| Background Synthetic chemical BPA (Bisphenol A, BPA) has become one of the highest production volume in the world, whose output is more than 6 billion pounds per year. BPA is mainly used for the production of polycarbonate plastics and epoxy resins. Polycarbonate plastics are widely used in the production of beverage containers and food packaging, and epoxy resins are mainly used in the contacting surfaces of canned food. In heating, alkaline or acidic conditions, BPA monomer will be released from the packing container into the environment and food. It is thought that human exposure mainly occurs through consuming foods packaged with these containers. Biological monitoring data show that over 90% of the population are exposed to BPA, with BPA or its metabolite detected in their blood or urine. BPA is widely recognized as an environmental endocrine disrupting chemical with weak estrogenic activity, and can have adverse effects on endocrine, neurobehavior, reproduction and development.Many studies have shown that BPA exposure has a significant correlation with obesity and obesity-related insulin resistance. Obesity is systemic status related with chronic inflammation of low grade, characterized by infiltration of a large amount of adipose tissue macrophages (ATMs) in adipose tissue. It is well appreciated that infiltration and activation of macrophages in the visceral AT correlate with a chronic inflammatory state. These macrophages secrete cytokines and other factors that could impair the ability of adipocytes to secrete beneficial adipokines or store lipid. The infiltration of macrophage in adipose tissue and its polarization state was significantly correlated. Macrophages can be divided into classically activated macrophages (F4/80+CD11c+CD206-) and alternative activated macrophages (F4/80+CD11c-CD206+).However, until now, the exact mechanism of obesity related with insulin resistance caused by BPA remains unclear. The polarization of macrophages may play a key role in insulin resistance. As the effects of BPA on macrophage polarization, by far, hasn’t been reported so much, it is meaningful to explore the mechanism of insulin resistance caused by BPA.Objective The present study was to investigate the effects of BPA on polarization of macrophages in vitro through analyzing the influence of BPA at different concentrations on surface molecules of mice peritoneal macrophages subtypes (M1 and M2 type) of which polarization were induced in vitro, cytokine secretion, transcription factors IRF-4 and IRF-5 which could regulate the polarization of macrophages in vitro. This experiment could provide the pathogenesis for obesity and obesity-related metabolic disorders caused by BPA.Methods C57BL/6J 6-8 weeks-old healthy male mice SPF grade. Mice were killed by decapitation, Inject 5 ml of DMEM medium (contain 100 U/ml penicillin and 100μg/ ml streptomycin) into the peritoneal cavity under sterile conditions, collect the fluid about 4 ml with a syringe.1000 r · min-1 centrifugal 8 min, at room temperature, the supernatant was discarded。 Adjusting the cell to desired concentration, seeded in 6-well plates,37 ℃,5% CO2 incubator 3h. Washing away the non-adherent cells with PBS. After cultured 24h, cells were stimulated with murine (10 ng/ml) IFN-γ and LPS (500 ng/ml) for induction of Ml-type macrophages, and (10 ng/ml) IL-4 for M2-type, respectively. Meanwhile cells were treated with 0.1,1 and 10 μmol/L BPA. DMSO was used as Vehicle control.When induced to M1 type polarized, first with 10 ng/mL of IFN-γ induced 24h, then added 500 ng/mL of LPS 24h to stimulate cells, and then cells and supernatant were collecte. Proportion of M1 and M2 macrophages were detected by flow cytometry (FCM). Cytokine IL-6, iNOS, MCP-1, IL-10, and Arg-1 expression levels were detected by ELISA. Real time-quantitative polymerase chain reaction (RT-qPCR) were used to detect gene expression levels IL-6, iNOS, MCP-1, CD11c, IL-10, Arg-1, CD206, IRF4 and IRF5. SPSS 17.0 statistical package were used for statistical analysis of the experimental results.Results1. Results of polarization of mice peritoneal macrophages induced in vitro1.1 Morphological changesAfter induction with IFN-y, most of cultured macrophages are round (Ml phenotype) shape. After induction with IL-4, populations of cultured macrophages are fibroblast-like (M2 phenotype) shape.1.2 Proportions of M1 macrophages and M2 macrophages before and after inductionCompared with the pre-induction, proportion of M1 macrophages increased by 2.50 times after induction and M2 macrophages increased by 2.33 times after induction, detected by FCM, with the differences statistically significant (P<0.01, P<0.01).1.3 Changes of cytokinesCytokine iNOS secreted by M1 macrophages increased apparently, detected by ELISA, with the differences statistically significant (P<0.01), while cytokine Arg-1 secreted by M2 macrophages decreased apparently, detected by ELISA, with the differences statistically significant (P<0.01).2 The effects of different doses of BPA on the polarization of mouse peritoneal macrophages toward the M1 phenotype.2.1 Impact on the number of M1 macrophagesCompared with the blank control group, there was no statistically significant difference in M1 proportion in solvent control group and 0.1 μmol/L BPA groups, while the M1 proportion of 1μmol/L and 10μmol/L BPA group increased (P<0.05, P<0.01).2.2 Impact on mRNA expression of M1 macrophages surface molecules CD11cCompared with the blank control group, there was no statistically significant difference in mRNA expression of CD11c in solvent control group and 0.1μmol/L BPA groups, while the expression of CDllc in 1 μmol/L and 10 μmol/L BPA group significantly increased (P<0.01, P<0.01)2.3 Impact on M1 macrophages secreted cytokines2.3.1 Impact on the activity and content of M1 macrophages secreted cytokinesCompared with the blank control group, there was no statistically significant difference in M1 macrophages secreted anti-inflammatory cytokines IL-6, iNOS and MCP-1 in solvent control group and 0.1 μmol/L BPA groups, while the expression of IL-6, iNOS and MCP-1 in 1 μmol/L and 10 μmol/L BPA group increased (P<0.01, P<0.01).2.3.2 Impact on mRNA of M1 macrophages secreted cytokinesCompared with the blank control group,there was no statistically significant difference in mRNA of M1 macrophages secreted anti-inflammatory cytokines IL-6, iNOS and MCP-1 in solvent control group and 0.1 μmol/L BPA groups, while the expression IL-6, iNOS and MCP-1 in 1 μmol/L and 10 μmol/L BPA group significantly increased (P<0.01, P<0.01, P<0.01).3 The effects of different doses of BPA on the polarization of mouse peritoneal macrophages toward the M12phenotype.3.1 Impact on the number of M1 macrophagesCompared with the blank control group, there was no statistically significant difference in M2 proportion in solvent control group and 0.1 μmol/L BPA groups, while the M2 proportion of 1 μmol/L and 10 μmol/L BPA group decreased (P<0.05, P<0.01).3.2 Impact on mRNA expression of M2 macrophages surface molecules CD206Compared with the blank control group, there was no statistically significant difference in mRNA expression of M2 macrophages surface molecules CD206 in solvent control group and 0.1 μmol/L BPA groups, while the expression of CD206 in 1 μmol/L and 10 μmol/L BPA group significantly decreased (P<0.05, P<0.01)3.3 Impact on M2 macrophages secreted cytokines3.3.1 Impact on the activity and content of M2 macrophages secreted cytokinesCompared with the blank control group, there was no statistically significant difference in M2 macrophages secreted anti-inflammatory cytokines IL-10 and Arg-1 in solvent control group and 0.1μmol/L BPA groups, while the expression of IL-10 and Arg-1 in 1μmol/L and 10μmol/L BPA group decreased (P<0.01, P<0.01).3.3.2 Impact on mRNA of M2 macrophages secreted cytokinesCompared with the blank control group, there was no statistically significant difference in mRNA of M2 macrophages secreted anti-inflammatory cytokines IL-10 and Arg-1 in solvent control group and 0.1μmol/L BPA groups, while the expression IL-10 and Arg-1 in 1μmol/L (P<0.05, P<0.01) and 10μmol/L (P<0.05, P<0.05) BPA group significantly decreased.4 BPA may affect the polarization of mouse peritoneal macrophages in vitro via transcription factor IRF4 and IRF54.1 Effects of BPA on mRNA expression of mouse peritoneal macrophages IRF4 and IRF5M1-type polarization group:There was no statistically significant difference in mRNA expression of transcription factor IRF4. Compared with the blank control group, there was no statistically significant difference in mRNA expression of IRF5 in solvent control group and 0.1 μmol/L BPA groups, while the mRNA expression of IRF5 in 1 μmol/L and 10μmol/L BPA group significantly increased (P<0.01, P<0.01).M2-type polarization group:There was no statistically significant difference in mRNA expression of transcription factor IRF5. Compared with the blank control group, there was no statistically significant difference in mRNA expression of IRF4 in solvent control,0.1μmol/L and 1μmol/L BPA groups, while the mRNA expression of IRF4 in 10μmol/L BPA group significantly decreased (P<0.01).4.2 The correlation between cell proportion and transcription factor IRF4 and IRF5 Correlation analysis showed that transcription factor IRF5 and M1 macrophages are significant correlated (correlation coefficient r= 0.629, F=0.012)。Conclusion1 In vitro, IFN-γ can induce macrophage polarization toward the M2 phenotype, IL-4 can induce macrophage polarization toward the M1 phenotype.2 In vitro, low concentrations of BPA can promote the polarization of M1 macrophages, increase the number of M1 macrophages, increase mRNA expression of M1 macrophages specific surface molecules CDllc3 and promote the expression of M1 macrophages secreted cytokines TNF-α, IL-6, iNOS and MCP-1 at molecular and protein levels.3 In vitro, low concentrations of BPA can inhibit the polarization of M2 macrophages, reduce the number of M2 macrophages, reduce mRNA expression of M1 macrophages specific surface molecules CDllc3 and inhibit the expression of M2 macrophages secreted anti-inflammatory cytokines IL-10 and Arg-1 at molecular and protein levels.4 BPA may promote the polarization of M1 macrophages via transcription factor IRF5 in vitro. |
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