The Mechanism Of PM_2.5 Induced Bile Acid Metabolism Disorder In Hepatocytes Through Activation Of Farnesoid X Receptor （FXR） Pathway

Objective:The pollution of fine particulate matter(PM_2.5)in the air leads to many adverse health effects in human beings,not only limited to respiratory tract and lung damage,but also its toxic substances have invaded the body and caused harm to the liver.There has been evidence that PM_2.5can cause disruption of bile acid metabolism in liver cells,but the specific mechanism is still unclear.The objective of this study was to elucidate the specific molecular regulatory mechanism of PM_2.5induced bile acid metabolism disorder in hepatocytes.Methods:PM_2.5（fine particulate matter）was purified into PM_2.5extract,particulate free organic extract and water-soluble fraction respectively by Soxhlet extraction and vacuum freeze-drying;The disequilibrium of bile acid metabolism caused by PM_2.5extract,organic extract,water-soluble fraction,benzo（a）pyrene and cadmium was analyzed using sandwich culture model of rat and mouse primary hepatocytes;Oil red O staining,triglyceride,total cholesterol and total bile acid were used to demonstrate the effects of PM_2.5extract on lipid metabolism and bile acid metabolism in hepatocytes;The effects of PM_2.5extract,organic extract,water-soluble fraction,benzo（a）pyrene and cadmium on the bile acid transport capacity of hepatocytes were investigated by bile acid flow assay;KEGG assay pathway was used to explore potential bile acid-related dysexpression genes in hepatocytes;The specific activator GW4064 of FXR was used to screen potential bile acid-related genes affected by FXR;The binding effects of PM_2.5extract,organic extract and water-soluble fraction with FXR were demonstrated by receptor competitive binding experiments;The FXR-mediated dual luciferase reporter gene was used to verify the activation ability of PM_2.5extract,organic extract,water-soluble fraction,benzo（a）pyrene and cadmium to FXR.The effect of PM_2.5extract on bile acid-related gene expression was demonstrated by RT-q PCR and western blotting;The expression of FXR in primary mouse hepatocytes was knocked down by si RNA sequence to verify the key role of FXR in the disturbance of bile acid metabolism caused by PM_2.5.Results:After exposure of PM_2.5extract to rat and mouse primary hepatocytes for 48h,PM_2.5promoted accumulation of lipid droplets,triglycerides and total cholesterol in mouse primary hepatocytes in a dose-dependent way,and decreased the level of total bile acid in mouse primary hepatocytes.The bile acid flow experiment demonstrated that PM_2.5could enhance the bile acid transport ability of primary rat hepatocytes,indicating that PM_2.5interfered with lipid metabolism and bile acid metabolic balance in hepatocytes.Fourteen bile acid metabolisation-related genes were extracted by KEGG pathway analysis,and three bile acid metabolisation-related genes（CYP7A1,ABCB11 and ABCC2）were screened by FXR specific activators GW4064 and PM_2.5.RT-q PCR and western blotting showed that PM_2.5significantly inhibited the expression of bile acid synthesis gene CYP7A1,and promoted the expression of bile acid transporter genes ABCB11 and ABCC2.The competitive binding experiment showed that the PM_2.5extract could directly bind to the ligand domain of FXR,and the IC₅₀was about 21.5μg/m L.The FXR-mediated dual luciferase reporter gene experiment showed that PM_2.5extract could significantly improve the transcriptional activity of human and mouse FXR.The minimum effective concentration was 50μg/m L,and the activation effect was about 2.7times at 100μg/m L.After FXR knockdown,the perturbation trend of key target genes induced by PM_2.5was weakened.Receptor competitive binding experiments showed that both organic extracts（OEs）and water-soluble fractions（WSFs）could directly bind FXR receptor,with IC₅₀values of25.7μg/m L and 51.3μg/m L,respectively.Dual luciferase reporter gene assay showed that both organic phase and aqueous phase could promote the transcriptional activity of human and mouse FXR.The lowest effective concentrations of FXR in OEs and WSFs were 12.5μg/m L and 25μg/m L for human,and 12.5μg/m L and 50μg/m L for mouse.,respectively.At 100μg/m L,the activation effect of OEs and WSFs was about 6 and 1.8times that of human FXR,and about 4 and 1.3 times that of mouse FXR,respectively.Human and mouse FXR were not activated by carbon black.After exposure to rat and mouse primary hepatocytes for 48h,both OEs and WSFs interfered with the m RNA and protein expression levels of bile acid genes（CYP7A1,ABCB11 and ABCC2）,inhibited the total bile acid content in mouse primary hepatocytes,and promoted the increase of bile duct like structures in rat primary hepatocytes.Benzopyrene（Ba P）can directly bind to the ligand domain of FXR,with IC₅₀of167.28μM.Ba P and chromium chloride（Cd Cl₂）both activated FXR transcriptional activity in a dose-dependent manner.The lowest effective dose of Ba P was 10μM,and the highest activation effect was 1.9 times.The lowest effective dose of Cd Cl₂was 0.25μM and the highest activation effect was 1.5 times.Both Ba P and Cd Cl₂inhibited the expression of bile acid synthesis gene and increased the expression of bile acid transporter gene by RT-q PCR.The bile acid flow experiment showed that Ba P and Cd Cl2 enhanced the bile acid transport ability and promoted the bile acid secretion of rat primary hepatocytes.Conclusion:PM_2.5extract can directly bind to and activate FXR receptor,thus interfering with the expression level of key genes in Fx R-mediated bile acid pathway,and ultimately leading to the disorder of bile acid metabolism in hepatocytes.Benzopyrene in the OEs and cadmium in the WSFs play key roles in the disruption of bile acid metabolism induced by PM_2.5through the FXR receptor pathway.