| Objective: Respiratory syncytial virus(RSV)infection can cause acute encephalopathy in children,leading to neurological symptoms,but the exact underlying pathogenesis has not yet been elucidated.Both receptors Toll-like receptor(TLR4)and nucleolin(C23)are required for infection by respiratory syncytial virus.In this study,the purpose of our study was to elucidate the possible synergistic effects of these two receptors in central neurons of RSV infection and provide new ideas for future clinical treatment and disease prevention.Method: After transfecting 293 T cells with TLR4 and C23 overexpression plasmids,their co-localization and interaction were detected by laser scanning confocal microscopy and immunoprecipitation.Then,the human neuroblastoma SH-SY5 Y cells were transfected with TLR4 and C23 overexpression plasmids.The cells without virus infection was set as a normal control group,and the RSV infection group,the TLR4 overexpression group,the C23 overexpression group,and the TLR4+C23 overexpression group were established respectively.Protein and cell supernatants were collected from each group after RSV infection for 2 h,4 h,24 h,and 48 h.(1)Western blotting was used to detect the expression changes of p-NF-κB,TLR4 and C23 protein levels in different groups at different time points.(2)Real-time PCR was used to detect the changes of TLR4 and C23 m RNA expression at different time points in each group.(3)Enzyme-linked immunosorbent assay(ELISA)was used to detect the changes of IL-6,IL-8 and TNF-α in the supernatant of each cell culture medium.(4)Flow cytometry to detect apoptosis rate in each group.Result:(1)The results of Laser scanning confocal microscopy and co-immunoprecipitation showed that after transfection of 293 T cells with TLR4 and C23 overexpression plasmids,co-localization of the two receptors was observed by laser confocal.The cells were incubated with the corresponding antibodies and then immunized.After completion of the co-precipitation reaction,the expression of corresponding protein results were detected by immunoblotting.After incubation with the C23 protein antibody,the TLR4 can be pulled down,and the C23 protein is also pulled down after incubation of the TLR4 protein antibody.The results suggest that TLR4 and C23 have an interaction.(2)The results of western blot showed that in the normal control group,the expression level of p-NF-κB protein was low.After infection with RSV virus,the protein expression increased with the increase of infection time.TLR4 overexpression was compared with RSV infection group.The expression of p-NF-κB in the TLR4+C23 overexpression group was increased in the C23 overexpression group,and the difference was statistically significant from 2h(P<0.05).The TLR4 overexpression group was compared with the simultaneous point.The increase in the C23 overexpression group was more pronounced,and the expression of p-NF-κB was highest in the TLR4+C23 overexpression group.The TLR4 expression level was lower in normal control,and the expression increased after RSV infection.The difference was statistically significant at 2h(P<0.05).Compared with the RSV-infected group,the expression levels of TLR4 overexpression group,C23 overexpression group and TLR4+C23 overexpression group was increased at the same time.The expression level in the TLR4+C23 overexpression group was significantly higher than that in the overexpressed group alone.Similar results were detected in C23 protein.(3)The results of Real-time PCR showed that the content of TLR4 m RNA was lower in normal cells,and the expression of TLR4 m RNA increased after RSV infection.Compared with the RSV-infected group,the expression levels in the TLR4 overexpression group and the C23 overexpression group were significantly increased.In addition,the expression level of TLR4+C23 overexpression group was the highest.The difference was statistically significant from 2h(P<0.01).Expression of C23 m RNA detected similar results as TLR4 m RNA.C23 m RNA was lower in normal cells,and the expression of C23 m RNA increased after RSV infection,and the difference was significant(P<0.05).Compared with the RSV-infected group,the expression levels in the TLR4 overexpression group and the C23 overexpression group were significantly increased.Among them,the expression level of TLR4+C23 overexpression group was the highest.(4)The results of ELISA showed that the expression levels of IL-6,IL-8 and TNF-α were lower in the normal control group,and IL-6,IL-8 and TNF-α in the culture supernatant of SH-SY5 Y cells were infected after RSV infection.The content of IL-6,IL-8 and TNF-α increased with the prolongation of infection time,which was higher than that of the control group(P<0.05).The expression levels of IL-6,IL-8 and TNF-α in TLR4 overexpression group and C23 overexpression group were significantly increased(P <0.01),and the TLR4 overexpression group was significantly increased.Among them,the expression level of cytokines in the TLR4 + C23 overexpression group was the highest,which was significantly different from the other groups(P< 0.05).(5)The results of Flow cytometry analysisindicated that the apoptosis of SH-SY5 Y cells in normal group was less.After 24 hours of RSV treatment,the apoptosis of RSV-infected group increased,and the apoptosis was statistically significant(P<0.01).TLR4 overexpression group’s apoptosis was significantly higher in the RSV group,and C23 overexpression group is as the same.The apoptosis was statistically significant(P<0.01).The TLR4+C23 overexpression group was compared with the RSV group.The change of apoptosis was more obvious and had statistical significance(P<0.01).At 48 h after infection,the apoptosis of each group was similar to that of 24 h,but it was more significant than that at 24 h.Conclusions: TLR4 and C23 can interact to promote RSV activation of NF-κB,leading to increased expression of cytokines IL-6,IL-8,TNF-α and apoptosis in the downstream of the relevant signaling pathway,indicating that the two receptors are synergism in the injury caused by RSV infection of SH-SY5 Y cells. |
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