Evodiamine alleviates DEHP-induced hepatocyte pyroptosis, necroptosis and immunosuppression in grass carp through ROS-regulated TLR4 / MyD88 / NF-κB pathway

Di (2-ethylhexyl) phthalate (DEHP) is a neuroendocrine disruptor that can cause multi-tissue organ damage by inducing oxidative stress. Evodiamine (EVO) is an indole alkaloid with anti-inflammatory, antitumor, and antioxidant pharmacological activity. In this manuscript, the effects of DEHP and EVO on the pyroptosis, necroptosis and immunology of grass carp hepatocytes (L8824) were investigated using DCFH-DA staining, PI staining, IF staining, AO/EB staining, LDH kit, qRT-PCR and protein Western blot. The results showed that DEHP exposure upregulated reactive oxygen species (ROS) levels, promoted the expression of TLR4/MyD88/NF-κB pathway, increased the expression of genes involved in cell pyroptosis pathway (LDH, NLRP3, ASC, caspase1, IL-1β, IL-18 and GSDMD) and necroptosis-related genes (RIPK1, RIPK3 and MLKL). The expression of DEHP can also affect immune function, which can be demonstrated by variationsin the activation of antimicrobial peptides (LEAP2, HEPC, and β-defensin) and inflammatory cytokines (TNF-α, IL-2, IL-6 and IL-10). EVO regulates cellular antioxidant capacity by inhibiting ROS burst, reduces DEHP-induced cell pyroptosis and necroptosis to some extent, and restores cellular immune function, after co-exposure with EVO. The TLR4 pathway was inhibited by the co-treatment of TLR4 inhibitor TLR–IN–C34 and DEHP, which attenuated the expression of cell pyroptosis, necroptosis, and immunosuppression. Thus, DEHP induced pyroptosis, necroptosis and abnormal immune function in L8824 cells by activating TLR4/MyD88/NF-κB pathway. In addition, EVO has a therapeutic effect on DEHP-induced toxic injury. This study further provides a theoretical basis for the risk assessment of plasticizer DEHP on aquatic organisms.