TLR4 Deficiency Decreased Airway Inflammatory And Remodeling In OVA Induced Asthmatic Mice Model

Objective:To explore the effect of TLR4 gene knockout on airway inflammation and remodeling in OVA induced asthma mice.Methods:30 Female C57BL/10 wide type and TLR4^-/-mice?6-8 weeks?were divided into 5 groups(6 mice in each group,normal control group,TLR4^-/-group,OVA group,(OVA+TLR4^-/-)group,and?OVA+DXM?group).The last three groups were intraperitoneally injected with aluminum hydroxide adjuvant on days 0 and 7.After 7 days,mice were atomized with 1%OVA every other day for 14 minutes from day 14 to day 40.The?OVA+DXM?group was injected intraperitoneally with dexamethasone sodium phosphate injection?5mg/ml,diluted with 0.9%physiological saline?.The mice in the normal control group and the TLR4^-/-group received the same sensitization schedule and were administered at the same dose,replaced with 0.9%sterile physiological saline.Bronchoalveolar Lavage Fluid?BALF?was collected and centrifuged,after the pellet resuspended,cell smear was prepareed,and Swiss-Gemsa staining was used for inflammatory cells classification and counting.ELISA was used to detect IL-4,IL-5,IL-13 and IFN-?levels in serum.HE,PAS and Masson staining were performed to observe the pathological changes of lung tissue.Immunohistochemical and immunofluorescence staining were used to detect the expression of GATA-3,T-bet and?-SMA proteins in mouse lung tissue.Western-Blot was used to detect GATA-3 and T-bet protein expression levels in mouse lung tissues.RT-PCR was used to detect GATA-3,T-bet,Foxp3 and ROR?t m RNA expression levels in lung tissues.Results:BALF results showed that the total number of white blood cells,neutrophils,lymphocytes,and eosinophils in the OVA group increased significantly compared with the normal control group,and the differences between the two groups were statistically significant?P<0.05?.The number of white blood cells,neutrophils,lymphocytes,and eosinophils in BALF in(OVA+TLR4^-/-)group was significantly reduced compared with OVA asthma group,and the difference between the two groups was statistically significant?P<0.05?.HE and PAS results showed that TLR4^-/-can effectively alleviate airway inflammation,reduce goblet cell proliferation and mucus secretion.Compared with the OVA group,the expression of GATA-3 was significantly reduced in the immunohistochemical detection(OVA+TLR4^-/-)group,and the expression of T-bet was significantly increased.ELISA results showed that IL-4,IL-5,and IL-13 were significantly increased in the OVA group compared with the normal control group?P<0.05?;(OVA+TLR4^-/-)compared with the OVA group,IL-4,IL-5.IL-13 was significantly reduced?P<0.05?;IFN-?was significantly increased?P<0.05?.Compared with the normal control group,the m RNA and protein expression levels of GATA-3 and ROR?T in the OVA group were significantly increased?P<0.05?;(OVA+TLR4^-/-)Compared with the OVA group,the m RNA and protein levels of GATA-3 and ROR?T The protein expression level was significantly reduced?P<0.05?.Compared with the normal control group,the m RNA and protein expression levels of T-bet and Foxp3 in the OVA group were significantly reduced?P<0.05?;(OVA+TLR4^-/-)Compared with the OVA group,the m RNA and protein levels of T-bet and Foxp3 were significantly reduced.The expression level was significantly increased?P<0.05?.Masson staining and immunofluorescence results showed that TLR4^-/-can effectively reduce collagen deposition and airway wall thickness.Conclusion:TLR4^-/-can reduce airway inflammation and airway remodeling in asthmatic model mice,and its mechanism may be related to the balance of Th1/Th2 and ROR?t/Foxp3 cytokines.