| Objective:This study aimed to construct the DNA vaccine co-expressing Der p2 and A20,and to investigate the treatment effect of intranasal administration of the DNA vaccine on nasal inflammation in mice with allergic rhinitis.Methods:(1)The pVAX1-Der p2-A20 eukaryotic expression vector-loaded by poly lactic-co-glycolic acid(PLGA)nanoparticles(PpDA)was prepared by double emulsion-solvent evaporation method,and its morphological characteristics were observed by scanning electron microscopy.The expression of PpDA in cells was verified by in vitro experiments.(2)The recombinant Der p2 was induced and purified,its immunological characteristics were analyzed by ELISA and Western blot.(3)Fifty female Balb/c mice were randomly divided into 5 groups:the normal group(NC),the model group(AR),the PpDA treatment group(PpDA),the blank PLGA treatment group(PLGA),the naked plasmid DNA treatment group(pDA).Mice in groups AR,PpDA,PLGA,pDA were sensitized by intraperitoneal injection of recombinant Der p2(100?g)and cholera toxin(CT)(15?g)on day 0,7 and 14,respectively,and mice in group NC only received PBS.One week after sensitization,mice in groups AR,PpDA,PLGA and pDA were intranasally treated with 20?l of PBS,PpDA(containing 100?g pDA plasmid),blank PLGA nanoparticles and pDA(containing 100?g pDA plasmid),respectively,every 3 days for a total of 5 treatments.Mice in the latter four groups were intranasally challenged three times daily with recombinant Der p2(200 ug)after the final treatment,mice in group NC only received PBS.The frequencies of scratching nose and sneezing in each group were recorded within 30 minutes after challenging by two persons who were unknown of the experimental design.The histological changes of nasal mucosa tissue were evaluated by hematoxylin and eosin(H&E)staining.The serum concentration of Der p2-specific antibodies(IgE,IgG2a and IgG1)and cytokines(L-4,IL-10,IL-13,IFN-?,TGF-?1)in splenocyte culture supematant were determined by ELISA.The percentage of CD4~+CD25~+Foxp3~+Treg/CD4~+T in the spleen was detected by flow cytometry.The expression of A20 protein in nasal mucosa were measured by western blot.Results:(1)The particle size of PpDA was(292±2.34)nm and the encapsulation rate was(85.8±0.02)%.This nanoparticle could not only transfect 293 T cells in vitro but also maintained its biological activity.It can be used for the next step of immunotherapy.(2)The recombinant Der p2 was induced and purified successfully,which can specifically bind to IgE from positive serum of house dust mite allergy patients,the recombinant Der p2 has high immunological activity,and can be used to construct the model of AR.(3)(1)The subjective symptoms result showed that the frequencies of scratching nose and sneezing were significantly increased in AR,PLGA and pDA groups when compared with NC group(P<0.01);the frequencies of scratching nose and sneezing were reduced in PpDA and pDA groups when compared with AR and PLGA groups(P<0.01);however,the frequencies of scratching nose and sneezing were significantly reduced in PpDA group when compared with pDA group(P<0.05);there was no significant difference between AR group and PLGA group(P>0.05).(2)The histopathological results showed that the nasal mucosa inflammatory reaction were obvious in the AR,PLGA and pDA groups when compared with NC group;the nasal mucosal inflammatory response were significantly improved in PpDA and pDA groups when compared with AR and PLGA groups;however,the improvement in PpDA group was more obvious when compared with pDA group;there was no significant difference between AR group and PLGA group.(3)Serum levels of specific antibodies and spleen cell supernatant levels of cytokine results showed that serum Der p2-specific IgE levels and spleen cell supernatants levels of IL-4,IL-13 were significantly increased in AR,PLGA and pDA groups when compared with NC group(P<0.01),while in PpDA and pDA groups were significantly lower when compared with AR and PLGA groups(P<0.05);the levels of Der p2-specific IgE and IL-4,IL-13 were significantly reduced when compared with pDA group(P<0.05).Compared with AR and PLGA groups,serum levels of IgG1,IgG2a and spleen cell supernatants levels of IFN-?,IL-10 and TGF-?1 were significantly increased in PpDA and pDA groups(P<0.01);compared with pDA group,the increase in PpDA group was more obvious(P<0.01).(4)The percentage of CD4~+CD25~+Foxp3~+Treg/CD4~+T cells in spleen monocytes were significantly increased in PpDA and pDA groups when compared with AR group(P<0.05);while the increase in PpDA group was more significantly than that in PLGA and pDA groups(P<0.05).(5)Western blot results showed that the expression of A20 in nasal mucosa was increased in AR,PpDA,PLGA and pDA groups when compared with NC group(P<0.05),while in PpDA group the expression of A20 was higher when compared with AR,PLGA and pDA groups(P<0.05).Conclusion:(1)The DNA vaccine PpDA has been successfully constructed,the particle size was(292±2.34)nm and the encapsulation rate was(85.8±0.02)%.(2)The DNA vaccine PpDA alleviates nasal allergic inflammation in mice with allergic rhinitis by promoting Treg population and up-regulating the expression of A20 in nasal mucosa.(3)The DNA vaccine PpDA can significantly improve allergic inflammatory response in Der p2 allergen induced AR model mice,which is better than naked plasmid pDA. |
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