The Funtion Of TLR-nf-κB Passway In Rats Of Allergic Rhinitis (AR) And The Study Of Tripterygium Glycoside's Interference Mechanism

Objective:1. To establish the model of allergic rhinitis (AR) in rats.2. To assay the expression of TLR2and TLR4subtype in nasal mucosa for ordinaryand model rats.3. To discuss probable mechanism of TLR-NF-κBp50pathway cascaded in AR.4. To study the effect of TLR4agonist lipopolysaccharide (LPS) induced highexpression NF-κBp50with different concentration in AR.5. To grow primary nasal mucosa epithelial cells for detection of high expression ofTLR4, NF-κBp50.6. To study the effect of TLR4agonist lipopolysaccharide (LPS) induced highexpression NF-κBp50in primary culture nasal mucosa epithelial cells.7. To discuss probable mechanism of Tripterygium glycosides-interference in AR.Methods:1. Model of allergic rhinitis (AR) in rats was established by intraperitoneal injectionand nasal topic delivery of ovalbumin (OVA) in concentration of0.3mg/1mL/rat.By HE staining method, the change in histo-morphology of nasal musosa wasobserved and the number of neutrophil and eosinophile granulocyte in nasalcavity and mucosa was counted. The tissue IgE level was determined by ELISAand praxiological change on AR rat mode was assessed.2. After the model was interfered by nasal delivery of LPS and PNG, the expressionof TLR2and TLR4in nasal mucosa was detected by immunohistochemistry andReal-Time PCR.3. After the model was interfered by nasal delivery of LPS, the expression ofcytokines IL-4, IFN-γ, TLR and NF-κB p50in nasal mucosa was detected by immunohistochemistry and Real-Time PCR.4. After the model was interfered by nasal delivery of LPS in different concentration,the expression of cytokines IL-4, IFN-γ,TLR and NF-κB p50in nasal mucosawas detected by immunohistochemistry, Real-Time PCR and/or Western-Blot.5. Nasal mucosa epithelium cells of rats were cultivated by separation andpurification using selective plating technique and conditional medium.6. Based on cultured nasal mucosa epithelium cells of rats and the interference ofsaid cells by OVA and LPS, the expression of TLR and NF-κB p50in nasalmucosa was detected by immunohistochemistry and Real-Time PCR.7. Based on Tripterygium glycosides-interference of cultured nasal mucosaepithelium cells of rats, the expression of TLR and NF-κB p50, and equilibriumbetween cytokines Th1and Th2was detected.All data were represented as means±SEM. Statistical analysis was performed usingone-way ANOVA and Repeated measures with SPSS13.0software. A probabilityvalue of less than0.05was considered significant.Results:1. After intraperitoneal injection and nasal topic delivery of ovalbumin (OVA) inconcentration of0.3mg/1mL/rat, based on Wright's staining of various modelgroups, a dramatic thickening in nasal mucosa of rats, an obvious hemangiectasisand an infiltration of EOS in large amount was observed. A significant increase ofcytokines TH2, IL4and IgE levels in tissue was detected by ELISA. Thepraxiological score of group AR was much higher than that of ordinary group.2. After the model was interfered by nasal delivery of OVA and LPS, the expressionof TLR2and NF-κB p50in nasal mucosa was detected in each group byinmmunohisocheminstry and Real-time PCR, furthermore the relationshipbetween TLR2and NF-κB was presented.3. After the model was interfered by nasal delivery of LPS and PGN, the expressionof TLR2and TLR4in nasal mucosa was relatively up-regulated byinmmunohisocheminstry and Real-time PCR. Increasing amount of Neurphile andintensity of inflammination in nasal mucosa were found.. 4. After the model was interfered by nasal delivery of LPS, the expression of IL-4, IFN-γ, TLR,IgE and NF-κB p50were correlated with the concentration of the LPS. In the group ofPDTC (an inhibitor of NF-κB), decreased expression level of NF-κB p50, decreasedexpression of Th cytocines and decreased expression level of IgE was found.5. After being cultivated by separation and purification using selective platingtechnique and conditional medium, the nasal mucosa epithelium cells identified byIHC Staining is more than90%.6. Based on cultured nasal mucosa epithelium cells of rats and the interference ofsaid cells by OVA and different concentration of LPS, the expression of TLR andNF-κB p50in nasal mucosa was presented by the method ofimmunohistochemistry and Real-Time PCR. In the group of PDTC, In the groupof PDTC, relatively decreased expression level of NF-κB p50, increased ratio ofTh1/Th2and relatively decreased expression level of IgE was found.7. Based on Tripterygium glycosides-interference of cultured nasal mucosaepithelium cells of rats, the expression of TLR,NF-κB p50, IL-4, IFN-γ, and IgEwere down-regulated. The ratio between cytokines Th1and Th2was higher thanthe modle group.Conclusion:1. Model of AR in rats could be successfully established by intraperitoneal injectionand nasal topic delivery induction with OVA.2. TLR in nasal mucosa of ordinary rats was existed in two subtypes: TLR2andTLR4. The TLR2was widely distributed in normal rat nasal tissue. The TLR4wasdramatically changed in AR model rat, which may cast a principal role ininitiating AR.3. TLR may initiating the AR disease. Allergen may initiating the progression of ARvia TLR-NF-κBp cascade.4. The TLR4agonist LPS could give rise to the up-regulation of NF-κBp50with aconcentration correlated effect. High concentration of LPS could increased theexpression level of NF-κBp50, alter the equilibrium between cytokines Th1andTh2. 5. The nasal mucosa epithelium could be obtain by using selective plating technique,and purified by adding conditional medium.6. In successfully cultivated nasal epithelium, the high concentration TLR4agonistLPS may up-regulate the expression level of NF-κBp50, then interfering theequilibrium cytokines Th1and Th2,.7. Tripterygium glycosides could alter the progression of AR by regulating the TLRexpression, NF-κBp50and the ratio of Th1/Th2.