Mechanisms by which α-ketoglutarate alleviates intestinal injury caused by carbonate-alkaline stress in crucian carp (Carassius auratus): Insights from metabolomics and microbiomics

Carbonate alkalinity is a prevalent environmental stressor in inland waters and poses toxicological risks to aquatic organisms. While α-ketoglutarate (AKG) is known to alleviate the resulting intestinal injury in fish, its protective mechanisms remain unclear. Here, crucian carp ( Carassius auratus ) were exposed to carbonate-alkaline stress (20 and 40 mmol/L NaHCO 3 ) with or without exogenous AKG supplementation for 56 days. Histopathological examination, biochemical assays, 16S rRNA, and metabolomics were employed to investigate the effects of carbonate-alkaline stress on intestinal redox status, energy metabolism, and microbial community, and to evaluate the mitigating mechanism of exogenous AKG supplementation. Results indicated that carbonate-alkaline stress induced intestinal histopathological damage, with increased ROS and lipopolysaccharide, and decreased ATP levels. 16S rRNA sequencing revealed that carbonate-alkaline stress altered the gut microbial community, increasing Proteobacteria and decreasing Firmicutes abundance. Metabolomics indicated that energy pathways like glycolysis and TCA cycle were inhibited. Meanwhile, the activation of purine metabolism and inhibition of glutathione metabolism suggested intestinal redox imbalance. Notably, exogenous AKG supplementation alleviated these injuries. Metabolomics showed upregulation of key metabolites in glycolysis and TCA cycle, while abnormalities in purine and glutathione metabolism were mitigated. Additionally, the community structure shifted, decreasing Proteobacteria and increasing Firmicutes abundance. These findings indicate that carbonate-alkaline stress induces microbial dysbiosis and intestinal injury by disrupting energy and redox balance, a process mitigated by exogenous AKG supplementation which restores metabolic homeostasis. This study provides mechanistic insights into metabolic interventions alleviating intestinal toxicity under carbonate-alkaline stress and a theoretical basis for improving fish tolerance in carbonate-alkaline environments.