抑制 UBE2O 通过上调 BMAL1 减轻脓毒症急性肺损伤

Inhibiting UBE2O attenuates sepsis⁃induced acute lung injury by upregulating BMAL1 expression

摘要

目的：探讨泛素结合酶E2O（UBE2O）对脓毒症急性肺损伤（SI⁃ALI）的作用及机制。方法：动物实验部分，将雄性C57BL/6小鼠随机分为假手术（Sham）组、盲肠结扎穿孔（CLP）组和UBE2O敲低（CLP+AAV1）组（通过气管内注射AAV1⁃UBE2O敲低腺病毒构建小鼠UBE2O敲低模型），CLP术后24 h处死小鼠，取肺组织进行检测，HE染色观察肺组织病理学改变；Smith评分评估肺损伤程度；计算肺组织湿干质量比（W/D）；qPCR及Western Blot检测UBE2O在肺组织中的表达；ELISA检测肺组织中炎症因子IL⁃1β、TNF⁃α、IL⁃6的含量。细胞实验部分，将小鼠单核巨噬细胞白血病细胞（RAW264.7）分为PBS组、LPS组和LPS+si⁃UBE2O组，分别通过qPCR和Western Blot实验检测UBE2O及BMAL1的mRNA和蛋白表达水平；ELISA检测细胞上清液中炎症因子水平。结果：动物实验结果表明，相比于Sham组，CLP组小鼠的肺组织呈明显病理损伤，UBE2O表达显著上调，并伴随组织炎症因子水平升高；与CLP组相比，UBE2O敲低组肺损伤减轻，炎症因子水平下降；在细胞实验中，LPS刺激RAW264.7细胞后，与PBS组相比，UBE2O表达及炎症因子水平均显著升高；在LPS刺激基础上抑制UBE2O可使BMAL1表达增加，并显著减轻巨噬细胞炎症反应。结论：抑制UBE2O通过上调BMAL1的表达减轻脓毒症急性肺损伤。

Abstract

Objective: To investigate the role and mechanism of the ubiquitin⁃conjugating enzyme E2O (UBE2O) in sepsis⁃induced acute lung injury (SI⁃ALI). Methods: In vivo, male C57BL/6 mice were randomly divided into three groups: sham, cecal ligation and puncture (CLP), and CLP+AAV1 (UBE2O⁃knockdown via intratracheal AAV1 injection). Mice were euthanized 24 hours post⁃CLP, and lung tissues were collected for histopathological evaluation (HE staining, Smith score, wet/dry weight ratio) and molecular analyses (qPCR and Western Blot for UBE2O expression; ELISA for IL⁃1β, TNF⁃α, and IL⁃6 levels). In vitro, RAW264.7 macrophages were grouped into PBS, LPS, and LPS+si⁃UBE2O treatments. UBE2O and BMAL1 expression was assessed by qPCR and Western Blot, and inflammatory cytokines in supernatants were measured via ELISA. Results: Compared with the Sham group, the CLP group showed significantly aggravated lung injury, elevated UBE2O expression, and increased inflammatory cytokines. In contrast, UBE2O knockdown (CLP+AAV1) alleviated lung damage and reduced inflammatory mediator levels. In vitro, LPS stimulation upregulated UBE2O and promoted cytokine release in RAW264.7 cells. However, UBE2O inhibition further increased BMAL1 expression and attenuated the inflammatory response in LPS⁃treated macrophages. Conclusion: Inhibition of UBE2O attenuates sepsis⁃induced acute lung injury through upregulation of BMAL1.

关键词

脓毒症 / 急性肺损伤 / 泛素结合酶E2O / BMAL1

Key words

Sepsis / Acute Lung Injury / Ubiquitin⁃Conjugating Enzyme E2O / BMAL1

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基金资助

新发突发与重大传染病防控国家科技重大专项(2025ZD01902600)

国家重点研发计划(2023YFC2308404)

国家自然科学基金面上项目(82272226)

国家自然科学基金面上项目(82572461)

国家自然科学基金青年科学基金项目（C类）(82502662)