Combined exposure to MPs and DEHP exacerbated behavioral and cognitive impairments by inducing neuronal apoptotic damage through inhibiting the KLF2/IRF4/SRF pathway.

Microplastics (MPs) represent a novel category of pollutants accumulating in various human organs, particularly the brain. Di (2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, induces neuronal damage and cognitive behavioral disorders in mice. Nevertheless, whether their combined exposure further exacerbates neurotoxicity and the underlying mechanism remain unclear. To examine the effects of combined versus individual exposure, we established an in vivo ICR mouse model with four groups (control, MPs, DEHP, and combined group). Both single and combined exposures caused cognitive/behavioral impairments and apoptotic neuronal damage, with repression of the KLF2/IRF4/SRF pathway serving as an upstream regulator of apoptosis. Subsequent in vitro experimental data suggested that the toxicity in HT22 hippocampal neuron cells co-exposure to MPs and DEHP exceeded that observed with either substance individually. Moreover, the expression patterns of the KLF2/IRF4/SRF pathway were consistent with those observed in the animal experiments. Then we performed experiments utilizing apoptosis inhibitors. Our findings indicate that apoptosis inhibitors can reduce damage to HT22 neuronal cells but unable to alter the protein levels of its upstream KLF2 signaling pathway. Subsequently, when KLF2 overexpression was further induced，the decline in pathway proteins expression and apoptosis-related damage could be reversed partly. In conclusion, our results demonstrate that co-exposure to MPs and DEHP exacerbates cognitive impairment in mice by promoting neuronal apoptosis through synergistic inhibition of the KLF2/IRF4/SRF pathway. These findings elucidate the mechanisms by which combined exposure to MPs and DEHP induces neurotoxicity.