| [Objective] To explore whether SjCyPA and SjE16 proteins of Schistosoma japonicum can be used as PAMP to activate TLR4/NF-κB signaling pathway and activate and polarize macrophages,thus providing a new experimental basis for the pathogenesis of Schistosoma japonicum.[Method] 1.The prokaryotic expression plasmid of pET-28a/SjCyPA and pET-28a/SjE16 was constructed and induced by E.coli BL21.The expression levels of SjCyPA and SjE16 proteins were determined by SDS-PAGE.The recombinant proteins SjCyPA and SjE16 were purified by Ni-NAT column,and verified by western blot.The concentrations of purified proteins were determined by BCA method.2.Whether SjCyPA and SjE16 induced macrophage polarization was detected.Mouse macrophages RAW264.7 strain were cultured and stimulated with different concentrations of SjCyPA(0,1,20,40,60,80μg/m L)and SjE16(0,1,5,10,15,20μg/m L)for different time(0,12,24,36,48h).RT-PCR was used to detect the m RNA expression levels of i NOS,Arg1,IL-6,IL-1β,TNF-α and IL-10.Western blot was used to detect the expression of i NOS protein in macrophages stimulated by SjCyPA and SjE16 at different concentrations.Appropriate protein concentration and stimulation time were used to stimulate RAW264.7 cells,LPS stimulation was used as a positive control,and PBS stimulation was used as a negative control,and i NOS,CD86 and CD206 were detected by flow cytometry.3.To determine whether SjCyPA and SjE16 could activate the TLR4/NF-κB signaling pathway in macrophages.RAW264.7 cells were stimulated with different concentrations of proteins SjCyPA and SjE16,and the expression levels of TLR4 and My D88 proteins were detected by western blot.Macrophages were stimulated with SjCyPA and SjE16 at appropriate concentrations,and the phosphorylation of NF-κB(p65)was detected by western blot at different time points(0,15,30,60,120 and180 min).RAW264.7 cells were pretreated with different concentrations of TLR4/My D88 inhibitor TAK-242(0.1,1,10μM)and NF-κB inhibitor BAY 11-7082(1,10,20μM),and then co-incubated with SjCyPA or SjE16,to set up the DMSO control group,DMSO+SjCyPA and DMSO+SjE16 stimulation groups.Western blot was used to detect the expression of TLR4 and My D88 and the phosphorylation of p65,and to observe the inhibition efficiency of the two inhibitors.Macrophages were pretreated with the optimal inhibitory concentration of 10μM TAK-242,and then co-incubated with SjCyPA or SjE16 proteins,to set up DMSO control group,DMSO+SjCyPA and DMSO+SjE16 stimulation groups.The phosphorylation of p65 was detected by western blot,and to analyze whether the phosphorylation of p65 induced by SjCyPA and SjE16 proteins was mediated by TLR4.4.To determine whether SjCyPA and SjE16 could induce M1-type polarization of macrophages through TLR4/NF-κB pathway.Macrophages were pretreated with the optimal inhibitory concentration of 10μM TAK-242 and 20μM BAY 11-7082,respectively,and then co-incubated with SjCyPA or SjE16.DMSO control group,DMSO+SjCyPA group and DMSO+SjE16 group were set.The m RNA expression levels of i NOS,IL-6,IL-1β and TNF-α were detected by RT-PCR,the protein expression levels of i NOS were detected by western blot,and the expression levels of IL-1β and TNF-α in cell culture supernatant were detected by ELISA.5.To determine whether SjCyPA and SjE16 could induce macrophage activation through TLR4 pathway.Macrophages were pretreated with the optimal inhibitory concentration of TAK-242 and BAY 11-7082 respectively,and then co-incubated with SjCyPA or SjE16.LPS positive control group,DMSO control group,DMSO+SjCyPA and DMSO+SjE16 stimulated groups were set.Fluorescence probe DCFH-DA was used to detect ROS production by flow cytometry,and the level of NO in cell culture supernatant was detected by Griess.[Results] 1.The prokaryotic expression plasmid pET-28a/SjCyPA and pET-28a/SjE16 was successfully constructed.SDS-PAGE results showed that the prokaryotic expression plasmid could express 18 k D SjCyPA protein and 16 k D SjE16 protein.Western blot results showed that the purified recombinant protein could specifically bind to the corresponding antibody.2.SjCyPA and SjE16 induced M1-type polarization of macrophages.The effects of different concentrations of SjCyPA and SjE16 on macrophages were detected.RT-PCR results showed that compared with 0μg/m L group,SjCyPA(40,60,80μg/m L)and SjE16(15μg/m L and20μg/m L)significantly increased the m RNA expression levels of of M1-type macrophage related molecules and cytokines i NOS,IL-6,IL-1βand TNF-α(P<0.05).IL-10 m RNA expression was significantly increased in 15μg/m L and 20μg/m L SjE16(P<0.05).Western blot showed that the expression of i NOS protein induced by SjCyPA and SjE16 was significantly higher than that of 0μg/m L group(P<0.05).To detect the effects of SjCyPA and SjE16 stimulation on macrophages at different time points,RT-PCR results showed that,compared with the 0h group,m RNA expression levels of i NOS,IL-6,IL-1β and TNF-α after SjCyPA stimulation for 24,36 and 48 h and SjE16 stimulation for 12,24,36 and48 h were significantly increased(P<0.05).SjE16 induced significant increase in IL-10 m RNA expression at all time points(P<0.05).Flow cytometry results showed that SjCyPA and SjE16 significantly increased the expression of M1-type macrophages marker molecule i NOS and CD86 compared with PBS control group(P<0.05),while there was no significant change in the expression of M2-type marker molecule CD206(P>0.05).3.SjCyPA and SjE16 can activate TLR4 signaling pathway in macrophages.Effects of SjCyPA and SjE16 on TLR4 signaling pathway in macrophages.Western blot results showed that SjCyPA and SjE16 could induce the expression levels of TLR4 and My D88 in macrophages significantly increased(P<0.05).SjCyPA and SjE16 could induce the phosphorylation of NF-κB in macrophages,and the phosphorylation level of p65 was the highest at 60 min and 120 min of stimulation.Inhibitors TAK-242 and BAY 11-7082 significantly inhibited TLR4/My D88 expression and NF-κB phosphorylation that induced by SjCyPA and SjE16 in macrophages.4.SjCyPA and SjE16 can induce M1-type polarization of macrophages through TLR4 signaling pathway.ELISA results showed that SjCyPA and SjE16 significantly increased the expression levels of M1-type macrophage related cytokines IL-1β and TNF-α compared with DMSO control group(P<0.01).Under the action of TLR4 pathway inhibitors,the effects of SjCyPA and SjE16 on macrophage polarization were demonstrated by RT-PCR,western blot and ELISA results.Both TAK-242 and BAY 11-7082 significantly inhibited the m RNA expression of i NOS,IL-6,IL-1β and TNF-α in macrophages induced by SjCyPA and SjE16,as well as the expression of i NOS protein and IL-1β and TNF-α protein(P<0.05).5.SjCyPA and SjE16 can induce macrophage activation through TLR4 signaling pathway.The results of flow cytometry and Griess assay showed that SjCyPA and SjE16 significantly induced ROS and NO production in macrophages compared with DMSO control group(P<0.05),and under the action of inhibitors TAK-242 and BAY 11-7082,the expression levels of ROS and NO induced by SjCyPA and SjE16 were significantly lower than those stimulated by DMSO+SjCyPA and DMSO+SjE16 group(P<0.05).[Conclusion] 1.SjCyPA and SjE16 can act as PAMP to activate TLR4/NF-κB signaling pathway.2.SjCyPA and SjE16 can induce macrophage activation and M1-type polarization through TLR4/NF-κB pathway. |
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