| ObjectiveBisphenol A（BPA）is recognized as one of the environmental endocrine disruptors because of its mimic estrogenic and anti-androgen properties.Macrophages are one of the most important immune cells in innate and adaptive immunity,but the mechanism by which BPA affects macrophage function is still unclear.In this study,we studied the potential mechanism of AMPK/mTOR in the cytotoxicity of BPA to macrophages from the point of view of autophagy and apoptosis,and further explored the relationship between autophagy and apoptosis.This is helpful to provide new evidence for the prevention of immunotoxicity induced by environmental endocrine disruptors.MethodsMouse leukemia macrophages（RAW264.7）were used as target cells,and the control group（Con）was established.Other groups were divided into 0,10,100,200μmol/L BPA groups with 10 mg/L LPS as activator,and mTOR inhibitor or autophagy activator（1.25 μg/m L RAPA）,autophagy inhibitor（2.5 mmol/m L 3-MA）and Bcl-2inhibitor（2.5 μmol/L ABT-737）alone,or combined with 100 umol/L BPA for 12 h.Cell viability was detected by MTT,apoptosis rate was detected by Annexin V-FITC/PI,the mRNA and protein levels of secretory factors IL-6,IL-10,TNF-α,TGF-β and autophagy indexes ATG6,LC3,p62,ATG5,ATG12 and apoptotic index Bax,Bcl-2,Caspase-3,Caspase-9,Caspase-8 were detected by Real time PCR and Western blot.ATG6,LC3,p62 were further detected by immunofluorescence.The phagocytic ability of macrophages was measured by central red reagent,and the activity of Caspase-3/7was detected by Caspase-Glo3/7（?）kit.Results1.Effect of BPA on the viability of RAW264.7 cellsCompared with Con,0 μmol/L BPA increased the cell viability,and compared with 0 μmol/L BPA,the cell viability was decreased significantly in 100,200 μmol/L BPA groups.2.Effect of BPA on RAW264.7 cell functionCompared with Con,the mRNA levels of TNF-α and TGF-β and the protein levels of IL-10 and TGF-β were increased in 0 μmol/L BPA group（P<0.05）.Compared with 0μmol/L BPA group,the mRNA levels of IL-6 in 100,200 μmol/L BPA group and TNF-α in 10,100 μmol/L BPA group increased,and the protein levels of IL-6 and TNF-α also were increased in 100,200 μmol/L BPA group.However,the mRNA levels of IL-10 in 10,100 and 200 μmol/L BPA and TGF-β in 10 and 200 μmol/L BPA,and the protein levels of IL-10 and TGF-β in 10,100 and 200 μmol/L BPA were decreased（P<0.05）.In addition,the phagocytosis capacity of 100 and 200 μmol/LBPA was significantly inhibited in RAW264.7 cells（P<0.05）.3.Effects of BPA on autophagy in RAW264.7 cellsThe mRNA levels of ATG6 and p62 in 0 μmol/L BPA group was higher than that in Con group（P <0.05）.Compared with 0 μmol/L BPA group,the mRNA levels of p62,LC3 II in 100,200 μmol/L BPA group,ATG5 in 10,100,200 μmol/L BPA group and ATG6,ATG12 in 200 μmol/LBPA group were significantly increased.However,the protein level of ATG6 in the 100 and 200 μmol/L BPA groups and the LC3Ⅱ/Ⅰ ratio in the 100 μmol/L BPA group were reduced（P<0.05）.Further immunofluorescence detection showed that the fluorescence intensity of ATG6 and p62 in 0 umol/L BPA group was weaker than that in Con group.In contrast to the 0 μmol/L BPA group,LC3 fluorescence intensity was decreased in the 100 and 200 μmol/L BPA groups,while p62 fluorescence intensity was slightly enhanced in the 100 μmol/L BPA group.4.AMPK/mTOR involved in BPA affects autophagy in RAW264.7 cellsBoth the mRNA levels of AMPK and mTOR were decreased in the 0 μmol/L BPA group as compared to Con（P<0.05）.Compared with the 0 μmol/L BPA group,the protein levels of p-AMPK/AMPK were decreased in the 100 μmol/L BPA group,and p-mTOR/mTOR protein levels were increased in the 100 and 200 μmol/L BPA groups（P<0.05）.In contrast to the 100 μmol/L BPA group,the protein levels of p-AMPK/AMPK,ATG6,and LC3Ⅱ/Ⅰ were increased in the 100 mol/L BPA +1.25μg/m L RAPA group.However,the protein level of p-mTOR/mTOR was decreased in the 100 μmol/L BPA+1.25 μg/m L RAPA group（P<0.05）.Immunofluorescence detection showed that the fluorescence intensity of ATG6 and LC3 in 100 μmol/L BPA+1.25 μg/m L RAPA group was enhanced,while that of p62 was decreased,compared with the 0 μmol/L BPA group.5.AMPK/mTOR involved in BPA affects cell function in RAW264.7 cellsThe protein levels of IL-10 and TGF-β were increased in the 0 μmol/L BPA group when compared with Con（P<0.05）.The protein levels of IL-6 and TNF-α were increased in 100 μmol/L BPA group compared to 0 μmol/L BPA group,while the protein levels of IL-10 and TGF-β were decreased in 100 μmol/L BPA group（P<0.05）.Compared with the 100 μmol/L BPA group,the 100 μmol/L BPA+1.25 μg/m L RAPA and 1.25 μg/m L RAPA groups were decreased,while the protein levels of IL-10 and TGF-β were increased in the 100 μmol/L BPA+1.25 μg/m L RAPA group（P<0.05）.Moreover,compared with 100 μmol/L BPA,cell phagocytic capacity in 1.25 μg/m L RAPA and 1.25 μg/m L RAPA were increased,while in 100 μmol/L BPA+2.5 mmol/m L3-MA and 2.5 mmol/m L 3-MA were further decreased（P<0.05）.6.Effect of BPA on the apoptosis levels in RAW264.7 cellsIn contrast to the Con,the mRNA level of Caspase-8 was increased in 0 μmol/L BPA group.However,the mRNA level of Bcl-2 in the 0 μmol/L BPA group was decreased（P<0.05）.In contrast to the 0 μmol/L BPA group,the apoptosis rate of RAW264.7 cells were significantly increased in the 100 and 200 μmol/L BPA cells（P<0.05）.The mRNA levels of Bax,Caspase-3 and Caspase-9 in the 10,100 and 200μmol/L BPA groups and Bcl-2 in the 200 μmol/L BPA group were increased.However,the mRNA level of Bcl-2 was reduced in the 10 μmol/L BPA group（P<0.05）.7.Effect of autophagy on apoptosis in RAW264.7 cellsIn comparison to the Con,cell viability was increased in 0 μmol/L BPA（P<0.05）;Compared with the 0 μmol/L BPA,cell viability was decreased in the 100 μmol/L BPA group（P<0.05）.When compared with the 100 μmol/L BPA,the viability of RAW264.7cells in the 100 μmol/L BPA+1.25 μg/m L RAPA and 1.25 μg/m L RAPA group were improved.However,the viability of RAW264.7 cells was further reduced in the 100μmol/L BPA+2.5 mmol/m L 3-MA and 2.5 mmol/m L 3-MA group（P<0.05）.The Bax/Bcl-2 ratio was decreased in the 0 μmol/L BPA group,compare with the Con（P<0.05）.The Bax/Bcl-2 ratio and Caspase3/7 activity were increased in the 100 and200 μmol/L BPA groups as compared to the 0 μmol/L BPA group（P<0.05）.In contrast to the 100 μmol/L BPA group,the ratio of Bax/Bcl-2 in the 100 μmol/L BPA+1.25μg/m L RAPA group was decreased.However,the ratio of Bax/Bcl-2 in 100 μmol/L BPA+2.5 mmol/m L 3-MA was increased further（P <0.05）.Besides,in contrast to the100 μmol/L BPA group,Caspase3/7 activity was elevated in the 100 μmol/L BPA+1.25μg/m L RAPA,100 μmol/L BPA+2.5 mmol/m L 3-MA,and 2.5 mmol/m L 3-MA groups（P<0.05）.8.Effects of the Bcl-2 inhibitor ABT-737 on ATG6 in RAW264.7 cellsCompared with 0 μmol/L BPA,the concentration of ABT-737 significantly reduced cells viability above 2.5 μmol/L（P<0.05）.Compared with 100 μmol/L BPA,the cell viability of 100 μmol/L BPA+2.5 μmol/L ABT-737 was reduced（P<0.05）.The mRNA level of ATG6 in 100 μmol/L BPA+2.5 μmol/L ABT-737 and 2.5 μmol/L ABT-737 were increased（P<0.05）.The protein level of ATG6 in 100 μmol/L BPA+2.5μmol/L ABT-737 and 2.5 μmol/L ABT-737 were increased,and the protein level of Bcl-2 in 100 μmol/L BPA+2.5 μmol/L ABT-737 and 2.5 μmol/L ABT-737 were decreased（P<0.05）.9.Effect of BPA on oestrogen receptors in RAW264.7 cellsERα mRNA level was decreased in 0 μmol/L BPA group compared with Con（P<0.05）.The mRNA and protein level of ERα was increased in 200 μmol/L BPA group compared with 0 μmol/L BPA（P<0.05）.Conclusions1.BPA induced secretion of proinflammatory factors from RAW264.7 cells,suppressed secretion of anti-inflammatory factors,and decreased cellular phagocytic capacity.2.Autophagy and apoptosis were participated in BPA-induced cytotoxicity to RAW264.7 cells.3.BPA suppressed autophagy by AMPK/mTOR signal pathway and induced apoptosis through endogenous mitochondrial pathway.4.Bcl-2 and ATG6 were involved in the regulation of apoptosis and autophagy by BPA. |
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