Effects Of Bisphenol A On Circadian Clock And Glycolipid Metabolism In Mouse Liver

Bisphenol propane,also known as bisphenol A(BPA),is an important industrial compound commonly used in the manufacture of everyday plastic products.Studies have shown that BPA,as an environmental endocrine disruptor,can enter the body through the digestive tract,respiratory tract,skin and other pathways,and affect tissues and organs by binding to cell surface receptors,causing disorders of reproductive,metabolic and endocrine functions in mammals.As an important metabolic organ of mammals,the liver has the physiological functions of regulating glycolipid metabolism and secreting bile.Studies have found that long-term intake of BPA can cause steatosis in the animal liver,cause non-alcoholic fatty liver,and damage the glycolipid metabolism function of the mammalian liver.However,the specific mechanism of action by BPA exposure on liver glycolipid metabolism remains unclear.As a conserved life activity regulation system,the circadian clock is involved in regulating the rhythmic expression of a variety of key genes in glycolipid metabolism,thus playing an important role in maintaining the homeostasis of glucose and lipid metabolism in mammals.Previous research in our group found that BPA inhibits testosterone synthesis in mice by causing the decrease of the circadian clock gene Nr1d1,but whether BPA affects the liver clock and glycolipid metabolism function of mice is still unclear and further exploration is needed.In this study,we first established a BPA exposure model using wild-type male mice and mouse liver parenchymal cells(AML12 cell line),and then systematically explored the effects of BPA on the circadian clock system and glycolipid metabolism function of mice liver were systematically explored using a variety of experimental techniques such as flow cytometry,q PCR,BODIPY fluorescence staining,Western blot(WB),Kronos AB-2550 cell real-time biofluorescence measurement system,kit quantitative detection and other experimental techniques.The specific results are as follows:1.CCK8 and flow cytometry assays showed that the optimal BPA treatment concentration that did not significantly affect the viability and apoptosis of AML12 cells was 100 μM,which was subsequently used to treat AML12 cells.q PCR results showed that BPA treatment of unsynchronized AML12 cells significantly disrupted the expression of m RNA of circadian clock genes(Bmal1,Nr1d1,Per2,Dbp)and glycolipid metabolism-related genes(Srebp1c,Fasn,Hmgcr,Cd36,Lpin1,Pparα,Pparγ,Elovl6 and Glut2).The results of BODIPY fluorescence staining showed that BPA treatment had no significant effect on lipid accumulation in unsynchronized AML12 cells.The WB results showed that after BPA treatment of unsynchronized AML12 cells for 24 h,the expression of the circadian clock protein BMAL1 was significantly reduced.The results of the Kronos AB-2550 cell real-time biofluorescence assay system showed that BPA treatment significantly reduced the luciferase activity of Per2::Luc in the liver tissue block of Per2::Luc mice.After the AML12 cells were synchronized,the circadian clock gene(Bmal1,Nr1d1)m RNA of the control group and the BPA-treated group had significant rhythmic expression,and the expression of the circadian clock gene Bmal1 was significantly increased by BPA treatment.At the same time,BPA significantly inhibited the expression of Hmgcr m RNA in AML12 cells and promoted the expression of Pparαm RNA in AML12 cells.2.In order to further explore the effect of BPA on the circadian clock system and glycolipid metabolism function of mice,the modeling method of using 50 μg/kg/day BPA for 6 weeks was further determined by reviewing the references.During gavage,mice are recorded for weight(every other day),feed intake,and water intake(every two days).After 6 weeks of gavage,mouse activity rhythms are detected using the mouse runner rhythm monitoring system.Compared with the control group,the results of the mouse runner rhythm monitoring system,feed intake and water intake showed that compared with the control group,the endogenous cycle was prolonged,and the water intake and feed intake were significantly increased in the BPA treatment group.The results of allday blood glucose test,glucose tolerance and insulin tolerance test showed that the BPA treatment group had a significant decrease in the full-day blood glucose concentration compared with the control group.The metabolic rate and insulin sensitivity of glucose in the BPA-treated group were significantly lower than those in the control group.In the glucose and insulin tolerance tests,the area under the curve in the BPA-treated group was significantly higher than that in the control group.There were no significant differences in the content of liver glycogen and serum and liver triglyceride using glucose detection kit and triglyceride detection kit,respectively.The results of q PCR and WB showed that the expression of m RNA of liver clock genes(Bmal1,Nr1d1,Dbp and Per2)in the control group and BPA-treated were rhythmically expressed,but the amplitude and phase of m RNA expression of circadian clock gene in the BPA-treated group changed.BPA reduced the expression of the mouse liver clock gene Bmal1 and lipid metabolism genes Srebp-1c,Fasn,Hmgcr and Elovl6 m RNA,while BPA increased the expression of the circadian clock genes Dbp,Nr1d1 and glycolipid metabolism genes G6 pc,Cyp7a1 and Cd36 m RNA.BPA treatment significantly reduced BMAL1 and DBP proteins,but significantly increased the expression of NR1D1,FASN,and SREBP1 C proteins.In summary,BPA treatment significantly altered the expression of circadian clock genes and some glycolipid metabolism-related genes in AML12 cells.And BPA treatment caused significant prolongation of the endogenous cycle in mice at the in vivo level,causing a decrease in blood glucose with insulin sensitivity,and disrupting blood glucose homeostasis.Meanwhile,BPA treatment disrupted the expression of mouse liver clock genes(Bmal1,Nr1d1 and Dbp)and glycolipid metabolism-related genes(Srebp1c,Cd36,Elovl6,G6 pc and Glut2).This study provides a preliminary experimental basis for the in-depth analysis of the mechanism of BPA affecting the liver circadian clock and glycolipid metabolism,and provides some theoretical support for exploring the hepatotoxicity of BPA from the perspective of chronotoxicology.