| Objective:This topic uses C57BL/6 mice and HepG2 cells to study the effects of Benzo[a]pyrene(BaP)on liver lipid metabolism and gluconeogenesis,as well as BaP through the Aryl hydrocarbon receptor(AhR)the molecular mechanism that regulates liver lipid metabolism and gluconeogenesis.Methods:(1)Gavage C57BL/6 mice with BaP-corn oil solution to establish a BaP interference liver lipid metabolism model:60 mice were randomly divided into blank group,the solvent control group,BaP low,medium and high dose groups.Each group of 12 animals,the solvent control group was given corn oil by gavage at 0.1mL/10g/d,and the BaP low,medium and high dose groups were gavaged 0.45mg/kg/d,0.90mg/kg/d and 1.80mg/kg respectively/d BaP-corn oil solution,after 12 weeks of continuous modeling and administration,animal materials and related experiments were carried out.Take the liver tissues of C57BL/6 mice to detect the effects of BaP on Alanine aminotransferase(ALT)and Aspartate aminotransferase(AST)in the liver tissues of mice;observe the effects of BaP on C57BL/6 mice by oil red O staining accumulation of liver lipids;qPCR and Western blot methods to detect fat production and transport related factors(PPARγ,SCD1 and CD36),fatty acid β-oxidation related factors(CPT-la,PPARa and PGCla),and gluconeogenesis related factors in liver tissue the expression of factors(FBP1,G6Pase and PCK1)and CYP1A1 and FGF21.(2)HepG2 cells were given 0.1μmol/L,1μmol/L and 10μmol/L BaP,and then HepG2 cells were given 1μmol/L BaP in the presence of the AhR inhibitor CH223191,and the lipid content of the cells was observed by oil red O staining;qPCR The expression of PPARγ,SCD1,CD36,CPT-1α,PPARα,PGC1α,FBP1,G6Pase,PCK1,CYP1A1 and FGF 21 were detected by method and Western blot method;the effect of BaP on FGF21 in HepG2 cells was detected by immunofluorescence.Results:1.In vivo experimental results:(1)After 12 weeks,mouse liver tissues were taken for relevant experimental verification.Compared with the solvent control group,the levels of ALT in mice in the low,medium and high BaP dose groups were significantly increased,and the difference was statistically significant(P<0.05,P<0.01,P<0.001);the BaP medium and high dose groups were small the level of AST in mice increased significantly,and the difference was statistically significant(P<0.05,P<0.001).Through the analysis of HALO software,compared with the solvent control group,the lipid accumulation in the low,medium and high dose groups of BaP was obvious,and the difference between the medium and high dose groups of BaP was statistically significant(P<0.01,P<0.001).(2)The qPCR results showed that compared with the solvent control group,the expression of CD36 mRNA in the liver tissues of mice in the low,medium and high doses of BaP in the adipogenesis and transport related factors was significantly increased,and the difference was statistically significant(P<0.01,P<0.001);the mRNA expressions of PPARy and SCD1 in BaP medium and high dose groups were significantly increased,and the difference was statistically significant(P<0.01,P<0.001).Compared with the solvent control group,the low,medium and high doses of BaP in fatty acid β-oxidation related factors significantly increased the expression of CPT-1α mRNA,and the difference was statistically significant(P<0.01,P<0.001);in BaP The expressions of PPARα and PGC1α mRNA in the high-dose group were significantly increased,and the difference was statistically significant(P<0.01,P<0.001).Compared with the solvent control group,the expression of FBP1,G6Pase and PCK1 mRNA in the medium and high dose groups of BaP in gluconeogenesis-related factors were significantly increased,and the difference was statistically significant(P<0.001).Compared with the solvent control group,the expression of CYP1A1 and FGF21 mRNA in the BaP low,medium and high dose groups were significantly increased,and the difference was statistically significant(P<0.01,P<0.001).(3)The results of Western blot showed that compared with the solvent control group,the expression of PPARy,SCD1 and CD36 proteins in the low,medium and high doses of BaP in the lipogenesis and transport related factors increased and the difference was statistically significant(P<0.05,P<0.01,P<0.001).Compared with the solvent control group,the protein expressions of PPARα,PGC1α and CPT-1α in the middle and high dose groups of BaP in the fatty acid β-oxidati on related factors were statistically significant(P<0.01,P<0.001).Compared with the solvent control group,the expression of G6Pase and PCK1 protein in the low,medium and high doses of BaP in the gluconeogenesis-related factors increased and the difference was statistically significant(P<0.01,P<0.001);medium and high doses of BaP The expression of FBP1 protein in the group increased and the difference was statistically significant(P<0.05,P<0.001).Compared with the solvent control group,the expression of CYP1A1 and FGF21 protein in the low,medium and high BaP dose groups increased and the difference was statistically significant(P<0.01,P<0.001).2.In vitro experimental results:(1)HepG2 cells were given 0.1,1,10 μmol/L BaP,oil red O staining and HALO software analysis results showed that with the increase of BaP concentration,cell lipids aggregated,BaP 0.1,1,compared with the blank group,the difference of 10 μmol/L was statistically significant(P<0.01,P<0.001).(2)The qPCR results showed that the mRNA expression of the blank group was set to 1.Compared with the blank group,the mRNA expression of PPARy,SCD1 and CD36 increased to varying degrees after the administration of 1 μmol/L and 10 μmol/L BaP,and the difference was statistically significant.Significance(P<0.05,P<0.01,P<0.001);compared with the blank group,the expression of PPARa,PGC1α and CPT-1α mRNA in the BaP group of 1 and 10 μmol/L increased and the difference was statistically significant(P<0.05,P<0.01,P<0.001);compared with the blank group,administration of 0.1,1,10 μmol/L BaP increased the expression of FBP1,G6Pase and PCK1 mRNA to varying degrees,and 1,10 μmol/L the expression of FBP1,G6Pase and PCK1 mRNA in the BaP group increased,and the difference was statistically significant(P<0.01,P<0.001);compared with the blank group,the expression of CYP1A1 mRNA in the BaP group of 1,10μmol/L was significantly increased,the difference was significant It was statistically significant(P<0.01,P<0.001);compared with the blank group,the FGF21 mRNA expression in the 1,10 μmol/L BaP group was significantly increased,and the difference was statistically significant(P<0.01,P<0.001).(3)Western blot results showed that compared with the blank group,1μmol/L and 10μmol/L BaP could promote the protein expression of PPARy,SCD1 and CD36,and the difference was statistically significant(P<0.05,P<0.01);compared with the blank group,administration of 1 μmol/L BaP can significantly increase the protein expression of PGC1α and CPT-1α,and the difference is statistically significant(P<0.05);administration of 10 μmol/L BaP can significantly increase PPARα,the difference in protein expression of PGC1α and CPT-1α is statistically significant(P<0.01);compared with the blank group,1 and 10 μmol/L BaP can significantly increase the protein expression of FBP1,G6Pase and PCK1,and the difference is statistically significant Scientific significance(P<0.05,P<0.01,P<0.001);compared with the blank group,the CYP1A1 protein expression in the 1,10μmol/L BaP group was significantly increased,and the difference was statistically significant(P<0.05,P<0.01);compared with the blank group,the FGF21 protein expression in the 1,10 μmol/L BaP group was significantly increased,and the difference was statistically significant(P<0.05,P<0.01).(4)Immunofluorescence showed that with the increase of BaP concentration,the green fluorescence intensity increased,indicating that the expression of FGF21 increased significantly after BaP treatment of HepG2 cells.This topic then clarified the effect of BaP on lipid metabolism and gluconeogenesis by giving AhR inhibitors to HepG2 cells.(5)The qPCR results showed that the blank group mRNA expression was set to 1.The results showed that the BaP group compared with the blank,the CYP1A1 mRNA expression increased,and the difference was statistically significant(P<0.01);BaP+CH223191 group and BaP group In comparison,the expression of CYP1A1 mRNA was reduced,and the difference was statistically significant(P<0.01).Western blot results showed that the expression of CYP1A1 protein in the BaP group was significantly higher than that in the blank,and the difference was statistically significant(P<0.01);the expression of CYP1A1 protein in the BaP+CH223191 group was decreased compared with the BaP group,and the difference was statistically significant(P<0.01).(6)The results of Oil Red O showed that the lipid content of the BaP group was significantly higher than that of the blank group,and the difference was statistically significant(P<0.001).Compared with the BaP group,the lipid content of the BaP+CH223191 group was significantly lower,and the difference was statistically significant(P<0.01).(7)The qPCR results showed that compared with the blank group,the expression of PPARy,SCD1 and CD36 mRNA in the BaP group was significantly increased,and the difference was statistically significant(P<0.001);the BaP+CH223191 group compared with the BaP group,PPARy,SCD1 the expression of CD36 and CD36 mRNA was significantly reduced,and the difference was statistically significant(P<0.05,P<0.01).Compared with the blank group,the expression of PPARα,PGC1α and CPT-1α mRNA in the BaP group was significantly increased,and the difference was statistically significant(P<0.01,P<0.001).Compared with the BaP group,the expression of PPARα,PGC1α and CPT-1α mRNA in the BaP+CH223191 group was significantly reduced,and the difference was statistically significant(P<0.05,P<0.01).Compared with the blank group,the expression of FBP1,G6Pase and PCK1 mRNA in the BaP group was significantly increased,and the difference was statistically significant(P<0.001);compared with the BaP group,the expression of FBP1,G6Pase and PCK1 mRNA in the BaP+CH223191 group was significantly reduced,the difference was statistically significant(P<0.05,P<0.01).Compared with the blank,the expression of FGF21 mRNA in the BaP group was significantly increased,and the difference was statistically significant(P<0.01);compared with the BaP group,the expression of FGF21 mRNA in the BaP+CH223191 group was decreased,and the difference was statistically significant(P<0.05).(8)Western blot results showed that compared with the blank group,the expression of PPARy,SCD1 and CD36 protein in the BaP group was significantly increased,and the difference was statistically significant(P<0.01,P<0.001);the BaP+CH223191 group was similar to the BaP group compared with,the expression of PPARy,SCD1 and CD36 protein was significantly reduced,and the difference was statistically significant(P<0.05,P<0.01).Compared with the blank group,the BaP group had significantly higher protein expressions of PPARα,PGC1α and CPT-1α,and the difference was statistically significant(P<0.01,P<0.001).Compared with the BaP group,the BaP+CH223191 group had significantly lower protein expressions of PPARα,PGC1α and CPT-1α,and the difference was statistically significant(P<0.05).Compared with the blank group,the protein expression of FBP1,G6Pase and PCK1 in the BaP group was significantly increased,and the difference was statistically significant(P<0.01);compared with the BaP group,the protein expression of FBP1,G6Pase and PCK1 in the BaP+CH223191 group was significantly reduced.The difference was statistically significant(P<0.05).Compared with the blank group,the expression of FGF21 protein in the BaP group was significantly increased,and the difference was statistically significant(P<0.01);compared with the BaP group,the BaP+CH223191 group significantly inhibited the expression of FGF21 protein,and the difference was statistically significant(P<0.05).(9)The immunofluorescence results showed that after inhibiting AhR,the green fluorescence intensity of FGF21 in the BaP group was significantly increased compared with the blank group and the BaP+CH223191 group.Conclusion:(1)In vivo experiments have shown that BaP affects liver lipid metabolism and gluconeogenesis by promoting adipogenesis and transport,fatty acid β-oxidation,glucose metabolism,and the expression of CYP1A1 and FGF21 in the liver tissue of C57BL/6 mice.(2)In vitro experiments show that BaP promotes lipid aggregation,adipogenesis and transport,fatty acid β-oxidation,glucose metabolism,and the expression of CYP1A1 and FGF21 in HepG2 cells through AhR,thereby affecting the lipid metabolism and gluconeogenesis of HepG2 cells,and The results of in vivo experiments are consistent,which affects the occurrence and development of NAFLD. |
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