CTSB mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury.

Intestinal ischemia‑reperfusion (I/R) injury is a clinical condition that leads to severe intestinal damage, inflammation and oxidative stress. While cathepsin B (CTSB) has been implicated in these pathophysiological processes, its precise role in mediating I/R‑induced injury remains poorly understood. The present study aimed to elucidate how CTSB knockdown influences oxidative stress, inflammatory responses and the integrity of the intestinal epithelial barrier in intestinal epithelial Caco‑2 cells subjected to I/R injury. To identify key genes implicated in I/R injury, a comprehensive analysis was conducted using differential expression profiling and protein‑protein interaction network analysis of the GSE37013 dataset. To simulate I/R damage in vitro, an oxygen‑glucose deprivation/reoxygenation (OGD/R) model was employed in Caco‑2 cells. Subsequently, inflammation was induced by stimulating the cells with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). To investigate the role of CTSB in this context, small interfering RNA was utilized to knock down CTSB expression. In vitro assays were then performed to evaluate NLR family pyrin domain‑containing 3 (NLRP3) inflammasome activation, oxidative stress levels, inflammatory cytokine production and cell survival. The results revealed that intestinal tissues from the I/R group in the GSE37013 dataset showed markedly higher CTSB expression, and the Caco‑2 cells subjected to OGD/R model resulted in a considerable increase in CTSB expression. However, the expression levels of tight junction proteins were enhanced, cell survival was improved and lactate dehydrogenase release was reduced by CTSB knockdown. This reduction in CTSB levels also reduced malondialdehyde levels, and alleviated oxidative stress by increasing the activities of glutathione peroxidase and superoxide dismutase. Furthermore, pro‑inflammatory cytokine production was reduced, and NLRP3 inflammasome activation was inhibited by CTSB knockdown, although a modest increase was still observed after LPS + ATP stimulation. Notably, although CTSB knockdown significantly reduced the inflammatory response, LPS + ATP stimulation still elicited a modest reversal in cytokine levels, suggesting that a CTSB‑independent pathway of inflammatory activation may exist. In conclusion, CTSB knockdown effectively mitigates I/R injury by reducing inflammation, preserving barrier integrity and alleviating oxidative stress, positioning CTSB as a promising therapeutic target. Future work should validate these findings in in vivo models and explore CTSB‑targeted therapies to improve clinical outcomes in I/R‑related diseases.