Mechanism of silencing METTL3-mediated mitophagy in cerebral microvascular endothelial cells of mice with sepsis

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Mechanism of silencing METTL3-mediated mitophagy in cerebral microvascular endothelial cells of mice with sepsis

Basic Research | 更新时间：2025-06-17

- Mechanism of silencing METTL3-mediated mitophagy in cerebral microvascular endothelial cells of mice with sepsis
- JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY Vol. 34, Issue 6, Pages: 465-470(2025)
- 作者机构：
  
  1.福建医科大学附属第一医院麻醉科，福建福州 350000
  2.福建医科大学麻醉学研究所，福建福州 350000
- 作者简介：
- 基金信息：
- DOI：10.11659/jjssx.09E024027
  CLC： R631+.2
- Received：05 September 2024，
  
  Published：25 June 2025
- 稿件说明：
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林博,叶馨怡,李羽茜.沉默METTL3促进脓毒症小鼠脑微血管内皮细胞线粒体自噬的机制研究[J].局解手术学杂志,2025,34(06):465-470.

LIN Bo,YE Xin-yi,LI Yu-qian.Mechanism of silencing METTL3-mediated mitophagy in cerebral microvascular endothelial cells of mice with sepsis[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2025,34(06):465-470.
林博,叶馨怡,李羽茜.沉默METTL3促进脓毒症小鼠脑微血管内皮细胞线粒体自噬的机制研究[J].局解手术学杂志,2025,34(06):465-470. DOI： 10.11659/jjssx.09E024027.

LIN Bo,YE Xin-yi,LI Yu-qian.Mechanism of silencing METTL3-mediated mitophagy in cerebral microvascular endothelial cells of mice with sepsis[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2025,34(06):465-470. DOI： 10.11659/jjssx.09E024027.

摘要

目的

2

探讨甲基转移酶样3（METTL3）对脓毒症小鼠脑微血管内皮细胞中Sigma-1受体（Sigma-1R）mRNA的m6A甲基化和线粒体自噬的调控作用。

方法

2

将12只成年雄性C57BL/6J小鼠随机分为假手术组（不进行盲肠结扎穿孔）和脓毒症组（进行盲肠结扎穿孔术），每组6只。分别使用m6A Dot blot、RT-qPCR和Western blot检测脑微血管组织中的m6A甲基化水平、METTL3和Sigma-1R mRNA及蛋白相对表达量。体外培养bEnd.3细胞株，分为对照组（正常培养）、模型组[脂多糖（LPS）诱导体外脓毒症]、si-NC组（LPS处理联合转染si-NC）、si-METTL3组（LPS处理联合转染si-METTL3）。使用m6A Dot blot检测各组细胞中m6A甲基化水平；RT-qPCR检测METTL3和Sigma-1R mRNA的表达；Western blot检测METTL3、Sigma-1R以及自噬相关蛋白Bnip3、LC3B和Beclin1的表达。用m6A 免疫共沉淀-qPCR检测各组细胞中Sigma-1R的m6A甲基化水平。用JC-1探针法检测各组细胞线粒体膜电位，ATP检测试剂盒检测线粒体ATP含量。

结果

2

与假手术组比较，脓毒症组小鼠脑微血管组织中m6A甲基化水平和METTL3的表达增加（

P

<

0.05），而Sigma-1R的表达降低（

P

<

0.05）。与对照组比较，模型组和si-NC组细胞中m6A甲基化水平、METTL3表达和Sigma-1R mRNA的m6A甲基化水平增加，Sigma-1R表达、线粒体膜电位正常率和增加率降低，ATP含量减少，Bnip3、LC3B、Beclin1蛋白表达均下调，差异均有统计学意义（

P

<

0.05）。与si-NC组比较，si-METTL3组细胞中m6A甲基化水平、METTL3表达和Sigma-1R mRNA的m6A甲基化水平降低，Sigma-1R表达、线粒体膜电位正常率和增加率升高，ATP含量增加，Bnip3、LC3B、Beclin1蛋白表达均上调，差异均有统计学意义（

P

<

0.05）。

结论

2

沉默METTL3抑制Sigma-1R mRNA的m6A甲基化并促进脓毒症小鼠脑微血管内皮细胞的线粒体自噬。

Abstract

Objective

2

To explore the regulatory effect of methyltransferase-like 3 （METTL3） on m6A methylation of Sigma-1 receptor （Sigma-1R） mRNA and mitophagy in cerebral microvascular endothelial cells of sepsis mice.

Methods

2

Twelve adult male C57BL/6J mice were randomly divided into the Sham group （without cecal ligation or puncture） and the sepsis group （received cecal ligation and puncture），with 6 mice in each group. m6A methylation levels，and relative expression of METTL3 and Sigma-1R mRNA and protein in cerebral microvascular tissues were detected by m6A Dot blot，RT-qPCR，and Western blot，respectively.The bEnd.3 cells were cultured in vitro and divided into the control group （normal culture），model group ［lipopolysaccharide （LPS）-induced in vitro sepsis］，si-NC group （LPS treatment combined with si-NC transfection ），and si-METTL3 group （LPS treatment combined with si-METTL3 transfection）. m6A Dot blot was used to detect m6A methylation levels of cells in each group；RT-qPCR was used to detect the mRNA expression of METTL3 and Sigma-1R；and Western blot was used to detect the expression of METTL3，Sigma-1R，and autophagy-related proteins of Bnip3，LC3B，and Beclin1. m6A chromatin immunoprecipitation-qPCR was used to detect m6A methylation level of Sigma-1R of cells in each group. JC-1 probe was used to detect the mitochondrial membrane potential，and the ATP assay kit was used to detect the mitochondrial ATP content.

Results

2

Compared with the Sham group，the sepsis group showed increased m6A methylation level and METTL3 expression （

P

<

0.05），and decreased Sigma-1R expression in cerebral microvascular tissues（

P

<

0.05）. Compared with the control group，m6A methylation level，METTL3 expression and m6A methylation level of Sigma-1R mRNA increased，the expression of Sigma-1R，the normal rate and increase rate of mitochondrial membrane potential，and the ATP content decreased，and the protein expression of Bnip3，LC3B and Beclin1 downregulated in the model group and the si-NC group，with statistically significant differences （

P

<

0.05）. Compared w

ith the si-NC group，m6A methylation level，METTL3 expression and m6A methylation level of Sigma-1R mRNA decreased，the expression of Sigma-1R，the normal rate and increase rate of mitochondrial membrane potential elevated，the ATP content increased，and the protein expression of Bnip3，LC3B and Beclin1 upregulated in the si-METTL3 group，with statistically significant differences （

P

<

0.05）.

Conclusion

2

Silencing METTL3 can inhibit m6A methylation of Sigma-1R mRNA and promote the mitophagy in cerebral microvascular endothelial cells of septic mice.

关键词

Keywords

references

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