| Objective:By inoculated RSV and cytokines to microglia in vitro respectively, the possible mechanism of activated microglia and cytokines secreted by microglia in the damaged central neurons caused by RSV infection were investigated and clarified. It may provide the new ideas for clinical prevention and treatment of RSV infection of the central nervous system.Method:Neurons SY5Y lesion was detected by confocal microscopy after RSV infection. In addition, by RSV infection microglia CHME-5 and inoculated cytokines to microglia in vitro, cells and supernatants were collected after different post-infection times at 12,24,48,72 and 96 h, while uninfected group as normal group. (1) The result of virus through Transwell chamber was investigated by plaque forming unit assay (PFU). (2) The results of cytokines through Transwell chamber and the concentration of IL-6, IL-8 and TNF alpha in the supernatants of SY5Y cells in each group were determinated by ELISA. (3) Confocal microscopy assay was used to visualize cell damage and morphological changes after SY5Y cells inoculation of RSV and cytokines. (4) The expression level changes of TLR3/RIG-I proteins in each group and Hvl proteins in microglial cells at different point-infection times were detected by Western blot. (5) The total RNA of SY5Y cells was extracted by Trizol and the transcription level changes of the TLR3 and RIG-I mRNA were detected by real-time fluorescent quantitative PCR. (6) SY5Y cells apoptosis in each group was detected by flow cytometry.Result:(1) PFU showed that RSV could not pass the polycarbonate membrane in Transwell upper chamber which aperture is 0.1 μm.(2) ELISA showed that cytokines could pass the polycarbonate membrane in Transwell upper chamber which aperture is 0.1 μm. The concentrations of IL-6, IL-8 and TNF alpha in supernatant of normal SY5Y cells were lower than that in RSV group. After RSV infected, the expression of IL-6, IL-8 and TNF alpha were up-regulated markedly in a time-dependent manner with statistically significant. Compared with infection group, the expression of IL-6, IL-8 and TNF alpha in cytokines+CHME-5 Transwell group, RSV+CHME-5 Transwell group and RSV+cytokines Transwell group were increased, while the levels in RSV+cytokines Transwell group were more evidently with statistically significant from 24 h (P<0.01).(3) Uniform distribution with intact and high-density normal cells were observed by confocal microscopy assay. Whereas in RSV infected-SY5Y cells 48 h, the density of nucleus was low and the nuclei turned into irregular, fragmented or incomplete, cells body expansion and synaptic longer compared to that of normal control. Compared with infection group, the morphology changes in cytokines+CHME-5 Transwell group and RSV+CHME-5 Transwell group were obvious at 72 h, meanwhile RSV+cytokines Transwell group morphology change were more obviously with longer synaptic.(4) Low levels of TLR3/RIG-I proteins expression in the normal group were detected by Western blot. The expression of TLR3/RIG-I were markedly up-regulate in a time-dependent manner after RSV infection and the increase had statistically significant at 12 h. The expression in cytokines+CHME-5 Transwell group, RSV+CHME-5 Transwell group and RSV+cytokines Transwell group were elevated compared with infection group, which the levels in RSV+cytokines Transwell group were more evidently with statistically significant from 24 h. The expression of Hvl protein in normal microglial cells was scare, while it was increased in a time-dependent manner after RSV infection and the increase had statistically significant at 12 h. When cytokines were added to microglial cells, the Hvl protein was also showed increased in a time-dependent manner. In addition, the level in RSV+cytokines group was more evidently with statistically significant from 12 h.(5) Real-time PCR showed that the expression levels of TLR3/RIG-I mRNA were relatively low in normal group, while up-regulated levels in a time-dependent manner were detected in RSV infected group and the increase had statistically significant at 12 h. The expression of TLR3/RIG-I mRNA in cytokines+CHME-5 Transwell group, RSV+CHME-5 Transwell group and RSV+cytokines Transwell group were ascended compared with infection group, since the expression in RSV+cytokines Transwell group were more obviously with statistically significant from 24 h.(6) Prominent apoptosis in SY5Y cells at 48 and 72 h was observed by flow cytometry whereas the apoptosis in normal SY5 Y cells was low. Compared with infection group, the proportion of apoptotic ratio in cytokines+CHME-5 Transwell group and RSV+CHME-5 Transwell group were more apparently with 33.98% and 32.95%, meanwhile the proportion of apoptotic ratio in RSV+cytokines Transwell group morphology change were 43.02% with significant increase.Conclusions:Damaged SY5Y cells, longer synaptic, activated TLR3 and RIG-I and increased the downstream cell inflammatory cytokines like IL-6, IL-8 and TNF alpha secretion could be induced by virus and inflammatory factors could be generated by microglia which inoculated RSV in vitro accompanied by activated Hvl, then mediated the production of natural immune response, lead to neuronal injury and apoptosis accordingly. The above showed that microglia could promoted the central neurons damage infected by RSV. |
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