Unraveling the Protective Effect of Hesperetin In Experimentally Induced Colitis: Inhibition of NF‐κB and NLRP3 Inflammasome Activation

ABSTRACT This study aimed to investigate the protective effects of hesperetin (HES) against acetic acid (AA)‐induced colitis (AAC) in rats through suppression of nuclear factor kappa B (NF‐κB) and modulation of the NOD‐like receptor pyrin‐containing protein 3 (NLRP3) inflammasome. Forty‐eight rats were allocated into four groups: control, AAC, HES‐treated, and HES pre‐treatment followed by AAC. Disease activity index (DAI), macroscopic and histological colonic changes were assessed. Moreover, inflammatory markers, and signaling pathways were evaluated through qRT‐PCR, Western blot analysis, ELISA, and immunohistochemistry. HES pre‐treatment significantly decreased the DAI by 61.31%, macroscopic colonic damage by 61.25% and the histological score by 41.86% compared to the AAC group. HES also reduced the expression of miR‐155 by 73.79%, NLRP3 by 66.07%, Apoptosis‐associated speck‐like protein containing CARD (ASC) by 66.09%, cleaved caspase‐1 by 63.86%, and the pyroptosis marker gasdermin‐N (GSDMD‐N) by 61.29%. Concurrently, HES attenuated the NF‐κB pathway, reducing NF‐κB‐positive cells by 74.47% and p‐inhibitory κB kinaseα (IκBα)/IκBα and p‐Inhibitor of nuclear factor kappa‐B kinase subunit alpha (IKKα/β)/IKKα/β levels by 43.77% and 38.68%, respectively. Inflammatory cytokines IL‐1β and IL‐18 were diminished by 73.41% and 71.88%, respectively. HES pre‐treatment increased peroxisome proliferator‐activated receptors‐γ ( PPAR‐γ ) expression by 259.97%, while reducing CD68+ macrophage infiltration by 72.72%. In conclusion, HES alleviated AAC in rats by targeting the NF‐κB and NLRP3 inflammasome signaling pathways. This protective effect was mediated through the downregulation of miR‐155 expression and the concurrent enhancement of PPAR‐γ expression, resulting in reduced inflammation and pyroptosis. These findings highlight HES as a potential therapeutic protective agent for colitis.