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

doi:10.12122/j.issn.1673-4254.2026.07.18

Abstract

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

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.

Figures/Tables 9

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.

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

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.

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.

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.

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.

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.

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.

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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