Rutin alleviates heat stress-induced intestinal oxidative damage in broilers via AMPK/PINK1-Parkin signaling pathway

Oxidative stress is a primary pathogenic factor in heat stress (HS)-induced intestinal barrier dysfunction, causing substantial economic losses in poultry production and compromising animal welfare. Rutin, a natural flavonoid with potent antioxidant properties and prominent intestinal protective effects, represents a promising nutritional intervention. This study investigated the protective efficacy and underlying mechanisms of rutin against intestinal oxidative damage using both in vivo and in vitro HS models. One hundred sixty 21-day-old Arbor Acres broilers were randomly assigned to four treatments: control (C), rutin (R; 500 mg/kg diet), HS (H), and HS + rutin (HR), with four replicates of 10 birds each. Broilers in the H and HR groups were exposed to 34 ± 2°C for 8 h/d to induce HS. Concurrently, chicken small intestinal epithelial cells (CIECs) were assigned to the same four groups, with HS induced at 43°C for 3 h. Mechanistic validation was performed using compound C (an AMP-activated protein kinase [AMPK] inhibitor) and Parkin small interfering RNA (siRNA). The results showed that HS induced systemic and local oxidative stress and impaired intestinal mucosal barrier function, both of which were significantly mitigated by dietary rutin supplementation. In vitro evaluations revealed that rutin preserved cellular viability, restored mitochondrial membrane potential, and enhanced tight junction protein expression. These protective effects were driven by the activation of the AMPK/PTEN-induced kinase 1 (PINK1)-Parkin pathway, which up-regulated mitophagy to clear dysfunctional mitochondria. Conversely, inhibition of AMPK or knockdown of Parkin abolished rutin-mediated mitochondrial protection and barrier restoration in CIECs under HS. In conclusion, rutin alleviates HS-induced intestinal oxidative injury and barrier dysfunction by promoting AMPK/PINK1-Parkin-mediated mitophagy. These findings demonstrate that rutin is an effective dietary antioxidant that can optimize gut health and heat resistance in commercial broilers.