Terpine-4-ol alleviates lipopolysaccharide-induced intestinal injury in weaned piglets by regulating intestinal epithelial cell homeostasis via the Wnt/β-catenin signaling pathway

This study aims to elucidate intestinal injury from the perspective of intestinal epithelial cell homeostasis and to explore the modulatory roles of terpine-4-ol (a major bioactive component of tea tree oil). Forty 28-day-old piglets (7.82 ± 0.43 kg) were randomly divided into 5 groups with 8 pigs per group. Piglets in the control (CON) and lipopolysaccharide (LPS) groups were fed a basal diet, while those in the remaining 3 groups were fed the basal diet supplemented with 30 (LTP), 60 (MTP), or 90 mg/kg terpine-4-ol (HTP), respectively. Piglets in the LPS and terpine-4-ol supplementation groups were intraperitoneally injected with 100 μg/kg BW of LPS at the end of the 21-day growth period. Six hours after injection, 6 piglets from each group were randomly selected and slaughtered to detect intestinal cell homeostasis. The results showed that the average daily weight gain (ADG) and average daily feed intake (ADFI) of piglets in MTP and HTP groups were higher than those in the CON group (P < 0.05), whereas the feed conversion ratio (FCR) of piglets in LTP group was lower as compared to the CON group (P = 0.007). Terpine-4-ol supplementation also decreased the diarrhea rate of piglets. Lipopolysaccharide challenge decreased the villus length and the ratio of villus length to crypt depth (V/C) in the jejunum (P < 0.05), decreased the V/C ratio and increased the crypt depth in the ileum (P < 0.05). In contrast, terpine-4-ol supplementation led to increased villus length and V/C ratio in the jejunum, raised the V/C ratio in the HTP group, and decreased crypt depth in the LTP and MTP groups in the ileum (P < 0.05). Lipopolysaccharide reduced the number of antigen Ki-67 positive (Ki67⁺) cells and increased the number of caspase 3⁺ cells (P < 0.05) in both the jejunum and ileum, whereas terpine-4-ol increased the number of Ki67⁺ cells in the crypts of the jejunum and ileum, while decreasing the number of caspase 3⁺ cells of the ileum and in the jejunum of MTP and HTP groups (P < 0.05). Lipopolysaccharide down-regulated the expression of the intestinal stem cell marker leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) (P < 0.05, except for Lgr5 gene in the jejunum), while terpine-4-ol supplementation alleviated the effect. Lipopolysaccharide decreased the number of goblet cells and mucin 2 (Muc2) secretion (P < 0.05), whereas the number of goblet cells and Muc2 secretion in the small intestine of MTP group were increased (P < 0.05). In addition, LPS injection led to a reduction in the expression of endocrine cell marker chromogranin A and absorption cell marker villin (P < 0.05). Conversely, terpine-4-ol increased the abundance of endocrine cells and absorption cells in the small intestine (P < 0.05). Moreover, LPS inhibited the expression of genes and proteins of wingless-type MMTV integration site family (Wnt)/β-catenin signaling pathway (P < 0.05), whereas terpine-4-ol reversed this effect. In conclusion, terpine-4-ol supplementation improved growth performance, decreased diarrhea in piglets and alleviated the damage to the intestinal epithelial cell homeostasis under LPS challenge, probably via Wnt/β-catenin signaling activation. A dose of 60 mg terpine-4-ol per kilogram diet was recommended based on the present study.