Effects Of Low-dose DEHP Chronic Exposure Combined With High-Fat Diet On Cognitive Impairment Of Female Mice

ObjectiveDiethylhexyl phthalate(DEHP)is one of the most used plasticizers,and it readily released and migrated into water,soil,air and food during the processes of the production,storage,usage and disposal due to it is bound to plastic matrix by non-covalent bond.Human exposure to DEHP is mainly through the oral route,which accounts for 50-98%of the daily intake of adults.With the shift of Chinese traditional dietary pattern,dietary fat intake has been rising year by year,and high-fat diet might contribute to the risk of DEHP exposure.Human epidemiological data suggested that DEHP exposure was associated with the decrease of cognitive function,and laboratory studies have also evidenced that higher doses of DEHP exposure could lead to cognitive impairment in experimental animals.However,the effects of low-dose DEHP chronic exposure on cognitive function with or without high-fat diet(HFD)are unclear.In this study,mice were chronically exposed to environmentally relevant dose of DEHP under chow diet and HFD treatments respectively,the changes of cognitive function were investigated and the underlying mechanisms of damage were explored.MethodsC57BL/6J female mice were respectively fed with chow diet and HFD,meanwhile,orally treated with 50 μg/kg.bw DEHP dissolved in corn oil for 24 weeks.The glucose tolerance test(GTT)was performed at week 20 to indicate the changes of glucose tolerance,the neurobehavioral performances were evaluated by the open field test and new object recognition test at week 24.Then,part of mice from each group were fixed with transcardiac perfusion and the brain were taken out for preparation of brain sections.The immunohistochemical staining were performed to observe the changes of microglia,astrocytes and neuron.The remaining mice of each group were sacrificed,the brains were harvested and subjected to detection of related indicators.The levels of amino acid neurotransmitters in mice brain were detected by High Performance Liquid Chromatography(HPLC).Western blot was used to detect the changes of synaptic proteins,the aberrant activation of cyclin-dependent kinase 5(CDK5)and phosphorylation of its downstream peroxisome proliferator activated receptors γ(PPARγ),as well as the expression of insulin signaling pathway proteins,the levels of Aβ42 and its production and clearance of related proteins,inflammatory pathway proteins expression in the prefrontal cortex of mice brains.ResultsGTT revealed that glucose tolerance declined and serum insulin elevated in the DEHP or HFD group of mice,and a synergistic effect was observed in the HFD and DEHP co-treated mice.The mice in both of DEHP and HFD groups exhibited significant decreases of the activity time in the central area of open-field and new object recognition index compared to control mice,which indicated that both of DEHP and HFD treatments resulted in the anxiety-like symptom and cognitive impairment of mice.DEHP combined with HFD treatment showed synergistic effects on neurobehavioral impairment of mice.Morphological examination showed that the activation of microglia and astrocytes,accompanied by the damage and loss of neurons in mice treated with DEHP and HFD respectively,and the most changes were observed in HFD and DEHP co-treated mice.Western blot results showed that DEHP and HFD treatment decreased the synaptic proteins PSD95 and synapsin1 in prefrontal cortex of mice,with the most decreased in HFD combined with DEHP treated mice.DEHP and HFD treatments significantly increased the CDK5 expression in brain of mice,accompanied by obvious increase of p25 protein,suggesting CDK5 activation,and enhanced the phosphorylation of its downstream protein PPARγ at ser273 site,and the changes were the most pronounced in the DEHP and HFD co-treated group.In addition,the decreased expression of IRS1 protein,increased serine phosphorylation of IRS 1,decreased phosphorylation of PI3K and AKT,as well as mTOR,GSK3β and CREB in insulin signaling pathway were observed in the brain of DEHP and HFD respectively treated mice,all of which showed the synergistic effects of DEHP and HFD.Simultaneously,the results of western blot showed that DEHP and HFD respectively elevated the levels of Aβ42 in the prefrontal cortex of mice,accompanied by the increase of BACE1 protein expression,with the synergistic effects in HFD combined with DEHP.The decrease of IDE protein expression was only observed in DEHP exposed to mice.Both of DEHP and HFD treatments activated the NF-κB by phosphorylation,along with the increases of inflammasomes proteins such as NLRP3,pro-IL-1β and mature IL-1β expression,and the changes of these inflammatory proteins were also exhibited synergistic effects when co-treated with DEHP and HFD.Conclusions1.Both of low-dose DEHP chronic exposure and high-fat diet resulted in anxiety-like symptoms and cognitive dysfunction of mice,accompanied by the degeneration of neurons,with synergistic effects between DEHP and HFD treatment.2.Both of low-dose DEHP chronic exposure and high-fat diet could lead to the activation of CDK5 followed by its downstream PPARγ phosphorylation at ser273,damage the insulin signaling pathway and elevate the Aβ levels in the brain of mice.DEHP and HFD exhibited synergistic effect on the above changes.3.Both of low-dose DEHP chronic exposure and HFD could elicit the neuroinflammation,DEHP and HFD have synergic effects on neuroinflammation.