The Preliminary Study On Role And Mechanism Of TLRs Signal Pathway In Liver Eggs Granuloma Formation Of Schistosomasis Japonicum

Objectives:To establish a model of liver eggs granuloma of Schistosomasis japonicum in Balb/cmice and study the relationship between the expression of TLRs and the livergranuloma formation of schistosomiasis. SjSEA and SjSWA were used as the PAMPsof TLRs, to study schistosome antigen induced TLRs pathway activation andproduced proinflammatory cytokines in vitro.Methods：To establish the model of liver eggs granuloma of Schistosomasis japonicum inBalb/c mice, FCM and rea-ltime PCR were used to detect the expression levels ofTLR2and TLR4mRNA and protein in liver tissue at0w,2w,4w,6w and8winfection mice. The pathological changes of liver tissue were observed by HE andMasson staining, The expression levels of pro-inflammation cytokines (IL-1β、TNF-αand IL-6) in serum were determined by ELISA, The levels of ALT and AST wereassayed by rate method.The SjSEA and SjSWA were prepared and the peritoneal macrophages wereisolated and purified and cultured. The pro-inflammatory cytokines(IL-1β,TNF-αand IL-6) were detected by ELISA and the TLR2,TLR4mRNA by real-time PCRafter SjSEA, SjSWA, LPS and PBS stimulation pMφ at different time points. UsingSjSEA and SjSWA stimulated peritoneal macrophages, IκB were detected by westernblot.Results：The model of liver eggs granuloma of Schistosomasis japonicum in Balb/c micewas successfully constructed. The results of the pathological changes of liver tissue ininfection mice showed that size of egg granoma and the collagen deposition in micelivers increased with the infection time extension. The infection group（2w，4w，6w，8w）were compared with normal control group, the expression levels of TLR2and TLR4in liver tissues of the infected mice were significantly higher than the normalcontrol group,there were statistical significance (P<0.05), and their contents reachedthe peak at infection8week, TLR2was95.44±10.35%,TLR4was39.27±3.88%. Thereal-time-PCR results demonstrated that TLR2and TLR4in liver of infection micewas significantly higher than the normal control group(P<0.05), Infection8w reachedpeak,TLR2was7.14±0.96and TLR4was5.97±0.13. ELISA results showed thatthe levels of proinflammatory cytokines（IL-1β,TNF-αand IL-6）in infection miceserum were significantly higher than the normal control group(P<0.05), IL-1βcontent reached its peak in infection6w（234.81±31.7pg/mL）,TNF-α in4w(977.1±45.80pg/mL) and IL-6in8w(136.00±14.20pg/mL).The levels of serum ALT andAST in infection mice were significantly higher than the normal controlgroup(P<0.05), infection8w reached their peaks, ALT was149.60±15.30U/L andAST was163.50±21.80U/L. The correlation analysis showed that the levels ofTLR2and TLR4were positively correlated with the inflammation degree of livereggs granuloma and levels of pro-inflammatory factors and ALT and AST.The SjSEA and SjSWA groups compared with PBS group, the levels of theinflammatory cytokines (IL-1β,TNF-αand IL-6) increased significantly in SjSEAand SjSWA groups at6h and12h, there were the statistical significances (P<0.01),IL-1β, TNF-α and IL-6reached peak at12h, IL-1β, TNF-α�'�IL-6were91.90±15.28pg/mL,1350.90±160.28pg/mL and52.71±13.38pg/mL respectively afterSjSEA stimulation, and were107.03±25.19pg/mL,983.43±18.19pg/mL,48.03±11.19pg/mL respectively after SjSWA stimulated. Real-time PCR showed that theexpression level of TLR4mRNA increased significantly in SjSEA and SjSWA groups,TLR4mRNA reached peak at stimulating12h（10.91±1.32,10.14±1.23）.The levelof TLR2mRNA was no significant change in SjSEA and SjSWA groups. The IκBwas degraded significantly after SjSEA and SjSWA stimulation pMφ at30min bywestern blot analysis, but IκB levels was restored at60min.Conclusions：(1) The expression levels of TLR2/TLR4were positive correlated with theinflammation degree of liver eggs granuloma of Schistosomasis japonicum andlevels of pro-inflammatory factors (IL-1β，TNF-α and IL-6), ALT and AST. TLR2/TLR4pathway can take part in the inflammation of liver eggs granulomaof Schistosomasis japonicum.(2) SjSEA and SjSWA may be as PAMPs of TLR4to activate the TLR4signalpathway and induce pMφproducing pro-inflammatory cytokines(IL-1β,TNF-αand IL-6).