| Objective: To explore the effect of Th1 epitope peptide P25 from Schistosoma japonicum(SJ)soluble egg antigen Sj P40 protein on the production of IFN-? in splenic lymphocytes of mice infected with SJ cercariae,and investigate the inhibitory effect of P25 polypeptide with Freund's incomplete adjuvant(IFA)or aluminum adjuvant(Al)on OVA-induced allergic asthma.Methods: In vitro: Mice infected with SJ cercariae(n=3)were sacrificed and the spleen cells were isolated.The cells were divided into three groups including Medium control group(DMEM),P12 polypeptide control group(50 ug/ml P12 polypeptide in DMEM,P12 was non-Th1 epitope peptide from Sj P40),and P25 polypeptide group(50 ug/ml P25 polypeptide in DMEM,P25 was Th1 epitope peptide from Sj P40).Splenic lymphocytes proliferation potential was detected by cell counting and MTS analysis 72 h after treatment.Cell supernatant was collected 72 h after stimulation and the levels of IFN-? were analyzed by ELISA.Flow cytometric analysis was performed to determine the secretion of IFN-? in CD4+ T cells and CD8+ T cells.In vivo: BALB/c mice were divided into 5 groups randomly(n=6): Control group,OVA group,OVA + P25 group,OVA + P25 + Al group and.OVA + P25 + IFA group.BALB/c mice were immunized with P25 combined with IFA,Al or PBS respectively at 0d and 14 d.Asthma was induced in mice by injecting OVA at 7d and 21 d,and intranasally challenging OVA at 28 d and 35 d.The pathological change of lung tissue was observed after HE staining and measured by Underwood Score.The secretion of bronchial epithelial mucus was detected by AB-PAS staining.Bronchoalveolar lavage fluid(BALF)was collected and the eosinophils in BALF were counted.The levels of IL-4 and IFN-? in BALF and OVA-specific Ig E in serum were detected by ELISA.Results: In vitro: 1.Proliferation of splenic lymphocytes was detected by counting and MTS in P25 group,which was significantly increased compared with Medium group(P < 0.05,P < 0.001).There was no significant difference in cell proliferation between P25 and P12 group(P > 0.05).2.The secretion levels of IFN-? in splenic lymphocytes were detected by ELISA in P25 group,which was significantly increased compared with Medium group(P < 0.05).There was no significant difference in levels of IFN-? between P25 and P12 group(P > 0.05).3.The levels of IFN-? in CD4+ T cells and CD8+ T cells from splenic lymphocytes were detected by Flow Cytometry.The cell proportion of CD4+ T cells and CD8+ T cells secreting IFN-? in P25 peptide group was significantly higher compared with Medium group(P < 0.05).There was no significant difference in cell proportion between P25 and P12 group(P > 0.05).In vivo: 1.Pathological scores of lung tissue in Control group,OVA group,OVA + P25 group,OVA + P25 + Al group and OVA + P25 + IFA group were 1.01 ± 1.3,13.50 ± 1.04,13.00 ± 1.21,9.07 ± 0.97 and 7.80 ± 1.23,respectively.The pulmonary inflammation,bronchial mucus secretion and pathological score of lung tissue reduced in OVA + P25 + IFA and OVA + P25 + Al group compared with OVA group(P < 0.01).The pathological score of OVA + P25 + IFA group was lower than that of OVA + P25 + Al(P < 0 05).There was no significant difference in pathological score between OVA + P25 and OVA group(P > 0.05).2.The ratio in Control group,OVA group,OVA + P25 group,OVA + P25 + Al group and OVA + P25 + IFA group were 0.78 ± 2.3%,43.5 ± 0.84%,39.2 ± 3.31%,21.07 ± 1.37% and 15.8 ± 2.23%,respectively.Mucus secretion in OVA + P25 + IFA group and OVA + P25 + Al group were significantly decreased compared with OVA group(P < 0.01),and OVA + P25 + IFA group showed lower ratio when compared with OVA + P25 + Al group.There was no significant difference in ratio between OVA + P25 and OVA group(P > 0.05).3.Lower eosinophil counts in BALF were detected in OVA + P25 + IFA and OVA + P25 + Al group compared with OVA group(P < 0.01).The eosinophil counts of OVA + P25 + IFA group were less than that of OVA + P25 + Al(P < 0.05).There was no significant difference in eosinophil counts between OVA + P25 and OVA group(P > 0.05).4.There were lower levels of IL-4 and higher levels of IFN-? in OVA + P25 + IFA and OVA + P25 + Al group compared with OVA group(P < 0.05,P < 0.01).There was no significant difference in levels of IL-4 and IFN-? between OVA + P25 and OVA group,P > 0.05).5.The levels of OVA-specific Ig E were lower in OVA + P25 + IFA and OVA + P25 + Al group compared with OVA group(P < 0.05).There was no significant difference in level of serum Ig E between P25 + OVA group and OVA group(P > 0.05).Conclusion: 1.Th1 epitope peptide P25 from Schistosoma japonicum egg antigen could induce splenic lymphocytes proliferation and secretion of IFN-? in CD4+ T cells and CD8+ T cells in the mice infected with SJ cercariae,which suggests that P25 peptide may promote the expression of Th1 cytokine IFN-?.2.The combination treatment of Th1 epitope peptide P25 and IFA adjuvant or aluminum adjuvant could effectively inhibit OVA-induced allergic asthma through alleviating pulmonary inflammation,decreasing eosinophil infiltration,and reducing serum OVA-specific Ig E level.IFA showed better inhibitory effect than Aluminum.3.The combination treatment of Th1 epitope peptide P25 and IFA adjuvant or aluminum adjuvant could down-regulate the levels of IL-4 and up-regulate the levels of IFN-? levels in BALF,indicating that IFA adjuvant or aluminum adjuvant could facilitate Th1 immune response induced by P25 peptide,which might contribute in inhibiting allergic asthma by maintaining the balance of Th1/Th2. |
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