The Effects Of TLR4 And Melatonin In NLRP3 And Cell Autophagy Induced By RSV Infection Of Central Neuron

Objective On the basis of RSV infection of human neuron cells,inducing the generation of NLRP3 and promoting the occurrence of autophagy,to study the key role that TLR4 receptor plays in the RSV infection of human neuron cells.In this experiment,the TLR4 receptor in human neuron SY5Y cells was silenced by TLR4-siRNA and a proper amount of melatonin was given to the human neuron SY5Y cells infected by RSV in vitro,in order to study the regulation of TLR4 receptor on the central neurons infected by RSV and the possible protective mechanism of melatonin for the prevention which can provide new ideas for clinical prevention and treatment of RSV infection in the central nervous system.Methods RSV infected SY5Y cells in vitro,cells and cultured supernatant were collected respectively in 4,12,24 and 48 hours after infection.Establishing different groups and uninfected cells was used as control group to judge the effect of RSV infection on the human neuron;then interfering TLR4 receptor of SY5Y cells with different TLR4-siRNA sequences to select the most appropriate sequence of silence and setting up the TLR4-siRNA experimental group;finally using different concentrations of melatonin to culture human neuronal SY5Y cells,CCK8 detect neuronal activity situation and select suitable concentration of melatonin which was used as MT experimental group.?1?TCID50 to detect virus titer of virus;?2?Enzyme-linked immunosorbent assay?ELISA?to detect the expression of IL-1??IL-18 and TNF-?inflammatory factors in cultured supernatant;?3?Western-blot to detect the protein levels expression of TLR4?NLRP3?Caspase-1?NF-?B?LC3-?a t different time points;?5?The total RNA were extracted from SY5Y cells by TRIzol method and TLR4 and Caspase-1 transcription level of mRNA were detected by real time fluorescence quantitative PCR;?6?Apoptosis were detected by flow cytometry.Results?1?The results of detection showed that the TCID₅₀ of RSV virus was 2.5 x 10^-6.34/0.1ml.?2?ELISA assay showed that the expression of three inflammatory cytokines IL-1??IL-18 and TNF-?in normal control group were low;after RSV infection,the content of IL-6?IL-8 and TNF-?in the SY5Y cell cultured supernatant were increased with infection time,compared with RSV infection group the expression of IL-6,IL-8 and TNF-?were significantly decreased in TLR4-siRNA group and melatonin group and and the melatonin group decreased more obviously,the difference was statistically significant from 4 h?P<0.05?.?3?The test results of the Western blot method showed that TLR4,NLRP3,Caspase-1,NF-?B,LC3-II protein expression in normal control group were lower,after RSV infection,the expression of TLR4,NLRP3,Caspase-1,NF-?B,LC3-II were increased with the elongation of infection and the increase in 4h was statistically significant?P<0.01?;Compared with the RSV infection group,the expression of TLR4 protein in the TLR4-siRNA+RSV group and the MT+RSV group decreased,but the decrease of the expression of TLR4 protein in the TLR4-siRNA+RSV group was more obvious;At the same time,MT+RSV group and TLR4-siRNA+RSV group were compared with RSV infection group.The result showed the decrease of NLRP3,Caspase-1 and LC3-II expression were more obvious in MT+RSV group,and the difference was statistically significant from 4 h?P<0.01?.?4?At the mRNA transcriptional level,the mRNA expression of TLR4 and Caspase-1 in the normal group was lower by real-time fluorescence quantitative PCR detection.The expression of TLR4 and Caspase-1 increased gradually in the infected group,and increased with the prolongation of the infection time.The increase was statistically significant in the 4h respectively?P<0.01?;Compared with the RSV infection group,the expression of TLR4 and Caspase-1 decreased in TLR4-siRNA+RSV group and MT+RSV group,but TLR4 mRNA decreased more obviously in TLR4-siRNA+RSV group than in MT+RSV group,and the difference from 12 h began to be statistically significant?P<0.01?.While Caspase-1 mRNA decreased more significantly in MT+RSV group,and the difference was statistically significant from 4 h.It was found from the protein and gene levels that the related indexes of TLR4 receptor decreased after silencing the TLR4 receptor,suggesting that TLR4 plays a key regulatory role in RSV infected neurons,and melatonin also plays a significant protective role through inhibiting the expression of TLR4 and Caspase-1.?5?Flow cytometry showed that normal cell apoptosis was less,and RSV infected SY5Y cells after 24 and 48 h,cells showed significant apoptosis.When comparing TLR4-siRNA group and melatonin treated group with RSV infection group in 24h,apoptosis was significantly decreased,respectively 7.83%and 11.95%,compared with the RSV group apoptosis was statistically significant?P<0.05?;in 48h,melatonin group compared with RSV infection group,apoptosis is lower,9.6%,there was a statistically significant?P<0.05?.Conclusion After RSV in vitro acting on human neuronal 5Y5Y cells,activating TLR4expression and the downstream signal pathway,promoting the formation of NLRP3inflammatory corpuscle and inducing the activation of Caspase-1,promoting related precursor protein pro-IL-1?to hydrolyze into active IL-1?,which increases the secretion of IL-1?,IL-18 and TNF-alpha in the downstream inflammatory cytokines and leads to autophagy and apoptosis of neurons.Blocking the TLR4 receptor and giving additional melatonin can significantly regulate inflammatory corpuscle and autophagy.These suggest that the TLR4 receptor plays a key role in the damage of RSV infected central neurons.