| Objectives: Study on the role and mechanism of Th17/Treg imbalance in the livereggs granuloma formation of schistosomiasis japonica, improving the pathogenicmechanism of schistosoma and providing new strategy and target for theschistosomiasis treatment.Methods：Schistosoma japonicum infection model was constructed with6~8weeksold Balb/c mice, the number and proportion of Th17/Treg cell in the the liver andspleen tissues of infection mice was assayed by FCM. After extracting the mRNA ofliver tissue by Trizol, the expression levels of the IL-6, IL-10, IL-17, IL-23, RoR-γt,Foxp3mRNA were detected by real-time PCR. The expression levels of IL-6, IL-17,IL-23and TGF-β in serum of mice were detected by ELISA; the pathological changesof the mice liver were observed by HE and Masson staining. The SjSEA was extractedand purified from Schistosoma japonicum cercaria infected New Zealand rabbits. Thespleen mixed cells from Balb/c mice were extracted in vitro, then stimulated bySjSEA, and the levels of RoR-γt and Foxp3mRNA at different time points weretested by real-time PCR, then the expression levels of cytokines IL-6, IL-17, IL-23,TGF-β in culture supernatant were assayed by ELISA.Results： The mice model of schistosomiasis japonicum was establishedsuccessifully, the results of pathological changes in liver tissue of mice showed thathepatic granuloma size and the percentage of liver collagen deposition were extendedwith the infection time,the hepatic granuloma sizes of the normal group and infection2w mice were0, infection4w,6w and8w were0.098±0.010,0.156±0.017and0.219±0.020respectively, the percentage of liver collagen deposition of the normal group(0w)was1.50±0.20, and2w,4w6w and8w were2.05±0.34,12.6±1.30,18.30±2.03and23.47±2.94respectively,8w reached to the maximum value. FCM results showed: the ratio of IL-17A+Th17cells in CD4+T cells of normal mice liver tissue was0.74±0.09%,infection groups(2w,4w,6w) were4.87±1.03%,9.64±0.98%,5.24±2.75%respectively. The ratio of IL-17F+Th17cells in liver CD4+T cell of the normal groupwas1.19±0.51, and2w,4w and6w infection groups were5.54±1.64%,9.05±0.89%,6.58±3.99%respectively, the proportions of Th17cells of infection groups mice weresignificantly increased compared with control group (p<0.05). In infection8w group,the ratio of IL-17A+Th17cells in liver CD4+T cell was1.64±0.99%, and IL-17F+Th17cells was2.43±1.79%,the group compared with normal group, there were nostatistical differences. The percentages of IL-17A+Th17and IL-17F+Th17cells werereached to its peak in the liver of the infection4w mice.The ratio of Treg cells in liverCD4+T cells of the normal control group mice was0.07±0.02%, and infection groupmice(4w,6w,8w) were11.27±3.05%,12.28±6.52%,6.51±2.15%respectively, theratio of Treg cells were increased significantly compared with the normal group(p<0.05). The ratio of IL-17A+Th17cells in spleen CD4+T cells of normal group micewas1.73±0.39%and infection4w group mice was13.50±2.14%. The ratio ofIL-17A+Th17cells infection4w group mice increased significantly compared with thenormal group（p<0.05）. The ratio of IL-17F+Th17cells in spleen CD4+T cells ofnormal group mice was1.95±0.47%and infection groups mice (2w and4w) were3.98±0.64%and12.92±2.01%respectively, they were increased significantlycompared with the normal group (p<0.05), but there were no significant differenceswhile infection6w and8w group compared with normal group. The ratio of Treg cellsin spleen CD4+T cells of normal group was0.52±0.20%, infected2w and4w groupswere2.68±0.81%and10.53±1.57%respectively, the they increased significantlycompared with the normal group (p<0.05), the ratio reached its peak in infection6w.The ratio changes of Th17/Treg in liver (0w~8w) were from0.63±0.10to27.14±6.90, but the ratio ranges were from2.51±0.11to11.19±0.42in spleen, and thecorrelation coefficient which the percentages of Th17and Treg cells were in CD4Tcells of spleen is0.99, the correlation coefficient is only0.45in the liver tissue, theresults revealed that Th17/Treg imbalance was very serious in the liver tissue ofschistosomiasis japonicum.The results of real-time PCR showed that the expression content of ROR-γt mRNA in liver tissue of infected4w group reached to its peak as1.66±0.38, compared with normal control group (1.00±0.02), the difference ofROR-γt mRNA content was statistically significant (P<0.05). The expression contentsof Foxp3mRNA in infection group (4w,6w,8w) were64.8±13.44,107.72±26.19and35.49±5.97respectively, compared with the normal group (10.12±4.08),thedifferences of Foxp3mRNA were statistically significant (P<0.05), the Foxp3mRNAreached to the maximum value in infection6w. The expression levels of IL-6mRNAin liver of infection6w and8w mice were21.37±8.87and20.34±12.59respectively,they compared with the normal group (0.79±0.26), P<0.05. The expression levels ofIL-10mRNA in liver of infection4w,6w and8w mice were5.98±1.08,6.03±1.06and11.21±4.79respectively, compared with the normal group(0.46±0.50), P <0.05. TheIL-17mRNA in liver of infection4w,6w and8w mice were7.30±1.35,17.69±2.07and39.74±7.27respectively, compared with the normal group(0.99±0.18), P<0.05.TheIL-23mRNA in liver of infection4w,6w and8w mice were313.64±59.57,298.29±77.71and240.46±58.38respectively, compared with the normal group (4.41±1.41),P<0.05. The cytokines were detected in serum by ELISA, the results showed thatexpression levels of IL-6in infection groups (2w and6w) were1582.44±135.98and810.23±79.28respectively, compared with the normal group (560.99±95.51),P<0.05, the levels of IL-6reached its peak in infection2w mice. IL-17levels ininfection groups (2w and4w) were773.67±51.90and950.24±37.94respectively,compared with the normal group (631.28±64.26), P<0.05, the level reached its peakin infection4w. IL-23levels in infection groups (2w,4w,6w and8w) were1530.31±176.96,1761.824±90.57,2585.37±390.41and2938.73±317.24respectively,compared with the normal group (798.03±192.94), P<0.05. TGF-β levels in infectiongroups(4w,6w and8w) were107.39±7.07,150.04±12.26and188.87±32.10respectively, compared with the normal group (59.67±2.83), P<0.05.SjSEA could induce expression of RoR-γt and Foxp3mRNA in spleen mixed cellsfrom Balb/c mice. The RoR-γt mRNA reached its peak at36h after SjSEA stimulation,the expression content was3.207±0.600, the Foxp3mRNA reached its peak at12h(1.903±0.090), they compared with PBS group, the difference of mRNA contents was statistically significant p<0.05. The SjSEA could induce IL-6, IL-23, IL-17andTGF-beta cytokines expression in cells culture supernatant, the levels of IL-6was913.83±90.0at36h after SjSEA stimulation, IL-23was1387.81±110.00at24h afterSjSEA stimulation,TGF-beta level reached its peak at24h (202.27±39.80), theexpression content of IL-17that Th17cell secreted the function cytokine reached amaximum at48h after SjSEA induction, the content was1150.07±108.22. Theresults evidenced that the SjSEA could induce Th17differentiation and activity invitro.Conclusions：(1) The inmmue response of Th17cell can be induced in infection mice ofSchistosoma japonicum, Th17/Treg imbalance in the liver plays an important role inliver eggs granuloma formation of schistosoma japonicum.(2) Th17/Treg imbalance in the liver was more serious than in the spleen inSchistosoma japonicum infection mice. The results indicated that Th17cells playedmain role in immunopathological damage of the liver tissue of schistosomasisjaponicum.(3) The spleen mixed cells after SjSEA stimulation could express the IL-6, IL-17,IL-23and RoR-γt that they are relative to Th17differentiation and activation.Theresult evidenced that SjSEA could induce Th17differentiation in vitro. |
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