Thymol Improves Barrier Function and Attenuates Inflammatory Responses in Porcine Intestinal Epithelial Cells during Lipopolysaccharide (LPS)-Induced Inflammation

Abstract

It is well known that essential oil thymol exhibits antibacterial activity. The 13 protective effects of thymol on pig intestine during inflammation is yet to be investigated. In this 14 study, an in vitro lipopolysaccharide (LPS)-induced inflammation model using IPEC-J2 cells was 15 established. Cells were pre-treated with thymol for 1 h and then exposed to LPS for various assays. 16 Interleukin 8 (IL-8) secretion, the mRNA abundance of cytokines, reactive oxygen species (ROS), 17 nutrient transporters, and tight junction proteins was measured. The results showed that LPS 18 stimulation increased IL-8 secretion, ROS production, and tumor necrosis factor alpha (TNF-α) 19 mRNA abundance (P < 0.05), but the mRNA abundance of sodium-dependent glucose transporter 20 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1) and H + /peptide cotransporter 1 (PepT1) 21 were decreased (P < 0.05). Thymol blocked ROS production (P < 0.05) and tended to decrease the 22 production of LPS-induced IL-8 secretion (P = 0.0766). The mRNA abundance of IL-8 and TNF-α 23 was reduced by thymol pre-treatment (P < 0.05), but thymol did not improve the gene expression 24 of nutrient transporters (P > 0.05). The transepithelial electrical resistance (TEER) was reduced 25 and cell permeability increased by LPS treatment (P < 0.05), but these effects were attenuated by 26 thymol (P < 0.05). Moreover, thymol increased zonula occludens-1 (ZO-1) and actin staining in 27 the cells. However, the mRNA abundance of ZO-1 and occludin-3 was not affected by either LPS 28 or thymol treatments. These results indicated that thymol enhances barrier function and reduce 29 ROS production and pro-inflammatory cytokine gene expression in the epithelial cells during 30 inflammation. The regulation of barrier function by thymol and LPS may be at post-transcriptional 31 or post-translational levels.32 Intestinal epithelial cells (IECs) are continuously lined monolayer cells, which play important 36 roles in the animal's physical defense. Normally, IECs function as the first line of defense against 37 the invasion of pathogenic agents in the external environment of gut lumen. 1 The maintenance of 38 the barrier function of IECs contributes to the gut homeostasis and health of animals. Gut disorder 39 and dysfunction might be harmful to the growth performance of food-producing animals, and may 40 induce gut diseases such as inflammatory bowel diseases and diarrhea, possibly due to complex 41 interactions among immunologic, genetic, microbial and environmental factors. 2 For instance, 42 diarrhea is a common gut disorders, which causes almost 5% mortality per year in weaned piglets. 3 43 Therefore, it is necessary to prevent gut disorder and diseases by maintaining proper barrier 44 function of IECs in animals. 45 In addition to the physical barrier, IECs also function as an extrinsic barrier. Under certain 46 circumstances, IECs secrete signaling molecules like mucins, cytokines, and chemokines to 47 prevent the invasion of harmful microorganisms in the gu...

Dietary supplementation withLactobacillus plantarummodified gut microbiota, bile acid profile and glucose homoeostasis in weaning piglets

“…Cells were seeded in twelve-well plate. When grown to confluence, 5 μM of LCA or dimethyl sulphoxide was used to treat cells for 12 h and then each well was added with LPS (10 μg/ml) or saline for another 4 h. The concentrations of LCA and LPS used were based on previous studies (22,23) . Cell culture medium was collected for measurement of IL-8.…”

Eugenol attenuates inflammatory response and enhances barrier function during lipopolysaccharide-induced inflammation in the porcine intestinal epithelial cells

“…Nutrient transporters are essential for gut homeostasis and proper absorption of nutrients. B 0 system neutral amino acid co-transporter 1 (B 0 AT1), system ASC sodium-dependent neutral amino acid exchanger 2 (ASCT2), apical sodium-dependent glucose transporter 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1) and peptide transporter 1 (PepT1) are main nutrient transporters and chemosensing systems which are located on the apical side of IECs, and they play important roles in the transport of glucose, amino acids and peptides (Omonijo et al, 2019). Paracellular permeability is mainly regulated by tight junctions localized at the apical-lateral membranes to prevent leakage of macromolecules from the gut lumen (Laukoetter et al, 2008).…”

Effects of Thymol and Thymol α-D-Glucopyranoside on Intestinal Function and Microbiota of Weaned Pigs

“…It remains unclear why the gluco-conjugated form of thymol was unable to strengthen the GI integrity, especially because the luminal concentrations of both thymol and TαG in the distal jejunum were too low (<10 mg/kg) to be reliably detected with the current analytical method. Still, the effective thymol concentration needed to enhance the barrier function might be below our detection limit, as Omonijo et al (2019) [47] found already increased TEER values at only 50 µM or 7.5 mg/kg.…”

“…Unfortunately, no compositional analysis was performed for the colon. Otherwise, improved fecal scores potentially relate to the improved barrier function in the small intestine, which might indicate a reduced inflammatory status of thymol-supplemented pigs [47].…”

“…Essential oils can regulate the barrier function by acting upon the gene expression and synthesis of TJ proteins [7]. Indeed, it was demonstrated in vitro that pretreatment of IPEC-J2 cells with thymol (50 µM) could alleviate the negative effects of lipopolysaccharide-induced damage to the barrier function by increasing the trans-epithelial electrical resistance and reducing the FD4 flux [47]. Likewise, pigs fed a diet with oregano EO (25 mg/kg) during 4 weeks showed higher jejunal expression levels of the TJ proteins occludin and zonula occludens-1 and had longer villi, implying an improved intestinal integrity [56].…”