Abstract

Background

Liver fibrosis can progress to cirrhosis and hepatic carcinoma without treatment. CircDCBLD2 was found to be downregulated in liver fibrosis. However, the precise underlying mechanism requires further investigation.

Methods

qRT-PCR, Western blot, and immunohistochemistry assays were used to detect the related molecule levels. HE, Masson’s trichrome, and Sirius Red staining were used to assess the pathological changes in mice’s liver tissues. Flow cytometric analysis and commercial kit were used to assess the levels of lipid reactive oxygen species (ROS), malonaldehyde (MDA), glutathione (GSH), and iron. Cell viability was assessed by MTT. Immunoprecipitation was used to study the ubiquitination of PARK7. Mitophagy was determined by immunostaining and confocal imaging. RIP and Co-IP assays were used to assess the interactions of circDCBLD2/HuR, HuR/STUB1, and STUB1/PARK7. Fluorescence in situ hybridization and immunofluorescence staining were used to assess the co-localization of circDCBLD2 and HuR.

Results

CircDCBLD2 was downregulated, whereas PARK7 was upregulated in liver fibrosis. Ferroptosis activators increased circDCBLD2 while decreasing PARK7 in hepatic stellate cells (HSCs) and mice with liver fibrosis. CircDCBLD2 overexpression reduced cell viability and GSH, PARK7, and GPX4 expression in erastin-treated HSCs while increasing MDA and iron levels, whereas circDCBLD2 knockdown had the opposite effect. CircDCBLD2 overexpression increased STUB1-mediated PARK7 ubiquitination by promoting HuR-STUB1 binding and thus increasing STUB1 mRNA stability. PARK7 overexpression or HuR knockdown reversed the effects of circDCBLD2 overexpression on HSC activation and ferroptosis. CircDCBLD2 reduced liver fibrosis in mice by inhibiting PARK7.

Conclusion

CircDCBLD2 overexpression increased PARK7 ubiquitination degradation by upregulating STUB1 through its interaction with HuR, inhibiting HSC activation and promoting HSC ferroptosis, ultimately enhancing liver fibrosis.

中文翻译：

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CircDCBLD2 通过促进 STUB1 介导的 PARK7 泛素化降解来调节铁死亡，从而减轻肝纤维化

摘要

背景

如果不治疗，肝纤维化可进展为肝硬化和肝癌。 CircDCBLD2被发现在肝纤维化过程中下调。然而，确切的潜在机制需要进一步研究。

方法

采用qRT-PCR、Western blot和免疫组织化学检测相关分子水平。采用HE、马森三色和天狼星红染色评估小鼠肝组织的病理变化。使用流式细胞术分析和商业试剂盒评估脂质活性氧（ROS）、丙二醛（MDA）、谷胱甘肽（GSH）和铁的水平。通过MTT评估细胞活力。使用免疫沉淀法研究 PARK7 的泛素化。通过免疫染色和共聚焦成像测定线粒体自噬。 RIP 和 Co-IP 测定用于评估 circDCBLD2/HuR、HuR/STUB1 和 STUB1/PARK7 的相互作用。使用荧光原位杂交和免疫荧光染色来评估 circDCBLD2 和 HuR 的共定位。

结果

在肝纤维化过程中，CircDCBLD2 下调，而 PARK7 上调。在肝星状细胞 (HSC) 和肝纤维化小鼠中，铁死亡激活剂增加了 circDCBLD2，同时减少了 PARK7。 CircDCBLD2过表达降低了erastin处理的HSC中的细胞活力以及GSH、PARK7和GPX4表达，同时增加了MDA和铁水平，而circDCBLD2敲低则具有相反的效果。 CircDCBLD2 过表达通过促进 HuR-STUB1 结合增加 STUB1 介导的 PARK7 泛素化，从而增加 STUB1 mRNA 稳定性。 PARK7 过表达或 HuR 敲低逆转了 circDCBLD2 过表达对 HSC 激活和铁死亡的影响。 CircDCBLD2 通过抑制 PARK7 减少小鼠肝纤维化。

结论

CircDCBLD2过表达通过与HuR相互作用上调STUB1，增加PARK7泛素化降解，抑制HSC活化并促进HSC铁死亡，最终增强肝纤维化。