Dietary Zanthoxylum bungeanum leaves supplementation enhances antioxidant capacity through activation of Nrf2 signalling pathway in pigs

The present study investigated the effects of dietary supplementation with Zanthoxylum bungeanum leaf (ZBL) on growth performance, intestinal health, and antioxidant capacity in growing pigs. Forty-eight barrows (initial body weight 18.55 ± 0.44 kg; age 54 ± 1 d) were randomly assigned to four dietary treatments: a basal diet (control), basal diet supplemented with 1.5% ZBL, 3.0% ZBL, or 4.5% ZBL. Each treatment included 12 replicate pens with one pig per pen, and the experimental period lasted 50 d. Dietary ZBL supplementation resulted in a linear decrease in average daily gain ( P = 0.042). The mRNA expression levels of tight junction–related genes, including claudin-1, occludin, and mucin-2, in the jejunal mucosa increased linearly with increasing dietary ZBL levels ( P < 0.05). Plasma antioxidant indices, including total antioxidant capacity (T-AOC), CAT, SOD, and GSH-Px, were also linearly increased by ZBL supplementation ( P < 0.05). In contrast, plasma malondialdehyde (MDA) concentrations were reduced, with the lowest value observed in pigs fed the 1.5% ZBL diet ( P = 0.004). Furthermore, the jejunal mRNA expression of Nrf2 , CAT , and HO-1 was significantly upregulated, with peak expression observed at the 1.5% ZBL inclusion level ( P < 0.05). Analysis of gut microbiota using 16S rRNA gene sequencing revealed that dietary ZBL increased microbial diversity and altered microbial composition. Specifically, ZBL supplementation linearly decreased the relative abundance of Christensenellaceae , Clostridium_sensu_stricto_1 , Christensenellaceae_R-7_group , Family_XIII_AD3011_group , Clostridium , and Clostridia_UCG-014 ( P < 0.05), while linearly increasing the abundance of Coriobacteriales ( P < 0.001). To further elucidate the regulatory mechanisms underlying the antioxidant effects of ZBL, an in vitro oxidative stress model was established using intestinal porcine epithelial cells (IPEC-J2). ZBL extract markedly alleviated H 2 O 2 -induced oxidative damage by restoring cell viability and Nrf2 expression and by reducing intracellular reactive oxygen species, LDH release, MDA levels, and protein carbonyl content ( P < 0.05). These protective effects were abolished by the Nrf2 inhibitor ML385. In addition, ZBL extract reversed the H 2 O 2 -induced reductions in T-AOC, CAT activity, GSH, GSSG, as well as Keap1 and CAT expression, effects that were likewise blocked by ML385 ( P < 0.05). In conclusion, dietary supplementation with ZBL linearly reduced growth performance in growing pigs. However, supplementation at a lower inclusion level (1.5%) improved intestinal barrier function and enhanced antioxidant capacity, likely through activation of the Nrf2 signalling pathway.