Study On The Effect Of Combined Exposure Of DEHP And DBP On Glucose Metabolism Of Female Rats Via Mitochondrial Apoptosis Pathway

Objective To observe the serum insulin level of female rats,changes in oxidative stress and apoptotic proteins by exposing phthalate（2-ethylhexyl）（di-2-ethylhexyl phthalate,DEHP）and di-n-buty phthalate（di-n-buty phthalate,DBP）,to explore the possible mechanism of phthalate combined exposure to glucose metabolism disorders in rats,for the study of metabolic diseases such as type 2 diabetes（Type 2 Diabetes mellitus,T2DM）and prevention provides new ideas.Methods Forty three-week-old healthy and clean-grade female SD rats were selected and randomly divided into 4 groups with 10 rats in each group.They were the control group（corn oil group）、the DEHP group(1/40LD₅₀,750mg/kg)、the DBP group(1/40LD₅₀,500mg/kg)and the DEHP+DBP（750mg/kg+500mg/kg）group.In the drug-exposed group,gavage was given at 8 o’clock in the morning every day for 5 days a week for 8 consecutive weeks.Weighing the body weight of the rats every week;weighing the food intake and water intake every two weeks,and cut the tails of the rats,using a blood glucose meter to measure fasting blood glucose.Weighing the pancreas and organ coefficients at the end of the 8th week of exposure.Elisa method was used to measure Insulin level of serum,calculating the insulin resistance index（HOMA-IR）and insulin sensitivity index（ISI）;and measuring SOD activity and MDA content of oxidative stress indicators.q RT-PCR method was used to determine the mRNA expression levels of Bax,Bcl-2,Caspase9 and Caspase3 in pancreatic tissue;Western blot method was used to determine the protein expression levels of Bax,Bcl-2,Caspase9 and Caspase3 in pancreatic tissue.Results（1）During the exposure process,the back hair skin of the rats in the DEHP、DBP、DEHP+DBP exposure group did not feel shiny and some had slight hair loss,while the control group had a good mental state during the whole experiment without hair loss.（2）After8 weeks of exposure,there was no significant difference in body weight and food intake of rats in each exposure group（P>0.05）;while the water intake of rats in the DEHP、DBP and DEHP+DBP exposure groups was increased（P<0.05）.（3）After 8 weeks of exposure,there was no significant difference in the weight and organ coefficient of the pancreas tissue of rats in each group（P>0.05）.（4）In the DEHP+DBP combined treatment group,the fasting blood glucose level increased significantly at the 4th week of exposure,and the insulin level and HOMA-IR at the 8th week of exposure were increased（P<0.05）,and ISI was significantly lower than the control group（P<0.05）;compared with the DEHP+DBP combined exposure group,the difference between DEHP and DBP alone exposure was significantly different（P<0.05）.（5）Compared with the control group,the SOD activity of the DEHP,DBP,DEHP+DBP exposure group decreased,and the MDA content increased（P<0.05）.（6）Compared with the control group,the expression levels of Bax、Caspase9 and Caspase3mRNA in the DEHP、DBP and DEHP+DBP-exposed groups increased,while the expression levels of Bcl-2 mRNA and the ratio of Bcl-2/Bax decreased（P<0.05）;compared with the DEHP+DBP combined exposure group,the Caspase3 mRNA expression levels of DEHP and DBP alone were significantly different（P<0.05）.（7）Compared with the control group,the protein expression levels of Bax,Caspase9,cleaved-Caspase9,Caspase3,and cleaved-Caspase3 in the DEHP、DBP and DEHP+DBP-exposed groups increased,while the the protein expression levels of Bcl-2 and the ratio of Bcl-2/Bax decreased（P<0.05）;compared with the DEHP+DBP combined exposure group,DEHP and DBP alone had significant differences in the protein expression levels of cleaved-Caspase9,Caspase3,and cleaved-Caspase3（P<0.05）.Conclusion DEHP+DBP combined exposure can cause glucose metabolism disorders in rats;this process may be caused by the destruction of the balance of oxidation-antioxidant system,which activates the expression of apoptosis proteins related to cell mitochondrial apoptosis pathway and induces pancreaticβcells to undergo apoptosis.