Study On The Mechanism Of Substance P SiRNA In The Gene Therapy For Allergic Rhinitis

Allergic rhinitis (AR) is one of the more frequently encountered diseases in the otolaryngology department. Its prevalence is 5% to 15% in the global population. Previous studies on the pathogenesis have proved that there are many genes express abnormally in AR. For example, in AR, Substance P (SP) mRNA expression increase and which have positive correlation with levels of AR.Modulation of the expression of abnormal genes is a potential method in curing the gene related disease. The small interference RNA (siRNA) technique provides a good method in treating those diseases. siRNA technique:1) As to the target mRNA, design the matched oligonucleotide sequence (21-25 base pair); 2) Import the dsRNA or shRNA to the cells; 3) The imported dsRNA was decomposed by Dicer or shRNA translated to specific siRNA; 4) siRNA combination with the nucleotidase and form RNA-induced silencing complex (RISC); 5) Degrade the target mRNA and inhibit the expression of target gene. As an effective and specific method in inhibit the target gene expression, siRNA technique is not only used in the study of specific gene function, but also in the treatment of gene diseases.In order to study the role of SP in the pathogenesis and gene therapy position in AR, we investigated the function of SP-siRNA in AR in vitro and in vivo.Firstly, we compared the consensus sequence of SP mRNA sequence between rat, mouse and human. We designed 3 different SP specific siRNA sequences using online siRNA design software on the website of Whitehead Institute for Biomedical Research. We chose the RBL-2H3 mast cell as the in vitro model to validate the SP-siRNA. Real time-PCR, RT-PCR and immunofluorescence were used in testing the SP expression in RBL-2H3 cells. We found that, after DNP-IgE activation, SP expression increased significantly. We constructed SP specific shRNA expression plasmid using psilencer CMV neo vector. After colon and extraction, we gained 3 groups of SP-shRNAs. Then, RBL-2H3 cells were transfected using electro-transformation. Transfected RB1-2H3 cells were activated by DNP-IgE and the SP expression were dectected using real time PCR and immunofluorescence. The most effective SP-shRNA (SP-shRNA2) was chosen. The SP-shRNA2 transfected RBL-2H3 cells were used for the beta-hexosaminidase assay to detect the degranulation ability of mast cells. The results showed that, compared with control, SP-shRNA transfection decreased the degranulation ability of RBL-2H3 cells.After the in vitro assays, we dripped SP-siRNA2 which sequence correspondence to SP-shRNA2 intra-nasal to study the role of SP-siRNA in vivo. The BALB/C mice were divided to 3 groups:1) SP-siRNA treated AR model,2) NC-siRNA treated AR model, and 3) saline treated AR model. After 4 times i.p. sensitization using OVA,4 times interference was processed as the sequence:siRNA treatment/i.n OVA challenge/i.n OVA challenge/i.n OVA challenge. After the last i.n OVA challenge, the mice were anesthesia; serum and nasal mucosa were collected. ELISA, immunohistochemistry, pathology study, real time-PCR and antibody array were used in studying the SP expression in mucosa, eosinophil infiltration and the expression of inflammation cell factors. The results showed that, after SP-siRNA treatment, SP exression in nasal mucosa in AR decreased significantly. However, the IgE content in serum has no significant difference in 3 groups. SP-siRNA treatment alleviated the AR symptoms. HE and Luna's stains proved that SP-siRNA treatment inhibited infiltration of eosinophil in nasal mucosa. The inflammation cell factors antibody array showed that, after SP-siRNA treatment, many Th2 related cell factors including IL-1?, IL-1?and IL-4, IL-6 and eosinophil chemotactic factors including Eotaxin and Eotaxin-2 decreased significantly. And we analyzed the role and mechanism of SP-siRNA in AR mouse model.In conclusion, the present studies proved SP-siRNA modulate the AR pathogenesis in vitro and in vivo. SP-siRNA inhibited the SP expression in mast cells, and then, decreased the degranulation of mast cells and reduced the release of inflammation factors. The dripped SP-siRNA in the nasal mucosa inhibited the SP expreesion in AR mice mucosa. After SP-siRNA treatment, eosinophil infiltration decreased, IL-4 expression and eosinophil chemotactic factors decreased, then, the allergic inflammation decreased in nasal mucosa. And finally, the allergic rhinitis symptom alleviated. The study proved that the SP-siRNA interference is a perspective gene therapy in allergic rhinitis treatment.