC1

DNA methyltransferases (Dnmts)

and methyl CpG binding protein 2 (Mecp2) at mRNA and protein levels

respectively. Immunofluorescence assay was employed to observe the distribution and expression of UQCRC1 in the hippocampus. Bisulfite sequencing PCR (BSP) was applied to measure the methylation in the UQCRC1 promoter region. Results‍　‍Compared with the Con group

the mRNA and protein levels of UQCRC1 were down-regulated (

0.05)

and the mRNA level of Dnmts was up-regulated (

0.05) in both the Sev-6 h and Sev-24 h exposure groups

while the methylation level in the UQCRC1 promoter region was enhanced in the Sev-24 h exposure group (

0.05). Additionally

the Sev+5-AZA group had obviously increased mRNA and protein levels of UQCRC1 (

0.05)

and notable improvement in cognitive impairment (

0.05) when compared with the Sev+DMSO group. Conclusion‍　‍Hypermethylation of UQCRC1 promoter region and thus down-

regulating its mRNA and protein expression might be the main mechanism by which repeated neonatal sevoflurane exposure induces cognitive impairment later in life.

关键词

Keywords

references

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