Supplementary Material for: Role of melatonin in intestinal mucosal injury induced by chronic restraint stress in mice

Introduction: A growing body of evidence demonstrates that gastrointestinal motility disorder (GIMD) and gastric stress ulcers can be induced by chronic stress, whereas melatonin (MT) elicits anti-inflammatory and antioxidant effects. The present study investigated the mechanisms of MT-mediated protection against chronic restraint stress-induced GIMD. Methods: Sixty 8-week-old male ICR mice were divided into four groups: control, restraint stress, restraint stress + MT and MT (positive control). MT (20 mg/kg) or vehicle was administered intraperitoneally 60 minutes before chronic restraint stress (5 hours/day) once daily for 30 days. Biochemical parameters, intestinal mucosal integrity, tissue antioxidant ability and autophagic protein levels were determined. Results: Mice subjected to restraint stress presented elevated CORT and NE levels of 141.41% and 151.24%, respectively, and a decreased plasma MT content of 37.12%. Consistent with the decrease in MT levels, we observed a reduction in antioxidant ability and autophagic proteins and increased apoptotic protein levels in the gut, resulting in injury to the intestinal mucosa, which manifested as reductions in the villus height and the V/C ratio; the number of goblets; the number of mast and PCNA-positive cells; and the expression of tight junction proteins (ZO-1, occludin and claudin-1). In contrast, MT reversed these changes caused by chronic restraint stress and improved intestinal mucosal injury. However, there was no significant difference between the MT (positive control) and control groups. Conclusion: Our results suggest that MT effectively mitigates chronic psychological stress-induced intestinal mucosa injury, providing evidence demonstrating the potential for the use of MT as a therapy for intestinal impairment associated with chronic psychological stress.