The Impact Of Downregulation Of Orail Expression In Airway On Murine Allergic Rhinitis

Objective:To construct and produce the recombinant lentivirus Lenti-Orai1-shRNA which can inhibit the transcription of murine ORAI1gene through RNA interference, and to detect its infection efficacy and inhibition efficacy of ORAI1transcription of the recombinant lentivirus. To establish the murine model of Lenti-ORAI1-shRNA infection in the nasal cavity in vivo, and to determine the kinetics of the inhibition efficacy of Orail transcription in the nasal mucosas by this method. To set up the establishment of the model of Lenti-ORAI1-shRNA infection in the nasal cavity of AR mice and assess its impact on AR symptoms, functions in the immune cells in the nasal mucosa and the spleen and to study its mechanisms primarily.Methods:The interfering sequence that corresponded to the ORAI1gene was designed, chemically synthesized and then inserted into the lentiviral vector pLVTHM which had been restriction digested by the restriction enzymes MluI and Clal. The resultant clones were verified by sequencing. The recombinant lentiviruses were produced by co transfection of293T cells with the interferential plasmid, the pRsv-REV plasmid, the pMDLg-pRRE plasmid, and the pMD2.G plasmid according to standard protocols. the PANC cells were infected with lenti-ORAI1-shRNA and the viral infection efficacy was determined by fluorescence-activated cell sorting for GFP expressed in the transfected cells. The viral inhibition efficacy of ORAI1transcription was analyzed by Real-time RT PCR. Twenty-one BALB/c mice were randomly devided into seven groups, one of which was normal control group and the other6groups were Lenti-ORAI1-shRNA intervention groups. On day0, after the mice in the intervention groups were anesthetized,8μl lysophosphatidylcholine (in PBS,0.3%w/v) was delivered into the nasal airway via inhalation-driven instillation. The lentivirus (1×109tu/ml) were delivered into the nostrils of the mice1h after LPC delivery. The transfected mice were sacrificed on day1, day3, day7, day14, day21, day28, the mRNA levels of ORAI1in their nasal mucosas were measured using real-time RT-PCR. The mice were ramdomly devided into five groups:normal group (normal), normal mice with Lenti-ORAI1-shRNA intervention (NLI), AR mice (AR), AR mice with Lenti-GFP intervention (AR-GFP), AR mice with Lenti-ORAI1- shRNA intervention (AR-LI). The mice in the last three groups were sensitized by intraperitoneal injection with0.2ml of0.5mg/ml OVA/20mg/ml Al(OH)3solution for3times, and then were challenged by inhalation-driven instillation with20μl of40mg/ml OVA solution for8times. NLI and AR-LI mice accepted nasal instillation with20μl of Lenti-ORAI1-shRNA (1×109tu/ml)3days before the first challenge. AR-GFP mice received nasal instillation with the same dose of Lenti-GFP. The nasal symptoms of each mouse were evaluated by counting the number of sneezes and episodes of nasal rubbing during the10min immediately after the last intranasal challenge. The level of ORAI1, LTC4S, EAR3, germline Cε and IL-4mRNA in the nasal mucosa and spleen in each mice were measured using real-time RT-PCR. The level of Orai1, IL-33, IL-17E and TSLP expressions in the nasal mucosas and Orail expression in spleen in each mice were detected using Western Blotting. The distribution and intensity of Orai1expression in the nasal mucosa, NALT, and spleen in each mice were determined using immunohistochemical staining. Total and differential cell (epithelial cell, lympocyte, eosinophil and neutrophil) numbers were counted in nasal lavage fluid (NLF) obtained from each mice. The concentration of LTC4, ECP, OVA-IgE and IL-4in the NLF and serum in each mice were detected using ELISA.Resutls:The expression of GFP could be detected in PANC cells48hours after infected with Lenti-ORAI1-shRNA. When the multiplicity of infection is10, the transfection efficacy of Lenti-ORAI1is98.28%, and the transfection efficacy of Lenti-GFP is98.75%; and the inhibition efficacy of Lenti-ORAI1is90.5%, and that of lenti-GFP is-1.4%. After nasal infection with Lenti-ORAI1-shRNA, the ORAI1mRNA in the nasal mucosas of the mice reached the lowest level3days after the infection, and was still lower than the normal control group1month after the infection. The levels of ORAI1mRNA in the nasal mucosas and spleens of AR-LI mice were lower compared with the AR mice, and the Orail expression level in the nasal mucosas was also lower than AR mice, and their counts of sneezes and nasal rubbing were also fewer compared to AR mice. The expressions of Orai1in the cells in the epithelial layer and lamina propria of nasal mucosa, NALT and the germinal center of spleen of the AR-LI mice were decreased compared to the AR mice. The levels of LTC4S, EAR3, germline Cε and IL-4mRNA in the nasal mucosas and spleens of AR-LI mice were reduced compared with the AR mice, and the concentrations of LTC4, OVA-IgE and IL-4in NLF, the concentrations of LTC4, ECP, OVA-IgE and IL-4in serum were also decrease compared to AR mice. The expression levels of IL-33, IL-17E and TSLP were all higer in AR mice compared with normal controls, and the expressions of IL-33and TSLP were reduced in AR-LI mice.Conclusion:The recombinant lentivirus Lenti-Orail-shRNA was successfully produced, and it could effectively infect PANC cells and inhibit the transcription of murine ORAI1gene through RNA interference. Innasal instillation with Lenti-ORAI1-shRNA could effectively infect the cells in the epithelial layer of nasal mucosa. The inhibition efficacy of ORAI1transcription in nasal mucosa reached the highest level3days after the infection. Lenti-ORAI1-shRNA intervention in the nasal cavities of AR mice could downregulate the Orai1expressions in their nasal mucosas and spleens, and alleviated their AR symptoms. Downregulation of Orai1expression in the nasal mucosa of AR mice not only could reduce the inflammatory cells in NLF, but also could reduce the inflammatory mediators synthesis and release in mast cells, eosinophils, B cells and Th2cells. The inhibition of IL-33and TSLP synthesis in the nasal epithelial cells by Lenti-ORAI1-shRNA might be the reasons for its effect of reducing the immune cells responses in AR mice.