Experimental Study Of Chaoyifang Lurong Dabu Decoction In Improving Cough Variant Asthma In Guinea Pigs Through TLR4/WNT-5A Signaling Pathway

Objetctive:By establishing a cough variant asthma model,the effect of Lurong Dabu Decoction on airway inflammation in cough variant asthma guinea pigs was observed,and whether its mechanism was related to TLR4/WNT-5A signaling pathway.Methods:Fifty healthy male guinea pigs were randomly divided into a normal group,a cough variant asthma model group,a low-dose group of Chaoyifang Lurong Dabutang,a high-dose group of Chaoyifang Lurong Dabutang,and a western medicine dexamethasone group,10 in each group.Except for the normal group,the other 4 groups were smoked 30 minutes a day for 4 weeks;On the 15th and 22nd days,intramuscular injection of 0.5 ml of 4%OVA solution and intraperitoneal injection of0.2 ml of 2%Al（OH）₃were carried out for a total of 2 times.On the 29th day,the model group,low-dose group,high-dose group and dexamethasone group were stimulated with 1%OVA solution by ultrasonic atomization,once every other day for4 consecutive weeks.The normal group was stimulated by ultrasonic atomization with the same dose of sodium chloride solution.On the 29th day,the normal group and the model group were given 5g/kg sodium chloride solution by gavage one hour before the challenge,the high-dose group 20g/kg,the low-dose group 5g/kg each by gavage;the dexamethasone group was given 0.5mg/kg administered by gavage,once a day for4 consecutive weeks.The number of coughs was measured 24 hours after the last administration,and the guinea pigs were killed the next day to check the relevant indicators.Observation indicators:（1）Capsaicin induced cough to measure the number of coughs in guinea pigs;（2）The levels of IFN-γ,IL-4,IL-5 and IL-13inflammatory cytokines in guinea pig BALF were detected by ELISA;（3）HE,PAS and MASSON staining were used to study the pathological changes of guinea pig lung tissue.（4）The protein expression of TLR4 and WNT-5A in guinea pig lung tissue was detected by immunohistostaining,immunofluorescence staining and western blotting.Results:（1）Compared with normal group,the number of cough induced by capsaicin increased significantly in model group.Compared with the model group,low-dose and high-dose groups and dexamethasone group were significantly reduced（P<0.05）;（2）Inflammatory cells:Compared with the normal group,the total number of cells and the number of various inflammatory cells in the model group were significantly increased;Compared with the model group,the total number of cells and the number of various inflammatory cells in the low-dose and high-dose groups and the dexamethasone group were significantly reduced（P<0.05）;（3）ELISA:Compared with normal group,IFN-γlevels in model group were significantly decreased,while IL-4,IL-5 and IL-13 levels were significantly increased;Compared with the model group,the levels of IFN-γin low-dose and high-dose groups and dexamethasone group were significantly increased,while the levels of IL-4,IL-5 and IL-13 were significantly decreased（P<0.05）;（4）HE staining results:Compared with the normal group,extensive inflammatory cell infiltration,smooth muscle thickening,and bronchial mucosal congestion appeared in the lung tissue and around the bronchi of the model group;Compared with the model group,the infiltration of inflammatory cells in low-dose and high-dose groups and dexamethasone group were alleviated to varying degrees,and the airway injury was alleviated（P<0.05）;（5）PAS staining results:Compared with the normal group,the goblet cells of the guinea pig bronchus in the model group were significantly proliferated,and the PAS staining process was positive;Compared with model group,the hyperplasia of goblet cells could be significantly inhibited in low-dose and high-dose groups and dexamethasone group（P<0.05）;（6）MASSON:Compared with the normal group,the subepithelial collagen deposition of the airway in the model group was significantly increased;Compared with the model group,the peritracheal collagen deposition in low-dose and high-dose groups and dexamethasone group were significantly reduced（P<0.05）;（7）Immunohistochemical results:Compared with the normal group,TLR4 and WNT-5A protein expressions in the lung tissues of guinea pig model group were strongly positive,and TLR4 and WNT-5A were significantly aggregated around the airway after OVA stimulation;Compared with the model group,the expression levels of TLR4 and WNT-5A proteins in the lung tissue of the low-dose,high-dose and dexamethasone groups were all attenuated（P<0.05）;（8）Immunofluorescence results:Compared with the normal group,the expression of TLR4 and WNT-5A proteins in the guinea pig lung tissue of the model group was significantly enhanced;Compared with model group,the expression of TLR4 and WNT-5A protein in lung tissues of the low-dose and high-dose groups and dexamethasone group was significantly decreased（P<0.05）;（9）Western blotting results:Compared with the normal group,the expression of TLR4 and WNT-5A proteins in the guinea pig lung tissue of the model group was significantly increased;Compared with the model group,the protein contents of TLR4 and WNT-5A in lung tissues of low-dose and high-dose groups and dexamethasone group were significantly reduced（P<0.05）.Conclusions:（1）Lurong Dabu Decoction can relieve the cough symptoms of cough variant asthma,improve the pathological changes of the lungs,and play a therapeutic role;（2）Lurong Dabu Decoction can relieve inflammatory cell infiltration and regulate the release of inflammatory cytokines;（3）Lurong Dabu Decoction may be involved in airway inflammation in cough variant asthma through TLR4/WNT-5A signaling pathway.