Promotion Of DEHP On Obesity Of Female Mice Induced By High-fat Diet

ObjectiveObesity is the main risk factor of hypertension,diabetes,dyslipidemia,coronary heart disease,myocardial infarction,breast cancer and other diseases,It is recognized by the World Health Organization as the fifth risk factor affecting health.At present,it is generally believed that high-fat diet and low physical activity are the main causes of obesity,but it is still unable to explain the rapid increase of obesity and overweight population in the world in recent years.It is found that some endocrine disruptors may cause the disorder of energy metabolism and promote or induce the occurrence of obesity,which are called obesogens.Diethylhexyl phthalate,as a plasticizer,widely exists in the living environment,and it is easy to escape from plastic products into the air,soil,water and other environments,and enter people through diet,drinking water,respiration,skin and other ways,According to the study of population exposure assessment,the risk of DEHP long-term low-dose exposure may exist in the living environment.Animal experiments and population surveys have proved that DEHP has reproductive toxicity,immunotoxicity,embryotoxicity,neurotoxicity and carcinogenicity.As an obesogens,DEHP exposure is closely related to obesity,type Ⅱ diabetes,dyslipidemia and other metabolic syndrome.Experimental studies have proved that DEHP exposure can significantly improve the expression level of PPAR γ in adipose tissue,and increase the table of downstream related genes,As a result,the body weight,fat tissue,serum cholesterol and blood glucose were increased,which affected the metabolism of glucose and lipid and promoted the occurrence of obesity.It was also found that DEHP exposed mice showed significant increase in food intake and decrease in average body temperature during the day,and the expression of key neuropeptides in hypothalamus,the central organ regulated by energy metabolism,was disordered.It was suspected that DEHP exposure might induce obesity by interfering with the homeostasis of energy metabolism.In recent years,domestic and foreign scholars have studied the relationship between DEHP and its metabolites and obesity through animal experiments and epidemiological studies,but the research results have not been unified,and most of the doses used in animal experiments are higher than the environmental exposure concentration and the tolerable daily intake.In the actual living environment,people’s exposure is often long-term low-dose and through a variety of ways,and high-fat diet and DEHP are mostly exposed by mouth at the same time.Therefore,in this experimental study,DEHP of daily exposure dose of human body was exposed through mouth,simulating the normal diet and high-fat diet of human.To explore the effect of long-term low-dose DEHP exposure on the metabolic function of mice,and to explore the mechanism of DEHP promoting obesity by detecting the energy metabolism indexes such as glucose and lipid metabolism,as well as the effect on intestinal flora and short chain fatty acids,and to observe whether DEHP can promote obesity under the condition of high-fat diet.At the same time,we used DEHP to overexpress NIH in PPAR γ cultured in vitro-3T3 cells,further observed the relationship between PPAR γ and adipogenesis,in order to provide basic data for the effective intervention of obesity,and better evaluate the impact of DEHP on the environment and the population,provide scientific basis for its risk assessment and risk management,and reduce the potential risk of population exposure.Methods1.In vivo experiment1.1 Group and treatment of experimental animals:48 8-week-old(18-20g)female C57BL/6 mice were randomly divided into control group and 0.05mg/kg.bw DEHP group,HFD group,HFD+0.05mg/kg.bw DEHP group,12 in each group.The body weight and food consumption of the animals were recorded every week.After 28 weeks of DEHP exposure,the animals were dislocated and killed.1.2 Glucose tolerance test and insulin tolerance test:glucose tolerance test(GTT)and insulin tolerance test(ITT)were carried out at the end of the 16th and 28th week respectively.After 6 hours of fasting,blood samples were taken from tail vein to measure blood glucose,which was recorded as 0 min blood glucose,and then 2 g was injected intraperitoneally/kg.bw Glucose or 0.5u insulin/kg.bw 15,30,60,90,120 Blood glucose value at Min,draw the curve of blood glucose change,and calculate the area under the curve(AUC)1.3 Detection of energy metabolism:Six mice in each group were taken to monitor the metabolic cage at 25 weeks,and the metabolic data within 24 hours were recorded,including respiratory exchange rate(RER),oxygen consumption(VO2),carbon dioxide emission(VCO2)and heat production(heat)1.4 Body composition analysis:the mice in each group were anesthetized in 28 weeks.Lean mass and fat mass were measured by the dual energy X-ray absorptiometry(xr600).1.5 Morphological examination:paraffin sections of mouse adipose tissue and liver tissue were stained with he,cell morphology was observed under the microscope,cell oil red "O" staining,cell immunohistochemistry staining,microscopic observation and photo recording.1.6 Western blot:to detect the relative protein expression of UCP1,OXPHOS,PPARγ,Cdk5,p-ppar γ,adiponectin and perilipin in mouse adipose tissue;to detect the expression of PPAR γ,Cdk5 and p-ppar y in cells.1.7 Determination of gut microbiota and short chain fatty acids:the abundance,diversity and dominant microflora of intestinal microflora in mice faeces were detected by 16S rRNA amplicon sequencing,and the content of short chain fatty acids in mice faeces was determined by target metabolite detection.2.In vitro experiment2.1 Culture and experimental treatment of NIH-3T3 cells:NiH-3T3(PPARγ2 overexpressed)mouse embryonic fibroblasts were given DEHP with final concentration of 10μM,2μM,50μM,and 100μM,respectively.The blank group was given the same volume of culture medium,and the positive control group was added with troglitazone with final concentration of 0.1 μM.2.2 Index detection:Oil red"O" staining and immunohistochemical staining were performed after adding the subject for 4 day.The expression of PPAR,CDK5 and p-PPAR were detected by Western blot.Results1.In vivo experiment1.1 Changes of body weight and food consumption of each group:the body weight of DEHP exposed mice was higher than that of the control group,but there was no statistical difference(P>0.05).High fat feed 0.05mg/kg.bw The body weight of DEHP group was significantly higher than that of the high-fat control group(P<0.05)at the end of the 6th week,and then the body weight kept constant growth.The body weight continued to increase from the 12th week to the end of the experiment,which was significantly different from that of the high-fat control group(P<0.05).The food consumption and total calorie intake of DEHP exposed mice were significantly higher than those of the control group(P<0.05).In the two groups of animals fed with high-fat diet,the food utilization rate of DEHP exposed mice increased(P<0.01),and there was no statistical difference between the amount of food intake and total calories intake and the high-fat control group(P>0.05).1.2 Changes of body composition and fat content in each group:under the condition of conventional feed,the lean body weight and fat content in DEHP exposed group were higher than those in the control group,but the difference was not statistically significant(P>0.05).The body fat and lean body weight of DEHP exposed animals were significantly higher than that of the pure high fat feed group(P<0.05).Compared with the control group,the white fat including visceral fat/body weight and subcutaneous fat/body weight ratio in DEHP exposed mice increased,but the difference was not statistically significant(P>0.05).The visceral fat/body weight and subcutaneous fat/body weight of DEHP group were significantly higher than those of high-fat group(P<0.05).The weight/body weight of brown fat in DEHP exposed mice showed a decreasing trend under the condition of conventional feed and high fat feed,but the difference was not statistically significant(P>0.05).Under different dietary conditions,the fat cell volume of DEHP exposed mice increased significantly,and the fat drop vacuoles could be seen in the liver cells.1.3 Changes of glucose metabolism in each group:the results of GTT in the middle of the experiment showed that the blood glucose and AUC in the ordinary feed group were higher than those in the control group at 30min and 60min(P<0.05);in the high-fat feed group,the blood glucose and AUC in the DEHP combined exposure group at 15min,60min;90min and 120min were higher than those in the simple high-fat feed group,and the difference was statistically significant(P<0.05)Significance(P<0.05).The results of ITT in the middle period of the experiment showed that the blood glucose in the DEHP exposed group was significantly higher than that in the control group(P<0.05)at 60 minutes,but there was no significant difference in other time points and AUC(P>0.05).The blood glucose and AUC of the mice exposed to high-fat diet combined with DEHP at 60,90 and 120 minutes were significantly higher than those of the mice exposed to high-fat diet alone(P<0.05).At the end of the experiment,GTT results showed that there was no significant difference in blood glucose level and AUC between the conventional diet groups.The blood glucose and AUC of the mice in DEHP exposed group were significantly higher than those in non exposed group(P<0.01).At the end of the experiment,ITT results showed that there was no significant difference in blood glucose level and AUC at each time point between the conventional feed groups.The changes of blood glucose and AUC of DEHP exposed mice were significantly higher than those of DEHP non exposed mice(P<0.01).1.4 Changes of energy metabolism of animals in each group:DEHP exposure had no significant effect on RER in the conventional feed diet group,but the carbon dioxide emissions in the daytime and at night were reduced,and the oxygen consumption in the daytime was reduced compared with the control group,the difference was statistically significant(P<0.05),and there was no significant difference in heat production.In the high fat group,the RER,carbon dioxide emission,oxygen consumption and heat production of DEHP group were significantly lower than those of the control group(P<0.01).1.5 Changes of PPAR γ and energy metabolism related protein expression in adipose tissue of animals:compared with the control group,the expression of PPARγ protein in DEHP exposed group increased significantly(P<0.05),and there was no significant difference between the high-fat group and the high-fat group.The expression of p-ppar y(ser273)protein was significantly increased in DEHP exposure under different dietary conditions(P<0.05).Under the two dietary conditions,the expression of adiponectin decreased in DEHP exposed group(P<0.05).In the conventional diet group,the expression level of perilipin protein in the DEHP group increased significantly(P<0.01),but in the high-fat group,it decreased,but there was no statistical difference.1.6 The changes of intestinal flora and fatty acid in each group:DEHP and high fat diet can reduce the abundance and diversity of intestinal flora,reduce the number of dominant flora in intestinal flora,the F/b value of DEHP combined with high fat diet group is significantly higher than that of high fat control group(P<0.05).High fat diet and DEHP exposure can significantly reduce SCFA content except propionic acid,including acetic acid,butyric acid,valeric acid,isobutyric acid and isovaleric acid,among which butyric acid is the most obvious.2.In vitro experiment2.1 Effect of different doses of DEHP on the formation of lipid droplets:compared with the control group,the number of lipid droplets in DEHP group increased in a dose-dependent manner.2.2CEffects of different doses of DEHP on the expression of perilipin in cell fat:the expression of perilipin increased in different doses of DEHP group,especially in 100μM group.2.3 The effect of different doses of DEHP on the expression of PPAR γ,p-ppar γ(ser273),Cdk5 in cells:the effect of different doses of DEHP on the expression of PPAR γ,p-ppar y(ser273),Cdk5 in cells:Western blot showed that the expression of PPAR γ in cells of different doses of DEHP was not significantly different in each group(P>0.05),but the expression of p-ppar γ was significantly increased,with statistical differenceConclusion1.DEHP can increase the food consumption and the expression of PPAR γ and its downstream related proteins in adipose tissue after long-term low-dose exposure to normal diet.In vitro cell experiments showed that DEHP promoted the lipogenic differentiation of NIH-3T3(PPAR γ 2 overexpression)cells and affected the expression of PPAR γ and its downstream related proteins.2.High fat diet combined with DEHP long-term low-dose exposure can promote animal weight gain and fat accumulation,aggravate the abnormal metabolism of glucose and lipid,and aggravate the disorder of energy metabolism.3.DEHP exposure changed the abundance and diversity of intestinal microflora and the proportion of dominant microflora,and significantly reduced the SCFA content except propionic acid.