[Heat shock protein family A member 5 regulation of ferroptosis alleviates acute-phase mucosal injury in ulcerative colitis].

Objective: This study aimed to investigate the role of heat shock protein family A member 5 (HSPA5) in ferroptosis at its regulatory mechanisms in ulcerative colitis (UC), using both a dextran sulfate sodium (DSS)-induced mouse model of acute colitis and in vitro cell experiments. Methods: Differentially expressed genes in UC were identified using the GSE87466 dataset from the Gene Expression Omnibus, cross-referenced with the ferroptosis-related gene database FerrDB (version 2). A protein-protein interaction (PPI) network was constructed, identifying HSPA5 as a core hub gene. To validate its role in vivo, acute colitis was induced in C57BL/6 mice using DSS, followed by treatment with the ferroptosis inhibitor Ferrostatin-1 (Fer-1). Lipid peroxidation and ferroptosis levels were assessed by measuring malondialdehyde (MDA) and iron content in colon tissues. The expression of ferroptosis-related proteins, including prostaglandin-endoperoxide synthase 2 (PTGS2), glutathione peroxidase 4 (GPX4), ferritin light chain (FTL), activating transcription factor 4 (ATF4), and HSPA5, in addition to tight junction proteins ZO-1 and Occludin, were evaluated using immunohistochemistry and Western blotting. In vitro, an inflammatory model was established using lipopolysaccharide (LPS)-stimulated Caco-2 cells. Lentiviral knockdown of HSPA5 was performed to assess its regulatory effects on ferroptosis by assessing MDA levels, GPX4 activity, and the expression of related proteins. Statistical analyses were conducted with SPSS (version 29.1), with t-tests or one-way ANOVA for normally distributed data and the Mann-Whitney U test for ordinal data. Statistical significance was set at P<0.05. Results: Based on the PPI analysis and previous research, HSPA5 emerged as a key gene linking UC and ferroptosis. In DSS-treated mice, colonic injury was accompanied by elevated MDA levels (t=5.72, P<0.001) and iron accumulation (t=6.32, P<0.001). DSS also increased the expression of PTGS2 and proteins in the ATF4-HSPA5 pathway, while reducing the levels of GPX4, FTL, ZO-1, and Occludin. These findings could be partially reversed by Fer-1 (MDA: t=2.92, P<0.05; iron: t=5.84, P<0.001). In Caco-2 cells, LPS treatment elevated the expression of PTGS2, ATF4, and HSPA5, and elevated the MDA content (t=9.63, P<0.001), while reducing the expression of FTL, GPX4, ZO-1, and Occludin, as well as GPX4 enzyme activity (t=-11.20, P<0.001). Knockdown of HSPA5 further exacerbated these changes, significantly increasing MDA levels (t=4.15, P<0.01), decreasing GPX4 activity (t=-9.81, P<0.001), and altering ferroptosis-related protein expression. Conclusion: HSPA5 appears to protect against intestinal damage in UC by enhancing GPX4 expression and activity, thereby reducing ferroptosis and preserving epithelial barrier integrity through the maintenance of tight junction proteins.