Effects Of Down-regulating CCR3 Gene To Regulate PI3K/AKT Signaling Pathway On Mice With Allergic Rhinitis

Objectives:1.Conditional knockout mice of bone marrow cells CCR3 gene were used to establish the model of allergic rhinitis.The nasal symptoms and pathomorphological changes of nasal mucosa were observed in vivo,and the levels of Th1/Th2 inflammatory factors in serum and the levels of CCR3 protein and PI3K/AKT signaling transduction pathway protein in nasal mucosa were compared.2.The inhibitor was used to interfere with PI3K/AKT signaling pathway,and the model of allergic rhinitis was also established,and the above inflammatory indexes were detected in order to understand the mechanism of CCR3 gene and PI3K/ AKT signaling pathway in the pathogenesis of allergic rhinitis,and to lay a foundation for gene therapy of allergic rhinitis.Methods:1.Conditional knockout mice of bone marrow cells CCR3 gene were bred under the condition of SPF(Specific Pathogen Free),and the genotypes of offspring were identified by PCR amplification and agarose gel electrophoresis.The mice were divided into four groups: wild type negative control group(WT-NC group),wild type allergic rhinitis group(WT-OVA group),knockout CCR3 gene negative control group(KO-NC group),knockout CCR3 gene allergic rhinitis group(KO-OVA group).2.The mouse model of allergic rhinitis was established by intraperitoneal injection combined with intranasal instillation of ovalbumin(OVA),and the control group was replaced by sterile normal saline.The behavioral changes of mice in 10 min were observed after the last nasal drip.The histopathological changes of nasal mucosa in mice were observed by hematoxylin-eosin(HE)staining.3.The levels of CCR3,AKT and P-AKT in nasal mucosa of mice were detected by Western Blot.The protein contents of IL-4,IL-5 and IFN-γ in serum of mice were detected by enzyme-linked immunosorbent assay(ELISA).4.Ly294002,a specific inhibitor of PI3 K,was injected intraperitoneally into wild-type mice to interfere with the activation of PI3K/AKT signaling pathway.Experimental groups: Normal group,allergic rhinitis solvent group(OVA-DMSO group),allergic rhinitis low dose PI3 K inhibition group(OVA-1.5mg/kg Ly294002group),allergic rhinitis medium dose PI3 K inhibition group(OVA-3.0mg/kg Ly294002 group),high dose PI3 K inhibition group(OVA-6.0mg/kg Ly294002 group).The above inflammatory markers were detected as before.Results:Part one:(1)The results of PCR detection showed that the bone marrow CCR3 gene deficient homozygous mice could be obtained by breeding bone marrow CCR3 gene conditional knockout mice under the condition of SPF.(2)Behavioral observation showed that conditional knockout of bone marrow CCR3 gene could significantly improve nasal symptoms such as scratching and sneezing in allergic rhinitis mice.(3)The results of HE staining showed that conditional knockout of bone marrow CCR3 gene could improve the histopathological structure of nasal mucosa of allergic rhinitis mice,reduce local mucosal swelling and infiltration of eosinophil and other inflammatory cells,so as to reduce the destruction of nasal mucosa.(4)The results of ELISA detection showed that conditional knockout of bone marrow CCR3 gene could decrease the content of IL-4 and IL-5 in serum of allergic rhinitis mice,but increase the content of IFN-γ,thus correcte the immune imbalance of Th1/Th2 in allergic rhinitis mice.(5)The results of Western Blot detection showed that conditional knockout of bone marrow CCR3 gene could down-regulate both CCR3 and P-AKT protein levels in nasal mucosa of allergic rhinitis mice,indicating that PI3K/AKT signaling pathway may mediate the pathogenesis of CCR3 gene in allergic rhinitis.Part two:(1)Behavioral observation showed that intraperitoneal injection of PI3 K specific inhibitor Ly294002,could improve nasal symptoms such as nose scratching and sneezing in mice with allergic rhinitis,and the improvement was more obvious with the increase of concentration.(2)The results of HE staining showed that increasing the concentration of Ly294002 intraperitoneal injection could gradually improve the histopathological structure of nasal mucosa and reduce the damage of nasal mucosa in allergic rhinitis mice.(3)The results of ELISA detection showed that increasing the concentration of Ly294002 intraperitoneal injection could gradually decrease the level of Th2 inflammatory factors(IL-4,IL-5)in the serum of allergic rhinitis mice,while increase the level of Th1 inflammatory factors(IFN-γ),so as to correcte the imbalance of Th1/Th2 inflammation in allergic rhinitis mice.(4)The results of Western Blot detection showed that intraperitoneal injection of PI3 K specific inhibitor Ly294002,could effectively down-regulate the activation level of PI3K/AKT signaling pathway in nasal mucosa,but do not affect the expression level of CCR3 protein in nasal mucosa of allergic rhinitis mice.Conclusion:Conditional knockout of bone marrow CCR3 gene could decrease the activation level of PI3K/AKT signaling pathway by down-regulating the expression of CCR3,improve the histopathological structure of nasal mucosa,and alleviate the symptoms of allergic rhinitis mice by regulating the immune imbalance of Th1/Th2 in mice.Previous experimental studies have found that down-regulating CCR3 gene inhibits the proliferation,migration and degranulation of eosinophils in vitro by inhibiting PI3K/AKT pathway.Combined with previous experimental results,down-regulating CCR3 gene can play a role in the pathogenesis of allergic rhinitis by inhibiting PI3K/AKT signaling pathway,thus alleviating the symptoms of allergic rhinitis.