| ObjectiveBisphenol A is a common environmental endocrine disruptors,studies have shown that BPA can activate the release of cytokines by activating T nuclear factor(NFAT).NFAT is widely present in all kinds of cells and is a kind of transcription factor with important physiological function,which regulates the transcription and expression of cytokines.Interleukin-10(IL-10)is one of the most effective anti-inflammatory cytokines in immune cells,the secretion of which can reduce the secretion of inflammatory factors,leading to inflammatory response.In this study,mouse monocyte-macrophage leukemia cells(RAW264.7)were used as target cells,and different doses of lipopolysaccharide(LPS)were used as activators.From Ca2+/CaN and Raf/MEK/ERK to study the role of NFAT in the regulation of IL-10 secretion by macrophages by bisphenol A(BPA)to provide a theoretical basis for the further study of the mechanism of immunotoxicity of BPA.MethodsThe RAW264.7 cells were treated with 1,10,100 and 10000μmol/L BPA at 4h,12h and 24h,and the cell viability was detected by CCK-8 kit.The expression of CaN and IL-10 in the supernatant of the cells was measured by EL,ISA kit.The blocking agent Ver,FK506,PD9805 were added under the condition of 2,5μg/ml LPS as activator and the expression of CaN and IL-10 protein were measured at 100μmol/L BPA.The mRNA expression of IL-10,NFATp.NFATc,ppp3rl,ERK1/2,MEK,RafA/C was measured at 2,5 μg/ml LPS as activator and 1,10,100 μmol/L BPA for 4h and 12h,respectively.The mRNA expression of IL-10,NFATp,NFATc,ppp3rl and ERK1/2 was measured after adding the inhibitor Ver,FK506 and PD98059.2,5μg/ml LPS as activator,100μmol/L BPA with inhibitor Ver and FK506 acts on RAW264.7 for 24 hours,Western blot was used to detect protein expression.Results1.Compared with 1,2,5μg/ml Lps activator group,there were no effect on cell viability exposed to 1,10,100μmol/L BPA at 4h,12h and 24h(p>0.05),while BPA concentration was 1000p,mol/L had a significant inhibitory effect on RAW264.7 cell activity(P<0.01).2.1,10 and 100μmol/L BPA was used to treat RAW264.7 at 4h,compared with 2,5μg/ml LPS group,100μmol/L BPA could decrease the level of IL-10 mRNA(P<0.05).RAW264.7 was treated at 12h,the level of IL-10 mRNA was decreased by 100μmol/L BPA compared with 2,5μg/ml LPS.100μmol/L BPA with Ver,FK506,PD98059 was used to treat RAW264.7 at 4h and 12h,compared with 100μmol/L BPA group,the expression of IL-10 mRNA in Ca2+ blocker was significantly higher than that in control group(P<0.05);In the same condition,0.25 and 0.5μmol/L FK506 increased the level of IL-10 gene significantly(P<0.05);Compared with 100μmol/L BPA group,the expression of IL-10 mRNA in PD98059 was significantly lower than that in control group(P<0.05);Compared with 2,5μg/ml LPS activator group,the secretion of IL-10 protein was significantly inhibited when BPA concentration was 100μmol/L(P<0.05),100μmol/L BPA with the inhibitorVer(10,30μmol/L),FK506(0.25,0.5μmol/L)and PD98059(10,40μmol/L)compared with 100μmol/L BPA,Ver,Fk506 increased the expression of IL-10 protein,while PD98059 decreased the IL-10 protein secretion。3.Compared with the control group 2,5μg/ml LPS could increase the expression of NFATp/c gene.Compared with 2,5μg/ml LPS group,100μmol/L BPA could increase the gene expression of NFATp and NFATc(P<0.05).Compared with the control group,2p,g/ml LPS could increase the expression of NFATp and NFATc protein in the nucleus;5μg/ml LPS could increase the expression of NFATp and NFATc protein in the nucleus and decrease the expression of NFATp and NFATc protein in RAW264.7 cells at 100μmol/L BPA.The blocking agent Ver,FK506,PD98059 was added to 100μmol/L BPA,after expose to RAW264.7 for 4h and 12h,compared with 100μmol/L BPA,The expression of NFATp/c mRNA was decreased by blocking agent Ver;The expression of NFATp/c mRNA was decreased by 0.25 and 0.5μmol/L blocker FK506 under the same experimental conditions(P<0.05).Compared with 100μmol/L BPA group,the expression of NFATc mRNA were increased and the expression of NFATp mRNA were decreased by 10,40μmol/L PD98059(P<0.05).LPS 2μg/ml,100μmol/L BPA with Ver,FK506 compared to 100μmol/L BPA at 24h,the inhibitor Ver and FK506 group significantly decreased NFATp and NFATc protein expression.4.The expression of PPP3rl mRNA was significantly increased in RAW264.7 cells treated with 100μmol/L BPA for 4h and 12h compared with LPS(2,5μg/ml)(P<0.05);Compared with the control group,1,2,5μg/ml LPS could activate RAW264.7 secretion and significantly increase the secretion of CaN protein,after adding 10,30μmol/L Ver,the expression level of PPP3R1 mRNA was significantly decreased compared with 100μmol/L BPA at 4h and 12h.Compared with each activator group,the secretion of CaN protein was significantly increased when BPA concentration was 100μmol/L(P<0.05).After adding 10,30μmol/L Ver,the expression of CaN was significantly decreased compared with 100μmol/L BPA group(P<0.05).5.The expression of RafA gene was not change significantly at 4h and 12h when LPS was 2μg/ml contrasted with the 2,5μg/ml activator group,(P>0.05).When the LPS is 5μg/ml,the mRNA express of RafA was increased at 4h,12h exposed to 100μmol/L BPA(P<0.05).At each time point and the all activator dosages,the gene expression of RafC and MEK increase exposed to 100μmol/L BPA(except 2μg/ml LPS,the mRNA expression level of RafC,MEK increased significantly in the 100μmol/L BPA 4h exposure)(P<0.05).Compared with the 2.5μg/ml activator group,the expression level of ERK1/2 was increased at 100μmol/L BPA for 4h and 12h(P<0.05).After 10 and 40μmol/L blocker PD98059 exposure for 4h and 12h,Compared with 100μmol/L BPA,10,40μmol/L PD98059 could decrease the expression of ERK1/2 mRNA(P<0.05).Conclusions1.BPA induce the decrease of cytokine IL-10 level in macrophages by inhibiting the content of NFATp/c nuclear protein.2.NFAT plays an important role in IL-10 secretion of macrophages by BPA.3.BPA can promote the expression of Raf,MEK and ERK,and the inhibitor of ERK significantly increases the expression of NFATp and decreases the expression of NFATc and decreases the content of IL-10,which indicates that Raf/MEK/ERK is involved in the regulation of NFATp/c by BPA. |
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