C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白通过抑制内质网自噬并增强内质网-线粒体互作促进肝细胞焦亡

doi:10.12122/j.issn.1673-4254.2026.07.18

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (7): 1648-1659.doi: 10.12122/j.issn.1673-4254.2026.07.18

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C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白通过抑制内质网自噬并增强内质网-线粒体互作促进肝细胞焦亡

郭晓喻¹(), 吴家羽¹, 韩冰², 杨婷¹, 殷欢欢¹, 熊津怡¹, 谢汝佳¹()

^1.贵州医科大学基础医学院病理生理学教研室
^2.贵州省常见慢性疾病发病机制及防治全省重点实验室，贵州贵安新区 561113

收稿日期:2025-11-24 出版日期:2026-07-20 发布日期:2026-07-20
通讯作者: 谢汝佳 E-mail:1558009265@qq.com;592153968@qq.com
作者简介:郭晓喻，在读硕士研究生，E-mail: 1558009265@qq.com
基金资助:
国家自然科学基金(82260127);贵州省基础研究计划（自然科学）面上项目(黔科合基础MS2025-542);贵州省基础研究计划（自然科学）面上项目(黔科合基础MS2025-544)

CHOP promotes hepatocyte pyroptosis by downregulating endoplasmic reticulophagy and enhancing endoplasmic reticulum-mitochondria interaction

Xiaoyu GUO¹(), Jiayu WU¹, Bing HAN², Ting YANG¹, Huanhuan YIN¹, Jinyi XIONG¹, Rujia XIE¹()

^1.Department of Pathophysiology, School of Basic Medical Sciences, Guizhou Medical University
^2.Guizhou Provincial Key Laboratory of Pathogenesis and Prevention of Common Chronic Diseases, Guian 561113, China

Received:2025-11-24 Online:2026-07-20 Published:2026-07-20
Contact: Rujia XIE E-mail:1558009265@qq.com;592153968@qq.com
Supported by:
National Natural Science Foundation of China(82260127)

摘要/Abstract

摘要：

目的探讨C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白（CHOP）在二硫苏糖醇（DTT）诱导的大鼠正常肝细胞（BRL-3A）内质网自噬（ER-phagy）中的作用，并分析其通过内质网-线粒体互作调控肝细胞焦亡的潜在机制。方法 DTT作用于BRL-3A细胞0、12、36 h，Western blotting检测CHOP蛋白、内质网自噬、线粒体-内质网接触位点（MERCs）及焦亡相关蛋白的变化；DTT处理BRL-3A细胞36 h，采用染色质免疫共沉淀（ChIP）技术检测CHOP在ATG5、ATG12、LC3基因启动子区域的结合情况；使用自噬晚期抑制剂巴佛洛霉素A1（BafA1）预处理BRL-3A细胞，通过Hoechst 33342与碘化丙啶（PI）双染法测定细胞死亡水平，观察内质网自噬被抑制后对DTT诱导的肝细胞死亡的影响；采用过表达CHOP的慢病毒感染BRL-3A细胞，Western blotting检测CHOP基因过表达后各组细胞中内质网自噬、MERCs相关蛋白及焦亡相关蛋白的变化；免疫荧光双染观察过表达CHOP对内质网标志蛋白与自噬体标志蛋白共定位水平的影响；通过细胞器荧光探针，观察过表达CHOP对内质网与溶酶体及内质网与线粒体共定位水平的影响；检测过表达CHOP后线粒体内Ca²⁺浓度、线粒体膜电位及线粒体中焦亡蛋白GSDMD-N的变化。结果相较于0 h组，DTT处理BRL-3A细胞12 h后，CHOP蛋白表达无显著变化，而内质网自噬、MERCs相关蛋白及焦亡相关蛋白水平均上调；至36 h时，CHOP蛋白表达升高，MERCs相关蛋白及焦亡相关蛋白水平持续增加，而内质网自噬水平则有所下降（P<0.05）；ChIP结果显示，DTT处理细胞36 h后CHOP在自噬相关基因启动子区的富集减少（P<0.05）；采用自噬抑制剂预处理BRL-3A细胞后能显著增强DTT诱导的细胞死亡。过表达CHOP后，内质网自噬相关蛋白水平下调，而MERCs相关蛋白及焦亡相关蛋白水平上调（P<0.05）；激光共聚焦显微镜观察发现，过表达CHOP后内质网与自噬体及溶酶体的共定位区域减少，而内质网与线粒体的共定位区域增多并且线粒体内钙离子水平及GSDMD-N蛋白表达增加（P<0.05）；JC-1荧光染色实验证实，过表达CHOP后，线粒体跨膜电位降低（P<0.05）。结论 CHOP蛋白通过抑制内质网自噬并增强内质网-线粒体之间的相互作用，导致从内质网转移到线粒体的Ca²⁺增多，进而促进肝细胞焦亡。

关键词: C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白, 内质网自噬, 内质网-线粒体相互作用, 肝细胞焦亡

Abstract:

Objective To investigate the role of C/EBP homologous protein (CHOP) in dithiothreitol (DTT)‑induced endoplasmic reticulophagy in murine hepatocytes cells and its mechanism for regulating hepatocytes pyroptosis through endoplasmic reticulum (ER)‑mitochondria interactions. Methods The changes in CHOP, endoplasmic reticulophagy, mitochondrial-ER contact sites (MERCs)-associated protein, and pyroptosis-associated proteins in DTT-treated BRL-3A cells were examined using Western blotting, and CHOP enrichment in the promoter regions of ATG5, ATG12 and LC3 genes was detected using ChIP assay. The effect of bafilomycin A1 (an autophagy inhibitor) pretreatment on DTT-induced BRL-3A cell death was assessed using Hoechst 33342 and propidium iodide staining. The effects of lentivirus-mediated CHOP overexpression in the hepatocytes on ER-phagy, MERCs-associated proteins, pyroptosis-associated proteins, and colocalizations of ER with the autophagosomes, lysosomes and mitochondria were examined using Western blotting and immunofluorescence double staining; the changes in intramitochondrial Ca²⁺, mitochondrial membrane potential and intramitochondrial GSDMD-N levels were also analyzed. Results DTT treatment for 12 h did not significantly affect CHOP protein expression but significantly upregulated ER-phagy, MERCs-associated proteins, and pyroptosis-associated proteins in BRL-3A cells. DTT treatment for 36 h markedly increased CHOP protein expression and further increased MERCs-associated proteins and pyroptosis-associated protein levels, but moderately decreased ER-phagy levels. DDT treatment significantly downregulated CHOP enrichment in the promoter regions of ATG5, ATG12 and LC3 genes in BRL-3A cells. Pretreatment with bafilomycin A1 significantly increased DTT-induced BRL-3A cell death. CHOP overexpression in BRL-3A cells downregulated the levels of endoplasmic reticulophagy-related proteins, upregulated MERCs- and pyroptosis-related proteins, and reduced the colocalization of ER with autophagosomes and lysosomes, and increased its colocalization with the mitochondria, resulting in also elevated intramitochondrial Ca²⁺ levels and GSDMD-N protein and decreased mitochondrial transmembrane potential. Conclusion CHOP promotes hepatocytes pyroptosis by suppressing ER-phagy and enhancing ER-mitochondrial interactions, which leads to increased Ca²⁺ shuttling from the ER to the mitochondria.

Key words: C/EBP homologous protein, endoplasmic reticulophagy, endoplasmic reticulum-mitochondria interactions, hepatocyte pyroptosis

郭晓喻, 吴家羽, 韩冰, 杨婷, 殷欢欢, 熊津怡, 谢汝佳. C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白通过抑制内质网自噬并增强内质网-线粒体互作促进肝细胞焦亡[J]. 南方医科大学学报, 2026, 46(7): 1648-1659.

Xiaoyu GUO, Jiayu WU, Bing HAN, Ting YANG, Huanhuan YIN, Jinyi XIONG, Rujia XIE. CHOP promotes hepatocyte pyroptosis by downregulating endoplasmic reticulophagy and enhancing endoplasmic reticulum-mitochondria interaction[J]. Journal of Southern Medical University, 2026, 46(7): 1648-1659.

图/表 9

图1 DTT处理细胞后CHOP蛋白、内质网自噬、线粒体-内质网接触位点相关蛋白及细胞焦亡相关蛋白的表达变化

Fig.1 Expression of CHOP protein (A) and proteins associated with ER-phagy (B), mitochondrial-ER contact sites (MERCs) (C) and pyroptosis (D) in DTT-treated BRL-3A cells. **P<0.01, ***P<0.001 vs 0 h; #P<0.05,##P<0.01, ###P<0.001 vs 12 h.

图2 激光共聚焦显微镜观察自噬抑制对DTT诱导的细胞死亡的影响

Fig.2 Effect of bafilomycin A1 (BafA1) pretreatment on DTT-induced BRL-3A cell death (Laser confocal microscopy, scale bar=50 µm).

图3 ChIP检测ATG5、ATG12和LC3基因启动子区CHOP富集水平

Fig.3 Enrichment of CHOP in the promoter regions of ATG5, ATG12 and LC3 genes determined using ChIP assay. **P<0.01, ***P<0.001 vs 0 mmol/L DTT group.

图4 CHOP慢病毒感染BRL-3A 细胞后对CHOP mRNA和蛋白表达水平的影响

Fig.4 Effect of CHOP lentiviral infection on CHOP mRNA and protein expressions in BRL-3A cells. **P<0.01, ***P<0.001 vs LV-NC group.

图5 过表达CHOP对BRL-3A细胞中内质网自噬水平的影响

Fig.5 Effect of CHOP overexpression on the level of endoplasmic reticulophagy in BRL-3A cells (Scale bar=50 µm). **P<0.01, ***P<0.001 vs DTT group and LV-NC+DTT group.

图6 过表达CHOP对线粒体-内质网接触位点相关蛋白及内质网与线粒体共定位的影响

Fig.6 Effect of CHOP overexpression on expressions of MERCs-related proteins (A) and organelle colocalization levels (B) (Scale bar=50 μm). *P<0.05, **P<0.01, ***P<0.001 vs DTT group and LV-NC+DTT group.

图7 过表达CHOP对线粒体中钙离子水平的影响

Fig.7 Effect of CHOP overexpression on the level of Ca2+ in the mitochondria (Scale bar=50 μm). **P<0.01 vs DTT group and LV-NC+DTT group.

图8 过表达CHOP对内质网应激时线粒体膜电位的影响

Fig.8 Effect of CHOP overexpression on mitochondrial membrane potential in hepatocytes in response to ER stress. **P<0.01 vs DTT group and LV-NC+DTT group.

图9 过表达CHOP对BRL-3A细胞中焦亡相关蛋白及线粒体中GSDMD-N蛋白水平的影响

Fig.9 Effect of CHOP overexpression on levels of pyroptosis-associated proteins (A) and mitochondrial GSDMD-N protein (B) in BRL-3A cells (Scale bar=50 μm). *P<0.0, **P<0.01, ***P<0.001 vs DTT group and LV-NC+DTT group.

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C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白通过抑制内质网自噬并增强内质网-线粒体互作促进肝细胞焦亡

CHOP promotes hepatocyte pyroptosis by downregulating endoplasmic reticulophagy and enhancing endoplasmic reticulum-mitochondria interaction

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