Dietary chlorogenic acid mitigates intestinal barrier damage induced by heat stress in hybrid grouper (Epinephelus fuscoguttatus♀ ×Epinephelus lanceolatus♂)

Heat stress is a major environmental challenge in aquaculture that disrupts multiple physiological processes in fish, including gastrointestinal function. The intestine is increasingly recognized as a heat-sensitive organ. Chlorogenic acid (CGA) has intestinal microbiota-modulating properties, but its protective role against heat stress-induced intestinal barrier dysfunction remains unclear. In this study, hybrid grouper ( Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus♂ ) were fed diets with or without CGA (400 mg/kg) for 56 days, after which they were exposed to either 28°C (control) or 35°C (heat stress), resulting in four treatment groups as CON, CGA, CONHT and CGAHT, respectively. Compared with the control group, dietary CGA supplementation alone significantly increased intestinal villus height, upregulated the expression of tight junction genes and proteins, elevated anti-inflammatory cytokine levels, and improved the intestinal microbiota composition. Heat stress markedly reduced intestinal villus height from 641 to 343 μm, whereas dietary CGA supplementation increased villus height to 488 μm. Heat stress also significantly downregulated the expression of intestinal tight junction genes and proteins, and disrupted tight junction structure; these changes were alleviated by CGA supplementation. In addition, heat stress increased serum D-lactic acid, endothelin-1, and lipopolysaccharide levels, while decreasing diamine oxidase activity, whereas CGA supplementation reversed these effects. Heat stress also reduced intestinal trypsin (from 2098.16 to 1746.55 U/mg pro) and amylase activities, while CGA supplementation restored both. Moreover, heat stress increased pro-inflammatory gene and protein expression but decreased anti-inflammatory gene expression. CGA supplementation suppressed pro-inflammatory responses and enhanced anti-inflammatory gene levels. Heat stress also reduced the Simpson index and beneficial bacteria while increasing harmful taxa; these alterations were ameliorated by CGA supplementation. Overall, dietary CGA mitigated heat stress-induced intestinal barrier injury in hybrid grouper by improving digestive function, reducing inflammation, preserving structural integrity, and modulating intestinal microbiota, indicating its potential as a dietary strategy to alleviate heat stress-related intestinal damage. • CGA mitigates heat stress-induced damage to the intestinal histomorphology of hybrid grouper. • CGA mitigates the heat stress-induced suppression of intestinal digestive enzyme activity in hybrid grouper. • CGA mitigates heat stress-induced intestinal inflammation in hybrid grouper. • CGA mitigates heat stress-induced dysbiosis of the intestinal microbiota in hybrid grouper.