Aryl Hydrocarbon Receptors Regulate Pyocyanin-induced Inflammatory Factors Expression In Macrophages Via The P38MAPK Signaling Pathway

ObjectiveThis study explored the effect of Ah R on the expression of inflammatory factors in RAW264.7 macrophages induced by Pyocyanin(PCN)and their possible intracellular signal transduction pathways,so as to provide a new idea for the clinical treatment of Pseudomonas aeruginosa infection.MethodsThe RAW264.7 cells at logarithmic growth stage were infected with different concentrations of PCN 10,25,50,100 and 200 μM.In addition,The RAW264.7 cells were also incubated with aromatic hydrocarbon receptor inhibitor CH223191 10 μM and aromatic hydrocarbon receptor agonist FICZ100 n M(Finding the suitable dosages based on previously published studies)for 24 h.Then the CCK-8 method was used to detect the effect of drugs on cell viability.The experiments were grouped into control,PCN 10μM,PCN 25μM and PCN50μM groups.The expression of Ah R protein in the cells was detected by Western blot and immunofluorescence to determine the optimum PCN concentration to create infection models for subsequent experiments.Then,the experiments were grouped into control,PCN,Ah R inhibitor(CH223191)and PCN+Ah R inhibitor(CH223191)groups.The expression of Ah R protein and the phosphorylation of P38 MAPK in RAW264.7 induced by PCN were observed by Western blot.Western blot and ELISA were used to detect the expression of inflammatory cytokines IL-1β and TNF-ɑ in RAW264.7 induced by PCN.Last,the experiments were grouped into control,PCN,Ah R agonist(FICZ)and PCN+Ah R agonist(FICZ)groups.The expression of Ah R protein and the phosphorylation of P38 MAPK in RAW264.7 cells induced by PCN were observed by Western blot.Western blot and ELISA were used to detect the expression of inflammatory cytokines IL-1β and TNF-ɑ in PCN-induced RAW264.7 cells.ResultsAccording to CCK-8 data,PCN concentrations of 10,25 and 50μM at the same time had no effect on the viability of RAW264.7 cells compared to the control,which contained 0.1% dimethyl sulfoxide(DMSO),and the difference was not statistically significant(P>0.05).However,when the PCN concentration was 100μM,about 5% of the cells lost viability,and when the PCN concentration was 200μM,10%-20% of the cells lost viability.The difference was statistically significant when compared to the control group(P<0.05).So the final concentration of PCN was selected as 10,25,50μM.Compared with the control group(containing 0.1%Dimethyl sulfoxide DMSO)at the same time,CH223191 concentration of 10μM and FICZ concentration of 100 n M had no effect on cell viability,and the difference was not statistically significant(P>0.05).Western Blot and immunofluorescence results for the effect of PCN on Ah R show that Ah R could be activated by PCN compared with the control group,and the protein expression of Ah R increased with the increase of PCN concentration,the difference was statistically significant(P<0.05).Results for the aromatic hydrocarbon receptor inhibitor CH223191 :The western Blot results showed that the expression of Ah R was significantly reduced in RAW264.7 which treated with the Ah R inhibitor CH223191 compared to the control group,and the difference was statistically significant(P<0.05);PCN activates the P38 MAPK signaling pathway in macrophages,and the inhibition of Ah R leads to a further increase at the phosphorylation level of P38 MAPK,with a statistically significant difference compared to the control group(P<0.05);PCN stimulated cells to produce inflammatory factors IL-1β and TNF-ɑ and the inflammatory factors were added after the application of CH223191,which was statistically significant(P<0.05).The results of ELISA method showed that,as with Western Blot,the PCN+Ah R inhibitor(CH223191)group had a statistically significant increase in the production of the inflammatory factors IL-1β and TNF-ɑ compared with the PCN group,and the difference was statistically significant(P<0.05).Results of the action of the aromatic hydrocarbon receptor agonist FICZ: The western Blot showed that the expression of Ah R was significantly increased in the group treated with FICZ and PCN compared with the group treated with PCN alone,and the difference was statistically significant(P<0.05).FICZ could inhibit the phosphorylation of P38 MAPK,and this inhibition could be abolished by CH223191,with a statistically significant difference when compared with the control group(P<0.05).The inflammatory factors IL-1β and TNF-ɑ induced by PCN decreased after the RAW264.7 were stimulated by FICZ which was statistically significant(P<0.05).The results of ELISA showed that,similar to the Western Blot results,the protein levels of IL-1β and TNF-ɑ were lower in the FICZ and PCN combination group than in the PCN group,and the differences were statistically significant(P<0.05).ConclusionsAryl hydrocarbon receptors may inhibit the expression of inflammatory factors IL-1β and TNF-ɑ via suppressing the P38 MAPK signaling pathway in the macrophages induced by pyocyanin.