The Effects And Mechanism Of DEHP Combined With High Fat Diet On The Cognitive Function Of Experimental Animals

ObjectiveDiethylhexyl phthalate(DEHP)is a kind of environmental endocrine disruptors.It is a plasticizer used most in the world,which is used in plastic products such as medical instruments,household articles,cosmetics,building materials,etc.Globally,DEHP can be detected in indoor environments,drinking water,sediment,sludge,and air.Human body is easily exposed to DEHP through various ways such as respiratory tract,digestive tract and skin absorption,and oral route is the main route of human exposure to DEHP.Previous studies on DEHP focused on reproductive toxicity,immunotoxicity and hepatotoxicity.However,recent studies have shown that exposure to DEHP is associated with cognitive impairment,and the excessive accumulation ofβ-amyloid(AP)is the key pathological change of neurodegenerative diseases.Over-activation of CDK5 may be the main cause of neurodegenerative diseases.As a multifunctional kinase,CDK5,activated by p35,can phosphorylate a series of proteins,which are involved in the regulation of neuronal differentiation,axon growth and synaptic genesis.At the same time,the excessive accumulation of Aβ in the brain will cause a series of neurotoxicity,activate microglia,increase the release of pro-inflammatory factors,induce neuroinflammation and then lead to cognitive dysfunction.More attention has been paid on the relationship between obesity and cognitive function in recent years.Long-term HFD can induce neuroinflammation,cognitive dysfunction and other symptoms.The net population and growth rate of obesity in China ranks first in the world,and obesity has been a major public health problem.Traditional obesity-inducing factors can’t explain the rapid increase of obesity rate completely,and the exposure of environmental endocrine disruptors and the occurrence of obesity have gradually attracted persons’ attentionWith the continuous rise of our country’s economic level,the traditional dietary pattern of Chinese residents has undergone fundamental changes.The proportion of animal food and lipids in the diet has increased year by year,and the coexistence of environmental endocrine disruptor exposure and high-fat diet has become more and more common.Does DEHP affect the damage of the nervous system?Is there a synergistic effect among high-fat diet,obesity and DEHP to the nervous system?In this study,by simulating normal diet and high-fat diet,long-term oral exposure of DEHP with the human body daily exposure dose(0.05mg/kg.bw),observing the damage of DEHP combined with high-fat diet on cognitive function,and preliminarily exploring mechanism.Methods1.Grouping and treatment of experimental animals:sixty 20-22g SPF grade C57 mice were fed for 1 week and divided into 4 groups with 15 mice in each group randomly:control group,DEHP group,high fat diet group(HFD),and HFD with DEHP group(HFD+DEHP).During the experiment,DEHP solution diluted with corn oil was orall as gavage for 32 weeks with a dose of 0.05mg/kg.bw and a volume of 0.1 mL/10g.bw.Control group and HFD group were given equal volume of corn oil by gavage.The animals in control group and DEHP infected group were fed with normal maintenance diet,and the animals in HFD group and HFD+DEHP group were fed with HFD.The activity of mice was observed every day,and the body weight and feed consumption of mice were recorded weekly.2.Glucose tolerance test and insulin tolerance test:in the middle of the experiment,the glucose tolerance test(GTT)and the insulin tolerance test(ITT)were carried out at the 12th,the 13th week,with an interval of one week.At the end of the experiment,GTT test and ITT test were carried out again at the 30th week,the 31st week,with a week interval between the two experiments3.Neurobehavioral index detection:in the 32nd week,a new object recognition experiment was performed on experimental animals,and the learning and exploration time of each group of mice during the familiarization period and the test period were recorded and compared;the open field experiment was compared with each group of mice data such as the total distance of horizontal movement,the time of activity in the central area,the number of urinations,etc;the forced swimming experiment was recorded and compared the "immobility" time of each group of mice.4.Western blot:western blot was used to detect the protein expression of GLUT1、GLUT3、GLUT4、p35/25、CDK5、PPARγ、p-PPARγ(Ser273)、APP、BACE、Presenilin1、Presenilin2、Iba-1、GFAP、NeuN、caspase-3 and cleaved caspase-3 in the mouse prefrontal cortex level5.Histomorphological testing:prepare 40 μm frozen sections of cerebral cortex,and observe the morphology of microglia and neurons in the mouse cerebral cortex by immunohistochemical stainingResults1.Changes in body weight and glucose and lipid metabolism of animals1.1 The general condition of each group of mice and changes in body weight and feed consumptionCompared with the negative control group,the weight of the mice in the DEHP group increased significantly(P<0.05).The body weight and weight gain of HFD group and HFD+DEHP group were higher than those of the negative control group and DEHP group(P<0.05,P<0.01).The feed consumption of the DEHP group was significantly higher than that of the negative control group(P<0.01);the food utilization rate of the high-fat feed group was higher than that of the conventional maintenance feed group.1.2 The effect of DEHP and HFD on white fat and brown fat content on miceCompared with mice in the negative control group and DEHP group,the white fat(visceral fat and subcutaneous fat)content coefficients in the HFD group and HFD+DEHP group were increased(P<0.01).The coefficient of brown fat content in the DEHP group was lower than that in the negative control group(P<0.01).The coefficient of brown fat content in the HFD group and HFD+DEHP group was significantly higher than that of the negative control group and DEHP group respectively(P<0.01).1.3 The effect of DEHP and HFD on glucose metabolism on miceThe GTT test on the 12th week showed that the fasting blood glucose of the HFD group was significantly higher than that of the conventional maintenance feed group;the area under the curve(AUC)of the HFD group was also significantly higher than that of the conventional maintenance feed group(P<0.05,P<0.01).The results of the 13th week ITT test and GTT test showed similar trends.The fasting blood glucose of the HFD group was significantly higher than that of the conventional maintenance diet group(P<0.05,P<0.01).AUC of the DEHP group and HFD group were both higher than that of the negative control group,and the difference between the animals in the high-fat feed group and the negative control group was statistically significant(P<0.01).In the 30th week of the experiment,the GTT test showed that the fasting blood glucose of the animals fed with high-fat feed was significantly higher than that of the conventional maintenance feed group(P<0.05,P<0.01);AUC of the high-fat feed group was also significantly higher than that of the conventional maintenance group(P<0.05,P<0.01).The results of ITT in the 31st week of the experiment showed that the fasting blood glucose of the animals fed with HFD was significantly higher than that of the conventional maintenance feed group(P<0.05,P<0.01),and AUC of the animals in the high-fat feed group was also significantly higher than that of the conventional maintenance feed group(P<0.05,P<0.01).1.4 The effect of DEHP and HFD on the brain glucose transporter on miceThe western blot test results showed that both DEHP and HFD could reduce the expression levels of GLUT1,GLUT3,and GLUT4 in the brain.The reduction of GLUT1 and GLUT3 was statistically significant compared with the negative control group(P<0.05,P<0.01).The expression of GLUT4 under the combined action of DEHP and HFD was significantly lower than that of the negative control group,DEHP group and HFD group(P<0.05,P<0.01).2.Effects of DEHP and HFD on neurobehavioral indexes of mice2.1 Changes in new object recognition indicatorsThe experimental results showed that the learning time of animals in the DEHP,HFD and HFD+DEHP groups was lower than that of the negative control group,but the difference was not statistically significant(P>0.05).Compared with the negative control group,DEHP exposure significantly reduced the exploratory time of experimental animals(P<0.05),and showed a synergistic effect when fed with high fat,and the exploratory time was also significantly lower than that of the high fat group animals(P<0.05).Both DEHP exposure and high fat feed can reduce the recognition index of experimental animals.Among them,DEHP has a significant effect,and the recognition index is significantly lower than the negative control group(P<0.05);the recognition index of HFD+DEHP group is significantly lower than the negative control group and the HFD group(P<0.05,P<0.01).2.2 Changes in open field test indicatorsThe results of the open field experiment showed that there was no significant difference in the total horizontal movement distance of each group of mice,and the number of defecation and urination among mice in each group(P>0.05).The total time of exercise in the central area of the HFD+DEHP group was lower than that of the other groups,but the difference was not statistically significant(P>0.05).2.3 The effect of DEHP and HFD on depression behavior on miceCompared with the negative control group,the HFD group had longer "resting"time and the difference was statistically significant(P<0.05).Compared with the negative control group,the remaining groups of animals had no statistically significant difference in "resting" time(P>0.05).3.The effect of DEHP and HFD on the expression of Aβ-related protein on the prefrontal cortex of mice3.1 The effects of DEHP and HFD on IDE and BACE levelsCompared with the negative control group,HFD group can reduce the expression of IDE protein in the prefrontal cortex of mice(P<0.01);IDE in the brain of animals fed with DEHP combined with HFD was significantly lower than that of the negative control group and the DEHP exposure group(P<0.01).Compared with the negative control group,DEHP exposure significantly increased the BACE protein expression level in the prefrontal cortex of mice(P<0.05);when DEHP combined with HFD,the BACE expression level in the brain of the HFD+DEHP group was significantly higher than that in the negative control group,DEHP and HFD group(P<0.05,P<0.01).3.2 The effects of DEHP and HFD on the levels of Aβ and APPCompared with the negative control group,DEHP exposure and HFD can increase the levels of Aβ and APP protein in the prefrontal cortex(P<0.01),The levels of Aβ and APP in the brains of animals in the DEHP+HFD group were significantly higher than those in the negative control group and the HFD group(P<0.01).3.3 The effects of DEHP and HFD on the levels of Presenilin 1 and Presenilin 2Presenilin 1 in the brain of animals in the DEHP exposure,HFD and the combination of the two groups increased,and the HFD and HFD+DEHP groups were significantly different from the negative control group(P<0.01).There was no significant difference between DEHP exposure and HFD on the protein expression of Presenilin 2 in the brain of mice and the negative control group(P>0.05);DEHP combined with HFD significantly increased Presenilin 2 in the cerebral cortex of mice.The protein expression was significantly different from the negative control group and the simple DEHP and HFD group(P<0.01).4.The effects of DEHP and HFD on the expression of CDK5 and PPARγ protein4.1 The effects of DEHP and HFD exposure on CDK5 activationThere was no significant difference in the expression level of CDK5 protein in the cerebral cortex of mice in each group(P>0.05).However,compared with the negative control group and the DEHP exposure group,the DEHP and HFD feeding group had a significant increase in p35/25 cracking level(P<0.01).There was no significant difference between the HFD+DEHP group and the HFD group(P>0.05).4.2 The effects of DEHP and HFD exposure on PPARγ and its phosphorylation levelsThere was no significant difference in PPARγ protein expression between the different treatment groups(P>0.05),but compared with the negative control group,the DEHP exposure group,HFD group and HFD+DEHP group showed increased phosphorylation expression of PPARγ(Ser273)in the brain of mice(P<0.05).5.The effect of DEHP and HFD on the inflammatory response of the prefrontal cortex in mice5.1 The activation of DEHP and HFD on glial cells in the prefrontal cortex of miceThe immunohistochemical test showed that compared with the negative control group,the number of microglia in the DEHP exposure group,the HFD group and the combination group was significantly increased(P<0.01),the cell body became larger,and the pseudopodia disappeared in the form of "amoeba" changed.Western blot detection of Iba-1 protein expression in the prefrontal cortex of the mice brain is similar to morphological changes.The expression level of Iba-1 protein in DEHP exposure group,HFD group and combination group was significantly higher than that of the negative control group(P<0.05).In the routine maintenance feed group,there was no significant difference in the expression level of GFAP protein between the DEHP exposure group and the negative control group(P>0.05).Under the condition of HFD,the expression of GFAP protein increased in the HFD+DEHP exposure group compared with the HFD group(P<0.01).Compared with mice in the control group,the expression level of GFAP protein in the HFD group also increased significantly(P<0.01).5.2 The damage of DEHP and high-fat diet to neurons in the prefrontal cortex of miceImmunohistochemical examination showed that compared with the negative control group,the number of neurons in the prefrontal cortex of the DEHP exposure group,the HFD group and the combination group was significantly reduced(P<0.05,P<0.01);compared with the DEHP exposure group and the HFD group,the number of neurons in the prefrontal cortex of the brain was also significantly reduced(P<0.05,P<0.01).Western blot results showed that the expression of NeuN in the brain of mice in the DEHP exposure group and the HFD group was reduced.Compared with the negative control group,the NeuN level in the cerebral cortex of experimental animals in the HFD+DEHP group was significantly reduced(P<0.01),which was also significantly lower DEHP and high-fat feed group(P<0.05).The level of cleaved caspase-3 protein in the prefrontal cortex of mice in the DEHP exposure group and the HFD group was significantly increased.Conclusions1.Long-term low-dose exposure of DEHP and HFD can cause cognitive dysfunction of experimental animals.DEHP combined with HFD has a synergistic effect on the cognitive impairment of experimental animals.2.Long-term low-dose exposure to DEHP and HFD can cause inflammatory response and neuron loss in the prefrontal cortex of experimental animals.The combination of DEHP and HFD has a synergistic effect on the inflammatory response and neuron damage in the brain of experimental animals.3.Low-dose DEHP combined with HFD caused glucose transport obstacles in the brain of experimental animals,increased p35 cleavage and p25 levels,and promoted CDK5 activation and PPARy(Ser273)phosphorylation,increased BACE expression,decreased IDE expression,increased APP production,and Aβ accumulation may be an important cause of inflammatory reactions and neuronal damage in the brain.