Effects of Astragalus Polysaccharide and Isatis indigotica Extract Synergy on the Antioxidant Status, Inflammation, Autophagy, Apoptosis, and Intestinal Health of Larimichthys crocea Juveniles

This research aimed to examine the combined influence of Astragalus polysaccharide (APS) and Isatis indigotica extract (IIE) dietary supplements on oxidative-inflammatory status, cellular homeostasis, and intestinal integrity in large yellow croaker (Larimichthys crocea). Three replicates of experimental fish (n = 160) received one of five dietary regimens: a basal control (CON) diet, the CON diet containing 0.1% APS (AP), and the AP diet supplemented with 0.05%, 0.1%, and 0.15% IIE (AI1, AI2, and AI3) for 8 weeks. The combined supplements, particularly in the AI2 group, significantly improved intestinal morphology and enhanced the activities of key digestive enzymes. Gene expression analysis revealed that the APS-IIE combination consistently upregulated the intestinal mRNA abundance of major tight junction proteins (CLDN4, OCLN, ZO1, ZO2) compared to the CON or AP groups. Liver antioxidant capacity was strengthened (enhanced CAT capacity), as evidenced by a significant reduction in lipid peroxidation (MDA) levels. In the head kidney, the combination downregulated the expression of pro-inflammatory cytokines (IL8, TNF) and toll-like receptors (TLR1, TLR2, TLR5), and promoted the expression of anti-inflammatory cytokines (IL10, TGFB1). Furthermore, dietary supplementation modulated the crosstalk between autophagy and apoptosis, indicated by altered expression of key marker genes (e.g., increased MAP1LC3B and decreased CASP3/8/9). In conclusion, the simultaneous inclusion of APS and IIE in diets promotes intestinal health, strengthens antioxidant status, and alleviates inflammatory responses, with the 0.1% APS + 0.1% IIE (AI2) formulation demonstrating the most pronounced benefits.