| Objectives: At present,the reports on encephalopathy caused by RSV infection are mainly based on clinical features and hospitalized cases,and there are few studies on its pathogenic mechanism.The previous research results of our group have shown that TLR4 and nucleolin can synergistically mediate RSV infection of human central neuron cells,but the specific mechanism is still unclear.Therefore,the purpose of this project is to study whether the TIR domain of TLR4 and the GAR domain of NCL cooperate with each other and their related signaling pathways in the process of RSV infection of neurons.Development of control strategies,providing new ideas.Methods: According to the corresponding gene sequences,TIR(674-815aa),TLR4(del 674-815aa),GAR(645-707aa)and NCL(del 645-707aa)domain lentiviruses were synthesized by Genecare Bio.At the same time,according to the corresponding experimental groups,the constructed lentivirus was used to infect SY5 Y cells,and after drug screening,a stable overexpression cell line was constructed.In vitro cultured SY5 Y cells and stable overexpressed SY5 Y cell lines were infected with RSV at 4h,12 h,24h and 48 h,respectively.Using laser confocalmicroscopy,co-immunoprecipitation,western blotting,molecularbiology,enzyme-linked immunosorbent assay,flow cytometry and other methods and techniques to detect the co-localization and interaction of TIR/GAR domains,the expression levels of p-NF-?B and LC3 protein,TLR4/NCL m RNA,cytokines and apoptosis,etc.To clarify the synergistic effect of the TIR(674-815aa)domain of TLR4 and the GAR(645-707aa)domain of NCL in neuronal injury and elucidate the possible mechanisms and related signaling pathways of TIR(674-815aa)and GAR(645-707aa)in mediating RSV infection-induced nervous system inflammation.Results:(1)Laser confocal and co-immunoprecipitation results show:After coinfection of SY5 Y cells with TIR(674-815aa)lentivirus and GAR(645-707aa)lentivirus,the positioning phenomenon of TIR(674-815aa)domain and GAR(645-707aa)domain was observed under confocal microscopy.After RSV-infected cells,lysed cells were incubated with correspondingly labeled magnetic beads and then subjected to coimmunoprecipitation reaction.The results showed that there was an interaction between the TIR(674-815aa)domain and the GAR(645-707aa)domain.(2)Western Blot results showed:The expression levels of p-NF-?B and LC3 were low in the control group(NC group)without RSV infection.TIR(674-815aa)domain overexpression group,GAR(645-707aa)domain overexpression group,TIR(674-815aa)domain+GAR(645-707aa))domain co-overexpression group,as well as TIR(674-815aa)domain overexpression group and NCL(del 645-707aa)domain overexpression group,the expression of p-NF-?B increased after RSV infection and the difference was statistically significant at 4h(###P<0.001).Among them,the expression levels of p-NF-?B and LC3 in the TIR(674-815aa)domain + GAR(645-707aa)domain +RSV group increased most significantly,while the protein expression levels of p-NF-?B and LC3 in the TLR4(del 674-815aa)+RSV group and NCL(del 645-707aa)+RSV group were the same as those in the NC+RSV group,and the difference was not statistically significant.(3)Real-time PCR results showed: After RSV infection,the expression levels of TLR4/NCL m RNA in control group(NC+RSV group)and each overexpression group were increased,and the difference was statistically significant at 4h(###P<0.001).Compared with NC+RSV,the expression levels of TLR4/NCL m RNA in TIR(674-815aa)+RSV group,GAR(645-707aa)+RSV group,TIR(674-815aa)+ GAR(645-707aa)+RSV group were significantly increased to a certain extent.The increase was significantly different from 4h(*P<0.05,**P<0.01,***P<0.001).While TLR4(del 674-815aa)+RSV group and NCL(del 645-707aa)+RSVgroup had no significant difference in TLR4/NCL m RNA expression level with NC +RSV group.(4)ELISA results showed : The expression levels of IL-6,IL-1? and TNF-? in the cell control group(NC group)were not high.The concentration of TNF-? gradually increased,and the increase was statistically significant(#P<0.05,##P<0.01,###P<0.001).IL-6 and TNF-? were statistically significant from 4h,IL-1? was statistically significant from 12 h.When overexpressed TIR(674-815aa)domain,GAR(645-707aa)domain,TIR(674-815aa)domain and GAR(645-707aa)domain together,compared with NC+RSV group,we found that IL-6,IL-1? and TNF-? secreted by the three groups of cells were significantly increased to a certain extent,and the difference was significant(*P<0.05,**P<0.01,***P<0.001).Among them,TIR(674-815aa)and GAR(645-707aa)co-overexpressed at the highest level.However,when TLR4(del 674-815aa)and NCL(del 645-707aa)were overexpressed,respectively,they did not promote the secretion of IL-6,IL-1? and TNF-? compared with the NC+RSVgroup,which was not statistically significant differences in academic meaning.(5)Flow cytometry showed: The cell control group(NC group)had less apoptosis.After 48 h of RSV treatment,the apoptosis increased and was statistically significant(###P<0.001).Compared with the NC+RSV group,apoptosis was significantly increased in TIR(674-815aa)+RSV group,GAR(645-707 aa +RSV group and TIR(674-815aa)+GAR(645-707aa)+RSV group,there was a statistically significant difference in the increase in apoptosis(***P<0.001)in addition.Moreover,the apoptosis of cells in the co-overexpression group of TIR(674-815aa)and GAR(645-707aa)domains was the most obvious.However,the difference was not statistically significant in the TLR4(del674-815aa)+RSV group and the NCL(del 645-707aa)+RSV group comparable to the NC+RSV group.The above results indicate that the TIR(674-815aa)domain of TLR4 but not TLR4(del 674-815aa)and the GAR(645-707aa)domain of NCL but not NCL(del 645-707aa)can act synergistically in apoptosis occur.Conclusion: In neuronal cells SY5 Y,the TIR(674-815aa)domain of TLR4,but not the TLR4(del 674-815aa)is important for RSV infection.For NCL,its GAR(645-707aa)domain rather than the NCL(del 645-707aa)can mediate the infection of RSV.Our results suggest that the TIR domain and the GAR domain can synergize to mediate RSV infections,and activate the NF-?B signaling path to adjust the inflammation factor IL-6,IL-1? and TNF-?.Overexpression of TIR and GAR domains exacerbates apoptosis and autophagy,ultimately leading to neuronal damage. |
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