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

Abstract

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 L8824cells 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.