Caspase-1 knockout disrupts pyroptosis and protects photoreceptor cells from photochemical damage

• Published in: Molecular and cellular probes 2024-12, Vol.78, p.101991, Article 101991
• Main Authors: Yu, Xiaoping, Peng, Jiayuan, Zhong, Qian, Wu, Ailin, Deng, Xiaoming, Zhu, Yanfeng
• Format: Article
• Language: English
• Subjects: Animals; Caspase 1 - genetics; Caspase 1 - metabolism; Caspase-1 Gene; Cell Line; Cell Proliferation; Cell Survival - radiation effects; CRISPR-Cas Systems - genetics; CRISPR/Cas9 System; Gene Knockout; Gene Knockout Techniques; Light - adverse effects; Mice; Photoreceptor Cells - metabolism; Photoreceptor Cells - radiation effects; Pyroptosis; Pyroptosis - genetics; Reactive Oxygen Species - metabolism; Retinal Photochemical Damage
• ISSN: 0890-8508; 1096-1194; 1096-1194
• DOI: 10.1016/j.mcp.2024.101991

Retinal photochemical damage (RPD) plays a significant role in the development of various ocular diseases, with Caspase-1 being a key contributor. This study investigates the protective effects of Caspase-1 gene-mediated pyroptosis against RPD. Differentially expressed genes (DEGs) associated with RPD were identified through the analysis of two expression profiles from the GEO database. Correlation analysis was used to pinpoint pyroptosis-related genes (PRGs) linked to RPD. A Caspase-1 knockout 661 W cell line was generated via CRISPR-Cas9 gene editing, and single-cell colonies were screened and purified. Validation of knockout cells was performed through RT-qPCR, gene sequencing, and Western blot analysis. Comparative assays on cell proliferation, intracellular reactive oxygen species (ROS), and cytotoxicity were conducted between wild-type and Caspase-1 knockout cells under light exposure. Further RT-qPCR and Western blot experiments examined changes in the mRNA and protein levels of key pyroptosis pathway components. Significant alterations in Caspase-1 expression were observed among PRGs. Homozygous Caspase-1 knockout cell lines were confirmed through RT-qPCR, genomic PCR product sequencing, and Western blot analysis. Compared to wild-type 661 W cells, Caspase-1 knockout cells exhibited higher viability and proliferation rates after 24 h of light exposure, alongside reduced LDH release. The expression of downstream pyroptosis factors at both the mRNA and protein levels was markedly decreased in the knockout group. CRISPR/Cas9-mediated Caspase-1 knockout enhanced the resistance of 661 W cells to photochemical damage, suggesting that Caspase-1 may serve as a potential therapeutic target for RPD-related diseases. •CRISPR/Cas9-mediated Caspase-1 knockout improves 661W cells survival post-photochemical exposure.•Caspase-1 deficiency reduces pyroptosis, downregulating key pyroptosis markers in 661 W cells.•Knockout cells show increased viability and proliferation, decreased cytotoxicity under light damage.•Caspase-1 as a potential therapeutic target for treating retinal degenerative diseases.