The Study Of Perfluorinated Compound GenX On Liver Lipid Metabolism And Its Mechanism

Background and Objective:The liver is an important metabolic organ in the human body,and abnormal liver lipid metabolism can lead to a variety of diseases.The perfluorinated compound GenX is a novel compound,and the liver is the main target organ of GenX.With the widespread use of GenX,the effects of GenX on the liver are worth attention,so this study was conducted to investigate the effects and possible mechanisms of the perfluorinated compound GenX on abnormal hepatic lipid metabolism.Methods:1.Animal experiment:62 mice were randomly divided into normal group(NC group)(n=12),0.1 ppb group(n=12),10 ppb group(n=12),1 ppm group(n=12)and 100 ppm group(n=14).The corresponding concentration of GenX was added to the drinking water of each group for exposure treatment,and the body weight and dietary water intake of the mice were collected weekly after the start of the experiment.At the end of the experiment,serum levels of aspartate amino transferase(AST),alanine transaminase(ALT)and alkaline phosphatase(ALP)were measured to evaluate the extent of liver damage caused by GenX;Triglyceride(TG)and total cholesterol(TC)levels were measured.The tissue samples were stained with HE,oil red O and MASSON.qRT-PCR and Western-blot were performed to detect the expression levels of PPAR pathway-related genes and proteins in each group.For the KEGG and GO analysis of the effect of GenX on liver,the Gene Cards,OMIIM,TTD and Drug Bank databases were mined for relevant targets using "GenX" and "liver" and as search terms.After merging the targets of the four databases,the duplicate values were removed and the relevant targets were obtained.Based on DAVID database,GO and KEGG enrichment analysis was performed on the intersection targets of GenX and liver.The analysis results were sorted by Pvalue,and the top 10 results were selected for each analysis,and the results were visualized by R language programming.2.Cell experiment:HepG2 cells were treated with GenX for 24h and 48h,and the group of PPARa inhibitor GW6471 was also set.After exposure,the levels of TG and TC in the cells of each group were measured,and Bodiypy staining was used to observe the changes in lipid droplets in the cells.The expression levels of key genes and proteins of PPARa signaling pathway were detected.Results:Animal experiments:(1)GenX exposure caused damage to the liver of mice.The serum levels of glutamate aminotransferase(AST),glutamic acid aminotransferase(ALT)and alkaline phosphatase(ALP)in mice were significantly increased after GenX exposure;as the dose increased,the level of AST also increased,showing a dose-response relationship.(2)GenX exposure increased lipids in the liver of mice.In the 1 ppm and 100 ppm treated groups,the level of TG in mouse liver was significantly increased,and the level of TC in mouse serum and liver were also significantly elevated.HE staining of liver sections showed an increase in white fat vacuoles and irregular cell arrangement in the mouse liver after GenX exposure.Oil red O staining of liver sections revealed an increasing trend of red lipid droplets in the mouse liver tissue with increasing doses of GenX.In the high-dose group,there was a significant increase in lipid droplets,accompanied by obvious aggregation of the droplets.(3)GenX effects on the liver are associated with the PPAR signaling pathway.The results of KEGG pathway enrichment analysis and GO analysis showed that the effects of GenX on the liver were clustered in fatty acid catabolism and oxidation,enriched in the PPAR signaling pathway,and screened for 23 differential genes.(4)GenX caused disruption of liver lipid metabolism in mice.GenX exposure caused abnormal expression levels of fatty acid transporter-related genes and proteins.The expression levels of fatty acid transport-related proteins,such as fatty acid-binding protein 1(FABP1)and fatty acid-binding protein3(FABP3)were elevated.Cluster of differentiation 36(CD36)mRNA expression was also elevated in the treated group.Compared with the control group,the mRNA levels of malic enzyme 1(ME1),stearoyl-CoA desaturase 2(SCD2)and long-chain fatty acid transporter protein 1(SLC27A1)were significantly increased.The mRNA levels of stearoyl-CoA desaturase 1(SCD1)were not significantly different among the groups.Compared with the control group,the mRNA expression levels of acyl-coenzyme A oxidase 1(ACOX1),acyl-coenzyme A dehydrogenase long chain(ACADL),acetylcoenzyme A transferase 1(ACADL)and acyltransferase 1(ACAA1)were significantly higher in each treated groups.ACAA1b mRNA expression levels were significantly up-regulated in each treatment group;the expression levels of ACAA1 mRNA increased with the increasing dose,showing a dose-response relationship;enoyl-coenzyme A hydratase/3-hydroxyacylcoenzyme A dehydrogenase(EHHADH)mRNA expression levels did not change significantly;carnitine palmitoyl transferase 1B(CPT1β)mRNA expression levels decreased with increasing dose,showing a dose-response relationship.The expression levels of ACOX1 protein and SCD1 protein were significantly increased in each treatment group after GenX exposure,and the expression levels of ACOX1 protein showed a dose-response relationship in each treatment group.The expression levels of fatty acid synthase(FASN)protein,were significantly increased in the 0.1 ppb and 100 ppm treatment groups.Immunofluorescence results showed that CD36 protein levels were significantly elevated in the GenX-treated group;PPARa protein levels were significantly elevated in all other treatment groups compared with the control group;within the treatment groups,PPARa protein levels were highest in the 100 ppm-treated group and least different from the control group in the 1 ppm-treated group.Cell experiments:(1)GenX induced intracellular lipid deposition in HepG2 cellsAfter GenX exposure,intracellular TC levels in HepG2 cells were significantly higher compared with the control group,and TG levels were not significantly different compared with the control group;Bodipy assay revealed that the fluorescence intensity in the GenXtreated group alone became significantly stronger compared with the control group,and the fluorescence intensity was significantly weakened after the application of PPARα inhibitor GW6471.(2)GenX regulates lipid metabolism and fatty acid oxidation by activating PPARαThe mRNA level of peroxisome activator receptor(PPARα)was significantly increased in HepG2 cells after GenX treatment,and the mRNA expression levels ofACOX1,EHHADH,CYP4A12b,SCD1,A CADL,ACAA1b and CPT1B were up-regulated.The mRNA expression levels of ME1,SCD1,FASN and SREBP1 C were also up-regulated.The protein expression levels of FABP1 were significantly higher in the GenX group compared with the control group;the protein expression levels of FASN were also significantly higher in each treatment group compared with the control group;the protein expression levels of acetyl-coenzyme A carboxylase(ACC)were increased in each treatment group but not significantly different compare with the control group;the protein expression levels of carnitine palmitoyl transferase(CPT1B)were significantly higher in the GenX alone group compared with the control group.The protein expression level of carnitine CPT1β was significantly higher in the 640 μM group than in the control group,and no significant difference was found between the other groups and the control group.(3)PPARa inhibitor attenuates the effect of GenX on lipid metabolism in HepG2 cells.After adding GW6471 intervention(GW group),the TC level of HepG2 cells was significantly decreased compared with GenX-treated group,and the TG level showed a decreasing trend compared with GenX-treated group but no significance.The results of Bodipy neutral lipid droplet staining showed that the fluorescence intensity of GW group with the application of PPARa inhibitor GW6471 was still higher than that of the control group,but significantly weaker compared with the GenX-treated group.The mRNA expression level of CD36 and fatty acid-binding protein 5(FABP5)was significantly lower in the GW group compared with the GenX group.The mRNA expression level of FABP1 was not significantly different among the groups.The mRNA expression level of PPARa in the GW group was significantly lower compared with that in the GenX group;the level of RXRβ mRNA expression in the GenX group was significantly higher compared with that in the control group,and the level of RXRβ mRNA in the GW group showed a decreasing trend compared with that in the GenX group,but no significant difference was observed.The mRNA expression level of SLC27A1 and EHHADHwere significantly decreased in the GW group compared with the GenX group.CPT1β mRNA expression levels were significantly increased in the GenX group compared with the control group,and significantly decreased in the GW group compared with the control group;ACOX1 mRNA levels were significantly decreased in the GW group compared with the GenX group,and no significant difference was observed compared with the control group.CONCLUSIONS:(1)Exposure to GenX resulted in elevated intrahepatic cholesterol and triglyceride levels,and increased lipid aggregation in mice;disrupted lipid metabolism and increased fatty acid synthesis in the liver,but also enhanced intrahepatic fatty acid βoxidation,which may be due to the compensatory response of the organism.(2)GenX stimulated lipid synthesis and induced fatty acid β-oxidation by inducing PPARa expression in HepG2 cells;when the PPARα pathway was inhibited,the lipid disorder caused by GenX was ameliorated,and its effects on intracellular fatty acid transport,lipid synthesis and fatty acid β-oxidation were attenuated.