Dietary betaine alleviates corticosterone-induced intestinal barrier dysfunction in laying hens possibly by promoting tight junction protein expression via m6A methylation-mediated inhibition of the NF-κB pathway.

Excessive corticosterone (CORT) exposure impairs intestinal barrier function in laying hens by decreasing tight junction (TJ) proteins expression, whereas betaine has been known to exert protective effects on gut health. However, it is unknown whether and how betaine can mitigate intestinal barrier dysfunction caused by CORT in laying hens. A total of 72 laying hens were randomly allocated to three groups: control (CON), CORT, and CORT+betaine groups. The duration of the experiment was 35 d. Chickens in the CON and CORT groups received only the basal diet, whereas those in the CORT+betaine group were provided with the same diet plus 0.1% betaine throughout the 35-d period. Beginning on d 28 of the study, the CORT and CORT+betaine groups were treated with subcutaneous CORT (4 mg/kg BW daily for 7 d), and the CON group was handled identically but received the solvent vehicle in place of CORT. The results showed that CORT injection elevated plasma diamine oxidase (DAO) activity and endotoxin concentration. This was accompanied by the downregulated mRNA or protein abundances of Claudin-1 (CLDN1), Occludin (OCLN), JAMA, ZO-1, and Claudin-5 (CLDN5) in the duodenum; CLDN1, OCLN, JAMA, ZO-1, ZO-2, and Claudin-3 (CLDN3) in the jejunum; and CLDN1, OCLN, JAMA, ZO-1, and CLDN5 in the ileum. Betaine supplementation remitted (P< 0.05) CORT-induced partial downregulation of TJ proteins abundances in small intestine. Moreover, betaine supplementation remitted (P< 0.05) CORT-induced activation of the Nuclear factor-kappa B (NF-κB) subunit p65 signaling pathway, which coincided with modifications of RNA N6-methyladenosine (m6A) methylation on p65 mRNA. Furthermore, chromatin immunoprecipitation assays revealed that betaine suppressed the CORT-induced increase in p65 binding to the promoter regions of CLDN1 and OCLN. These results indicate that dietary betaine alleviates intestinal barrier dysfunction in laying hens, possibly through enhanced TJ protein expression associated with modulation of m6A methylation and inhibition of the NF-κB pathway.