Combined exposure to cypermethrin and sulfamethoxazole is associated with muscle injury in grass carp through oxidative stress, endoplasmic reticulum stress, MAPK activation, autophagy, and apoptosis.

The co-occurrence of pesticides and antibiotics in aquatic environments has raised increasing concern because of their potential effects on aquatic organisms. Cypermethrin (CMN) and sulfamethoxazole (SMZ) are representative pyrethroid pesticides and sulfonamide antibiotics, respectively, but their combined effects on fish muscle remain poorly understood. In this study, grass carp (Ctenopharyngodon idella) were exposed to CMN (0.65 μg/L), SMZ (0.30 μg/L), or their combination for 42 days. Network toxicology, histopathology, oxidative stress assays, Western blotting, protein-protein interaction analysis, and molecular docking were used to investigate muscle injury and related molecular responses. The MIX group showed more severe pathological alterations than the single-exposure groups, including extensive myofiber disruption, fragmentation, nuclear condensation, and nuclear displacement. It also increased malondialdehyde levels, decreased total superoxide dismutase activity, and markedly altered proteins associated with endoplasmic reticulum stress. Phosphorylation of P38, JNK, and ERK was elevated, indicating enhanced MAPK signaling. Suppression of the PI3K-AKT-mTOR pathway was accompanied by increased Beclin-1 expression, an elevated LC3-II/LC3-I ratio, and decreased P62 expression, suggesting enhanced autophagy-related responses. In addition, Bax, Caspase-9, and Caspase-3 expression increased, whereas Bcl-2 expression decreased, indicating enhanced mitochondria-associated apoptotic signaling. Network analysis and molecular docking identified HSP90, HSP70, and Caspase-3 as potential core targets. These findings show that combined CMN and SMZ exposure induces severe muscle injury in grass carp, with the MIX group exhibiting the most pronounced changes in oxidative stress, endoplasmic reticulum stress, MAPK activation, autophagy, and apoptosis.