| Objective:In this study,C57BL/6 mice and 3T3L-1 preadipocytes were used to study the effects of benzopyrene(Benzo[a]pyrene,BaP)on adipogenesis and decomposition in adipose tissue and adipocytes,and the mechanism of BaP regulating adipogenesis and decomposition through AhR.Methods:(1)Establishment of a model of lipid metabolism interfered by BaP in C57BL/6 mice by intragastric administration of BaP solution:60 mice were randomly divided into Control,Vehicle,Low-dose?Middle-dose and High-dose BaP groups with 12 mice in each group.The mice were fed with normal diet for 12 weeks.The Vehicle group was given corn oil according to 0.1mL/10g/d,and the low,middle and high BaP groups were given BaP-corn oil mixed solution of 0.45mg/kg/d,0.90mg/kg/d and 1.80mg/kg/d,respectively.The weekly weight changes of C57BL/6 mice were measured during the modeling period.After 12 weeks,orbital blood samples were taken to detect the contents of triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)in serum.Blood was taken from the tip of the tail for glucose tolerance test(GTT)and insulin tolerance test(ITT).The structure and morphology of adipose tissue in epididymis of C57BL/6 mice were observed by paraffin section and HE staining.The epididymal adipose tissue and subcutaneous adipose tissue of C57BL/6 mice were taken to detect the expression of adipogenesis-related factors C/EBP?,PPAR?,FABP4 and inflammatory factors NF-?B,MCP-1?TNF-? in adipose tissue and the expression of CYP1A1,an index reflecting the effect of BaP,in adipose tissue by qPCR and Western Blot methods.(2)The model of lipid metabolism of 3T3-L1 preadipocytes interfered by BaP was established by inducing the differentiation of 3T3-L1 preadipocytes and stimulating with different concentrations of BaP.3T3-L1 preadipocytes were stimulated with low,medium and high concentrations of BaP(0.1 ?M,1 ?M and 10 ?M),respectively,and then 1 ?M BaP was used to stimulate 3T3-L1 preadipocytes in the presence of AhR inhibitor CH223191.The lipid content of 3T3-L1 preadipocytes was observed by oil red O staining,and the expression of CYP1A1,adipogenesis related factors C/EBP?,PPAR?,FABP4 and inflammatory factors NF-?B,MCP-1,TNF-? mRNA were detected by qPCR method.The protein expressions of CYP1A1,C/EBP?,PPAR?,FABP4,mitochondrial ?-oxidation related factors PGC-1? and PPAR? were detected by Western Blot method Results(1)Compared with Vehicle group,the body weight of mice in the middle-dose BaP group and the high-dose BaP group increased slowly from the 8th week and the 6th week,respectively,and the weight of epididymal adipose tissue and subcutaneous adipose tissue in the middle-and high-dose BaP groups decreased significantly compared with the Vehicle group after 12 weeks(P<0.05).Compared with the Vehicle group,the content of TG in the serum of the middle and high dose groups of BaP increased significantly,the content of TC and LDL-C in the serum of the high dose group of BaP increased significantly,and the content of HDL-C decreased significantly.The difference was statistically significant(P<0.05).It is suggested that the middle and high dose of BaP can lead to dyslipidemia in C57BL/6 mice.The results of GTT and ITT showed that compared with the Vehicle group,middle dose and high dose of BaP could lead to abnormal glucose metabolism and insulin resistance in C57BL/6 mice(P<0.05).The results of HE staining showed that compared with the Vehicle group,the volume and lipid content of epididymal adipocytes in the low,middle and high dose groups of BaP were significantly lower than those in the Vehicle group(P<0.05).The results of qPCR showed that compared with the Vehicle group,the expression of C/EBP?,PPARy and FABP4 mRNA in the middle and high dose groups of BaP decreased significantly,while the expression of inflammatory cytokines NF-?B,MCP-1 and TNF-? mRNA increased significantly The results of Western Blot showed that compared with the Vehicle group,the expression of C/EBP? protein in the low,middle and high dose groups of BaP decreased significantly(P<0.05).The expression of PPARy and FABP4 protein in the middle and high dose groups of BaP decreased significantly(P<0.05).(2)the results of in vitro experiment:Part 1:The differentiated 3T3-L1 adipocytes were induced by different concentrations of BaP.The results of oil red O staining showed that 1 ?M BaP and 10 ?M BaP significantly decreased the lipid content of 3T3-L1 adipocytes compared with the control group(P<0.05).The results of qPCR detection showed that compared with the control group,the expression of C/EBP?,PPARy and FABP4 mRNA was significantly decreased and the mRNA expression of NF-?B,MCP-1,TNF-? and CYP1A1 was significantly increased in 1 ?M and 10 ?M BaP groups(P<0.05).The results of Western Blot showed that compared with the control group,the protein expression of PPARy,C/EBP?,FABP4,PGC-1? and PPAR? was significantly decreased and the expression of CYP1A1 protein was significantly increased in 1 ?M and 10 ?M BaP groups,and the difference was statistically significant(P<0.05).The results of glycerol content determination showed that BaP had no effect on isoproterenol-induced triglyceride decomposition compared with the control group.Part 2:After adding AhR inhibitor CH223191 to inhibit AhR in the differentiation of 3T3-L1 preadipocytes,the effect of 1 ?M BaP on lipid metabolism was observed.The results of Western Blot and qPCR showed that the expression of CYP1A1 mRNA and protein in BaP+CH223191 group was significantly lower than that in BaP group,and the lipid content in BaP+CH223191 group was significantly higher than that in BaP group(P<0.05).The results of qPCR showed that compared with BaP group,the expression of adipose differentiation related factors PPARy,C/EBP? and FABP4 mRNA in BaP+CH223191 group was significantly higher,while the mRNA expression of inflammatory factors NF-?B,MCP-1 and TNF-? was significantly lower in BaP+CH223 191 group than that in BaP group(P<0.05).Western Blot results showed that the protein expression of PPARy,C/EBP?,FABP4,PGC-1?and PPAR? in BaP+CH223191 group was significantly higher than that in BaP group(P<0.05).Conclusion:(1)BaP can slow down weight gain in normal mice,reduce the weight of epididymal adipose tissue and subcutaneous adipose tissue,reduce the volume of adipocytes and reduce fat content,interfere with glucose metabolism and lead to insulin resistance,dyslipidemia,reduce the expression of adipogenic factors in adipose tissue and increase the expression of inflammatory factors.(2)BaP interferes with adipose formation and decomposition and leads to pathological damage of adipose tissue by activating AhR in 3T3-L1 cells and inhibiting the expression of adipose differentiation and fatty acid ?-oxidation related factors while increasing the expression of inflammatory factors. |
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