| ObjectiveBisphenol A(BPA)is a widespread environmental endocrine disruptor that can enter the body through the digestive tract,respiratory tract and skin and cause immune system damage.Macrophages are a kind of innate immune cells,which can regulate inflammatory response and kill microorganisms,and are an important part of the body’s non-specific immunity.Macrophage polarization is a way of immune regulation in response to external stimuli.But the exact mechanism by which BPA-induced polarizes macrophages remains unclear.The purpose of this study was to explore the AKT signaling pathway and the role of m TOR-dependent autophagy in BPA-induced polarization of macrophages in combination with bioinformatics,as well as the different effects of AKT1 and AKT2 of the AKT subunits.This study provides a new clue for exploring the regulation of macrophage polarization by environmental endocrine disruptors and a new strategy for the regulation of immune response.To provide some theoretical basis for the research of diseases caused by M1/M2 imbalance and autoimmune diseases.MethodsMouse leukemia macrophages(RAW264.7)were used as target cells,control group(CON)was set up,and other groups were treated with 10μg/m L LPS as activator and divided into 0,10,100,200μM BPA groups for 12 h.The cytokines IL-6 and TNF-α were detected by Real time PCR,Western blot and immunofluorescence.The m RNA and protein expression levels of i NOS,Arg-1,CD86 and CD206 were detected.After determining that BPA can induce macrophage polarization,bioinformatics was used to screen the targets of BPA and macrophage polarization,and the GO,KEGG and PPI analyses were performed.KEGG found that the main signaling pathways involved were PI3K/AKT,m TOR,calcium channel,etc.PPI analysis showed that AKT1 was the highest degree in protein interaction.Based on the results of the bioinformatics analysis,m RNA and protein expression levels of AKT1 and AKT2 were detected by Real time PCR and Western blot.To determine the differential effect of AKT1 and AKT2,si AKT1 and si AKT2 were transferred into macrophages by si-mate transfection reagent,and transfection experiments were started when the cell density fusion was 50%-70%.Firstly,we screened the plasmids.m RNA expression levels of si AKT1 and si AKT2 were detected by Real time PCR respectively,and plasmids with silencing effect were screened for subsequent experiments.Then,48 h after transfection,the m RNA expression of AKT1 or AKT2,CD86,CD206,i NOS,Arg-1,TGF-β,TNF-α,IL-6 and IL-10 was detected by Real time PCR.72 h after transfection,100μM BPA was used for 12 h.Western blot was used to detect the protein expression levels of AKT1 or AKT2,i NOS,Arg-1,TGF-β and TNF-α.To further study AKT signaling and autophagy,a control group(CON)was set up,and the other groups were treated with LPS(10μg/m L)as the activator.They were divided into 0,100μM BPA,100μM BPA+5μM PE,5μM PE,100μM BPA+1.25 μg/m L RAPA,1.25 μg/m L RAPA,and treated for 12 h.The cytokines TGF-β and TNF-α,polarization markers i NOS,Arg-1,CD86,CD206,autophagy related indexes LC3 and P62,AKT and m TOR were detected by Real time PCR.The expression levels of i NOS,Arg-1,LC3,P62,AKT,m TOR and their phosphorylated proteins were detected by Western blot.The fluorescence intensity of CD86 and CD206 was determined by immunofluorescence.Results3.1.Effect of BPA on macrophage polarization Compared with 0μM BPA,m RNA and protein expressions of CD86 and i NOS in the 100μM group were significantly increased(P < 0.05).However,m RNA and protein expressions of CD206 and Arg-1 were significantly decreased(P < 0.05).3.2.BPA affects the expression of macrophage polarization related genes through bioinformatics analysisOnline database screening revealed that 88 target genes were expressed in both BPA and macrophage polarization,performing a bioinformatic analysis of the target genes.KEGG analysis showed that the potential targets were mainly involved in PI3K/AKT,chemokine signaling pathway,apoptosis and m TOR signaling pathway.GO analysis showed that it was mainly involved in inflammation,immune response and oxidative stress response.PPI analysis found that AKT1 ranked first with a degree of 41.3.3Effect of BPA on AKT subunits in macrophagesCompared with 0μM BPA group,200μM BPA significantly decreased the expression of AKT1 gene(P < 0.05),while 100μM and 200μM BPA groups significantly increased the expression of AKT2 gene(P < 0.05).Compared with0μM BPA group,AKT1 protein expression in 10μM,100μM and 200μM BPA groups was significantly decreased(P < 0.05).However,AKT2 protein expression was significantly increased in the 200μM BPA group(P < 0.05).3.4.Effect of downregulation of AKT1 on BPA-induced macrophage polarizationCompared with si AKT1+0μM BPA group,the TNF-α,i NOS,IL-6 and CD86 genes in si AKT1+100μM BPA group were significantly increased,while the TGF-β,Arg-1,IL-10 and CD206 genes were significantly decreased(P < 0.05).Compared with si AKT1+0μM BPA group,i NOS and TNF-α protein levels were increased in si AKT1+100μM BPA group,while Arg-1 and TGF-β were significantly decreased,respectively.3.5.Effect of downregulation of AKT2 on BPA-induced macrophage polarizationCompared with si AKT2+0μM BPA group,m RNA levels of CD86,TNF-α,IL-6and CD206 in si AKT2+100μM BPA group were significantly increased(P < 0.05).In addition,compared with NC+100μM BPA group,m RNA expressions of i NOS and IL-10 in si AKT2+100μM BPA group were significantly decreased(P < 0.05).However,protein expressions of Arg-1,TGF-β,i NOS and TNF-α were not significantly changed in si AKT2+100μM BPA group compared with si AKT2+0μM BPA group after transfection with AKT2.3.6.Effects of PE on the polarization and autophagy of macrophages induced by BPACCK8 showed that PE significantly decreased cell viability at 10-15μM(P <0.05).When PE was combined with 100μM BPA,5-15μM PE significantly inhibited cell viability(P < 0.05),5μM was selected for subsequent experimental study.Compared with the 100μM BPA group,PE significantly increased the m RNA levels of CD86,i NOS and TNF-α,but decreased the m RNA levels of CD206 and Arg-1(P< 0.05).In addition,PE also promoted the expression of i NOS protein and inhibited the phosphorylation of Arg-1 and AKT.PE decreased m RNA levels of LC3 II and P62,but had no effect on m RNA levels of m TOR(P < 0.05).Protein results showed that compared with the 100μM BPA group,PE significantly increased LC3 II/I ratio,inhibited the expression of P62 and phosphorylation of m TOR(P < 0.05).In addition,immunofluorescence results also showed that PE increased the expression of CD86 protein but decreased the expression of CD206 protein.3.7 Effects of RAPA on BPA induced macrophage polarization and autophagyCompared with the 100 μM BPA group,RAPA showed significantly increased m RNA expression of CD86 and IL-6,P62 and LC3 II,as well as decreased expression of TNF-α,TGF-β and m TOR in the 4-h intervention model(P < 0.05).However,RAPA significantly reduced the ratio of i NOS,P62 protein expression and p-m TOR / m TOR in the 12 h intervention model,and increased the ratio of Arg-1 and LC3 Ⅱ/I(P < 0.05).Conclusions1.BPA induces the imbalance of M1 / M2 polarization of macrophages activated by LPS,promotes M1 type polarization and suppresses M2 type polarization;2.AKT1 negatively regulates M1-type polarization and positively regulates M2-type polarization of macrophages,while AKT2 has the opposite effect.3.AKT1 is the AKT subunit that plays a major role in the polarization of macrophages induced by BPA.4.AKT is involved in BPA-induced macrophage polarization through m TORdependent autophagy... |
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