| ObjectiveThis study explored the effect of pyocyanin on the induction of IL-6expression in macrophages and the effect of aryl hydrocarbon receptor signal transduction pathways on IL-6 expression induced by pyocyanin,so as to provide a good theoretical basis for clinical treatment.MethodsA mouse mononuclear macrophage cell line(RAW264.7)in logarithmic growth phase was selected.Pyocyanin(PCN)were divided into group A,group B,group C and control group(CON)according to different concentrations.Group A,group B and group C were added with the final concentration of 25,50 and 100μmol/mL respectively,and the final concentration of the control group was 0,which acted together for 24 hours.Aromatic hydrocarbon receptor blocker(10,20μmol/mL)and NF-κB blocker(100μmol/mL)were set as the appropriate concentration selected according to the preexperiment results.CCK-8 method was used to detect the effect of drugs on cell viability.ELISA kit was used to detect the secretion of IL-6 in macrophages stimulated by PCN.Western blot was used to detect the expression of AhR,NF-κB and IL-6 protein in macrophages stimulated by PCN.AhR signal pathway was blocked with AhR blocker to detect the expression of NF-κB and IL-6 protein.NF-κB signal pathway was blocked with NF-κB blocker to detect the expression of IL-6 protein.Observe the immunofluorescence to observe and use spss16.0 for data analysis.The staining of AhR in cells was observed by immunofluorescence.Then,the data were analyzed by SPSS16.0.Results1.The results of CCK-8 show that: Set the PCN concentration of 10,25,50,100,200μmol/mL.At the same time,the concentration of control group and10,25,50,100 μmol/mL group had no effect on cell viability,while the cells in the200μmol/mL group had 10%-20% apoptosis.So the final concentration was selected as 25,50,100μmol/mL.Compared with the control group,the difference was not statistically significant(p ﹥ 0.05).The concentrations of Aromatic hydrocarbon receptor inhibitors were 5,10,20,50μmol/mL.At the same time,aromatic hydrocarbon receptor inhibitors at the concentrations of 5,10,20μmol/mL have no effect on cell viability.Compared with the control group,the difference is not statistically significant(p>0.05).The concentrations of aromatic hydrocarbon receptor inhibitor and NF-κB blocker were 10 and20μmol/mL and 50,100 and 200μmol/mL for the experiment.For the same time,the concentrations of NF-κB of 50 and 100μmol/mL both had no effect on cell viability,and the difference was not statistically significant compared with the control group(p>0.05).The concentration of NF-κB antagonist 100 μmol / mL was selected for the experiment,and with the prolongation of the action time of aromatic hydrocarbon receptor inhibitor and NF-κB blocker,there was no effect on activity compared with the control group,indicating that the selected concentration of PCN,aromatic hydrocarbon receptor blocker and NF-κB blocker had no toxic effect on cells.2.The results of ELISA method showed that PCN can make macrophages(RAW264.7)secrete IL-6.The expression of IL-6 increases with the increase of PCN concentration,indicating that PCN enhances the expression of IL-6 in macrophages(RAW264.7)in a concentration-dependent manner.When aromatic hydrocarbon receptor inhibitor(CH-223191)was used to inhibit aromatic hydrocarbon receptor signal pathway and NF-κB blocker(PDTC)is used to block the NF-κB signal pathway,PCN(50μmol/mL)stimulates the reduction of IL-6 expression in macrophages(p<0.05),which indicated that aryl hydrocarbon receptor signal transduction pathway may participate in this immune response by regulating the NF-κB signal pathway.3.The results of Western Blot showed that after 24 hours of stimulating macrophages(RAW264.7)with different concentrations of PCN,the expression of aryl hydrocarbon receptor protein,NF-κB,and IL-6 increased compared with the control group,and the difference was statistically significant(p<0.05).After being treated with aryl hydrocarbon receptor inhibitor(CH-223191),compared with the experimental group without aryl hydrocarbon receptor inhibitor(CH-223191),aryl hydrocarbon receptor protein,NF-κB,IL-6 expression decreased.And after blocking the NF-κB signaling pathway with NF-κB blocker(PDTC),the expression of IL-6 decreased.The difference was statistically significant(p<0.05),which further illustrates that the inhibition of aryl hydrocarbon receptors(CH-223191)can inhibit the expression of aryl hydrocarbon receptor protein,regulate the NF-κB signaling pathway,and down-regulate the expression of IL-6.4.Compared with the control group,the fluorescence staining of RAW264.7cells treated with different concentrations of PCN was significantly deeper than that of the control group,and dense and dense particles could be seen in the cells,especially in the 100μmol/mL group.The expression of aromatic hydrocarbon receptor protein increased with the increase of PCN concentration,suggesting that PCN induced the expression of aromatic hydrocarbon receptor in a concentration-dependent manner.Conclusion1.Pyocyanin induced the expression of IL-6 in RAW264.7 macrophages in a concentration-dependent manner.2.The mechanism of IL-6 expression in RAW264.7 macrophages induced by pyocyanin may be that the aryl hydrocarbon receptor signal transduction pathway acts on NF-κB signal pathway to regulate the expression of IL-6. |
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