| Background In recent years, the global prevalence of obesity increased rapidly and keeped persistently high status. Scientists proposed the "environmental induced obesity factors" hypothesis, the environment obesity factors are chemicals that have the ability to change the abnormal lipid metabolism, fat storage, metabolism, energy balance so that further promote lipid accumulation and obesity. In recent years, more and more evidences suggest that BPA in the environment is the environmental induced obesity factors. Humans can be exposed to BPA through the digestive tracts, respiratory, skin and other means. BPA can potentially affect the biological function of the adipocyte differentiation process and adipose tissue. BPA can potentially affect the biological function of the adipocyte and differentiation process of adipose tissue.Many studies have shown that low doses of BPA can not only promote adipocyte differentiation, but also can interfere with secretion of adipokines and inflammatory cytokines, which contributed to the formation of inflammatory pathological states. However, mechanisms of BPA promoting adipocyte differentiation and interferenceing adipokines and inflammatory factors is still unclear.Objective By detecting the differentiation degree, lipid accumulation, the levels of adpokines and inflammatory cytokines and the C/EBP and PPARγ expression levels in mice 3T3-L1 preadipocytes dealed with different low-doses BPA at different time-point, to explore the effect of low-dose BPA on differentiation of mouse 3T3-L1 preadipocytes and transcription factor expression.After mouse 3T3-L1 preadipocytes treated with PPARγ agonists and inhibitors after pre-treatmented with BPA for 6 days, detected cellular lipid deposition, adipokines and inflammatory cytokine secretion to discuss effct of PPARγ on BPA promoting mouse 3T3-L1 preadipocytes to differentiate.Methods After the mouse 3T3-L1 preadipocytes 90% merged in vitro, induced medium A cultured the mouse 3T3-L1 preadipocytes for 2 days and changed with induced medium B untill 8th. At the beginning of induction for differentiation, cells dealed with 10 nmol/L, 100 nmol/L or 1 000 nmol/L BPA, and blank control groups and DMSO groups. On 2, 4, 6, 8 day, the differentiation degree of cells detected by CCK8, lipid accumulation in cells detected by oil red O, the levels of adpokines and inflammatory cytokines detected by ELISA, the m RNA levels of adpokines,inflammatory cytokines, PPARγ, C/EBP detected by q PCR, PPARγ protein expression detected by IHC.Effect of PPARγ on BPA promoting preadipocyte to differentiate, when the cells were treated with the PPARγ agonist rosiglitazone or inhibitor GW9665, the cells also dealed with 1 000 nmol/L BPA untill the 6th day.lipid accumulation in cells detected by oil red O, the levels of adpokines and inflammatory cytokines detected by ELISA.the m RNA levels of adpokines, inflammatory cytokines, PPARγ, C/EBP detected by QPCR, PPARγ protein expression detected by IHC.Results1. The effect of low-dose BPA on cell viability and lipid deposition of mouse 3T3-L1 preadipocytes1.1 Cell viability change of 3T3-L1 preadipocytes Compared with the control group at the same time, CCK8 results showed that cell viability was significantly enhanced in 1000 nmol/L BPA group on 2 day,the difference statistically significant.on 4, 6, 8 day, cell viability decreased in BPA groups. The cell viability was significantly decreased in 100 nmol/L BPA groups on 6 day, the difference statistically significant.the cell viability was significantly decreased in 100 nmol/L BPA groups and 1000 nmol/L BPA groups on 8 day,the difference statistically significant.1.2 Lipid deposition change in 3T3-L1 preadipocytes Compared with the control group at the same time, oil red O staining showed that lipid droplet content increased in cells dealed with BPA. Compared with the control group, quantitative analysis showed that lipid droplets in 100 nmol/L BPA and 1 000 nmol/L BPAgroups significantly increased on 6 day and 8 day, and the difference was statistically significant.2. The effect of low-doses BPA on adipokines and cytokine secretion during mice 3T3-L1 preadipocytes differentiationCompared with the control groups, leptin, TNF-α, IL-6, IL-1β levels in medium and m RNA expressing in cells in 1 000 nmol/L BPA groups were increased on 4 day,and the differences were statistical significance.Leptin, TNF-α, IL-6, IL-1β levels in medium and m RNA expressing were increased on 6 and 8 day in cells in 100 nmol/L BPA groups and 1 000 nmol/L BPA groups, while adiponectin factor levels and m RNA expression decreased, the differences were statistically significant.3. The effect of low-dose BPA on the PPARγ and C/EBP m RNA during mouse 3T3-L1 preadipocytes differentiationCompared with the control group, m RNA levels of PPARγ and C/EBP in cells were increased in 1 000 nmol/L groups on 4 day, and the difference was statistically significant. The m RNA levels of PPARγ and C/EBP in cells were increased in 100 nmol/L BPA groups and 1 000 nmol/L BPA groups on 46 and 8day, and the difference was statistically significant.Immunohistochemical analysis showed that PPARγ expression was around the nucleus, compared with the control group, PPARγ expression in BPA groups was increased.4. The effect of PPARγ on BPA promoting mouse 3T3-L1 preadipocytes for mature4.1 Regulation of PPARγ agonists on BPA promoting mouse 3T3-L1 preadipocytes for matureCompared with the control groups, lipid accumulation in cells was increased significantly in rosiglitazone groups and rosiglitazone+1 000 nmol/LBPA groups, quantitative analysis showed significant difference. Leptin, TNF-α, IL-6, IL-1β levels in medium and leptin, TNF-α, IL-6, PPARγ, C/EBP m RNA expression in cells were increased,and adiponectin and adiponectin m RNA expression in cells decreased, the differences were statistically significant.Compared with 1 000 nmol / L BPA groups, lipid accumulation in rosiglitazone + 1 000 nmol/BPA groups was increased significantly, quantitative analysis showed significant difference.the leptin, TNF-α, IL6 levels in medium and leptin, TNF-α, IL-6, PPARγ, C/EBP m RNA expression in cells were increased, while adiponectin and adiponectin m RNA in cells was decreased, and the difference was significance. Compared with rosiglitazone, lipid deposition was increased in rosiglitazone + 1 000 mmol/L BPA groups, and quantitative analysis showed no significant difference.Leptin, TNF-α, IL-6, IL-1β expression in medium increased slightly, but not statistically significant; the m RNA expression of leptin, TNF-α, IL-6, PPARγ and PPARγ protein were increased, the differences were statistically significant.4.1 Regulation of PPARγ inhibitor on the promotion of mouse 3T3-L1 preadipocytes differention by BPACompared with the control groups, lipid accumulation in cells was decreased significantly in GW9665 groups and GW9665 + 1 000 nmol/L BPA groups, quantitative analysis showed significant difference. Leptin, TNF-α, IL-6, IL-1β levels in medium and the m RNA expression of leptin, TNF-α, IL-6, PPARγ, C/EBP in cells were decreased, and adiponectin and adiponectin m RNA expression in cells increased, the differences were statistically significant.Compared with 1 000 nmol/L BPA groups, lipid accumulation in GW9665 + 1 000 nmol/L BPA groups was decreased significantly, quantitative analysis showed significant difference.The leptin, TNF-α, IL6 levels in medium and the m RNA expression of leptin, TNF-α, IL-6, PPARγ, C/EBP in cells were decreased, while adiponectin and adiponectin m RNA in cells was increased, and the difference was significance.Compared with GW9665 groups, lipid deposition was increased in GE9665 + 1 000 mmol/L BPA groups,and quantitative analysis showed no significant difference.Leptin, TNF-α, IL-6, IL-1β expression in medium increased slightly, but not statistically significant; the m RNA expression of leptin, TNF-α, IL-6, PPARγ and PPARγ protein were increased, the differences were statistically significant.Conclusion 1.Low-dose BPA promoted the mouse 3T3-L1 preadipocytes to differentiate for mature, interfered expression of adipokines and inflammatory factors and increased C/EBP, PPARγ m RNA expresion in vitro.2. Low-dose BPA might throughed PPARγ pathway to promote differentiation of mouse3T3-L1 preadipocytes for maturation in vitro. |
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