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Effect of Yiqi Guben capsule on airway inflammation and airway remodeling in allergic asthma modelObjective: To explore the mechanism of Yiqi Guben capsule in reducing airway inflammation and airway remodeling of allergic asthma in vivo.Methods:1.OVA combined with AL(OH)3 sensitization and OVA challenge were used to prepare a mouse model of allergic asthma.2.Giemsa staining was used to detect the classification and counting of inflammatory cells in BALF of mice in each group.3.The lung tissues of mice in each group were stained with HE,PAS and Masson to determine the inflammation and infiltration of lung tissues,airway goblet cell metaplasia and airway subepithelial fibrosis.4.ELISA was used to detect the levels of IgE,IL-4,IL-5,IL-13,Eotaxin,GM-CSF,ECP,TLC4,LTD4,TGF-?1,PDGF and MUC5 AC in BALF of mice in each group.5.Immunohistochemical staining was used to observe the expression levels of TLR4 and?-SMA in the lung tissues of mice in each group.6.RT-PCR was used to detect the mRNA expression of IL-4,IL-5,IL-13,TGF-?1,PDGF,TLR4,MyD88,TRAF6,I?B?,p65 in the lung tissues of mice in each group.7.Western Blot was used to detect the protein expression of E-cadherin,Vimentin,Fibronectin,?-SMA,I?B?,p65,p-p65 in the lung tissue of mice in each group.Results:1.After OVA combined with AL(OH)3 sensitization and OVA stimulation,allergic asthma model showed typical symptoms during asthma attacks such as accelerated breathing,nodded breathing,standing instability,and restlessness.The levels of EOS and IgE in BALF increased,and lung tissue showed significantly inflammation,which indicating that the allergic asthma model was successfully constructed.With the interventionof Yiqi Guben capsule and Dexamethasone(positive control drug),the typical symptoms of asthma attacks such as accelerated breathing,nodded breathing,standing instability,and restlessness of allergic asthma models were significantly reduced,and the level of EOS and IgE in BALF was significantly reduced.Lung tissue inflammation infiltration was significantly reduced.2.The level of IL-4,IL-5,IL-13 in BALF and the mRNA expression of IL-4,IL-5,IL-13 in lung tissue were significantly increased of allergic asthma model.After intervention by Yiqi Guben capsule and dexamethasone(positive control drug),the level of IL-4,IL-5,IL-13 in BALF and the mRNA expression of IL-4,IL-5,IL-13 in lung tissue were significantly reduced of allergic asthma model.3.The level of Eotaxin,GM-CSF,ECP,TLC4,LTD4 in BALF of allergic asthma model was significantly increased.After intervention by Yiqi Guben capsule and dexamethasone(positive control drug),the level of Eotaxin,GM-CSF,ECP,LTC4,LTD4 in BALF was significantly reduced of allergic asthma model.4.Lung tissue showed goblet cell metaplasia significantly of allergic asthma model,and the level of MUC5 AC in BALF was significantly increased.After intervention by Yiqi Guben capsule and dexamethasone(positive control drug),the metaplasia of airway goblet cells was significantly reduced,and the level of MUC5 AC in BALF was significantly reduced of allergic asthma model.5.Lung tissue showed subepithelial fibrosis significantly of allergic asthma model,the level of TGF-?1 in BALF and the mRNA expression of TGF-?1 in lung tissue were significantly increased.The expression protein of E-cadherin in lung tissue was significantly reduced,and the protein expression of Vimentin and Fibronectin were significantly increased of allergic asthma model.After intervention by Yiqi Guben capsule and dexamethasone(positive control drug),the subepithelial fibrosis in lung tissue was significantly reduced,and the level of TGF-?1 in BALF and mRNA expression of TGF-?1in lung tissue were significantly reduced of allergic asthma model.The protein expression of E-cadherin in lung tissue was significantly increased,and the protein expression ofVimentin and Fibronectin was significantly reduced of allergic asthma model.6.The level of PDGF in BALF and the mRNA expression of PDGF in lung tissue were significantly increased of allergic asthma model,and the protein expression of ?-SMA was increased.After the intervention by Yiqi Guben capsule and dexamethasone(positive control drug),the level of PDGF in BALF and the mRNA expression of PDGF in lung tissue were reduced,and the protein expression of ?-SMA in the lung tissue was reduced significantly of allergic asthma model.7.The mRNA expression of TLR4,TRAF6,and p65 in the lung tissue of allergic asthma models was increased significantly,the mRNA and protein expression of I?B? were reduced significantly,and the protein expression of p65 and p-p65 was increased significantly.After intervention by Yiqi Guben capsule and Dexamethasone(positive control drug),the mRNA expression of TLR4,TRAF6 and p65 in the lung tissue of allergic asthma model was significantly reduced,the mRNA and protein expression of I?B? were significantly increased,and the protein expression of p65 and p-p65 were significantly reduced.Conclusion:1.Allergic asthma model was successfully constructed by using OVA combined with AL(OH)3 sensitization and OVA challenge.2.Yiqi Guben capsule can reduce airway inflammation of allergic asthma models by regulating the level of IL-4,IL-5,IL-13,Eotaxin,GM-CSF,ECP,TLC4,LTD4 and the mRNA expression of IL-4,IL-5,IL-13.3.Yiqi Guben capsule can reduce airway remodeling of allergic asthma model by regulating the level and mRNA expression of TGF-?1,PDGF,MUC5 AC and the protein expression of E-cadherin,Vimentin,Fibronectin,?-SMA.4.The mechanism of Yiqi Guben capsule in reducing airway inflammation and airway remodeling of allergic asthma models was related to the regulation of TLR4/NF-?B signaling pathway.Part ? Effect of Yiqi Guben capsule on PDGF-BB-induced Airway Smooth Muscle Proliferation and MigrationObjective: To explore the effect of Yiqi Guben capsule on PDGF-BB-induced airway smooth muscle proliferation and migration in vitro.Method:1.PDGF-BB-induced ASMC was used to construct airway smooth muscle proliferation and migration model.2.The MTT method was used to detect the effect of Yiqi Guben capsule on ASMC drug toxicity and proliferation of ASMC induced by PDGF-BB.3.The flow cytometry was used to detect the effect of Yiqi Guben capsule on cell cycle of ASMC induced by PDGF-BB.4.Transwell test was used to detect the effect of Yiqi Guben capsule on migration of ASMC induced by PDGF-BB.5.Cell scratch test was used to detect the effect of Yiqi Guben capsule on migration of ASMC induced by PDGF-BB.6.ELSIA was used to detect the effect of Yiqi Guben capsule on the level of IL-6,IL-8,IL-1?,TNF-? and TGF-?1 secreted by ASMC induced by PDGF-BB.7.RT-PCR was used to detect the effect of Yiqi Guben capsule on the m RNA expression level of I?B? and p65 of ASMC induced by PDGF-BB.8.Western blot was used to detect the effect of Yiqi Guben capsule on the protein expression level of I?B?,p65,p-p65 of ASMC induced by PDGF-BB.Result:1.Yiqi Guben capsule at the concentration of 0.05mg/m L,0.1mg/m L,0.2mg m L,and0.4mg/m L has no significant effect on the survival of ASMC and can be used in subsequent experimental studies.2.The ASMC induced by PDGF-BB after 24 h,48h and 72 h,its proliferation ability was significantly increased.After the intervention by Yiqi Guben capsule,the proliferation ability of ASMC induced by PDGF-BB was reduced.3.The G0-G1 phase of the cell cycle was shortened and the G2-M phase was prolonged of ASMC induced by PDGF-BB.After the intervention by Yiqi Guben capsule,the G0-G1 phase of the cell cycle was prolonged and the G2-M phase was shortened of ASMC induced by PDGF-BB.4.The migration ability was significantly increased of ASMC induced by PDGF-BB.After the intervention by Yiqi Guben capsule,the migration ability of ASMC induced by PDGF-BB was significantly reduced.5.The ASMC induced by PDGF-BB after 12 h and 24 h,its scratch repair ability was significantly increased.After the intervention by Yiqi Guben capsule,the scratch repair ability of ASMC induced by PDGF-BB was significantly reduced.6.The count of IL-6,IL-8,IL-1?,TNF-? and TGF-?1 secreted by ASMC induced by PDGF-BB was significantly increased.After the intervention by Yiqi Guben capsule,the count of IL-6,IL-8,IL-1?,TNF-? and TGF-?1 secreted by ASMC induced by PDGF-BB was significantly reduced.7.The m RNA and protein expression of I?B? were significantly reduced of ASMC induced by PDGF-BB,the m RNA expression of p65 and p50 was significantly increased,and the protein expression of p-p65 was significantly increased.After the intervention by Yiqi Guben capsule,The m RNA and protein expression of I?B? were significantly increased of ASMC induced by PDGF-BB,the m RNA expression of p65 and p50 was significantly decreased,and the protein expression of p-p65 was significantly decreased.Conclusion:1.After ASMC induced by PDGF-BB,its proliferation and migration ability was significantly increased,which suggested that the ASMC proliferation and migration model was successfully constructed.2.Yiqi Guben capsule could inhibit the proliferation and migration of ASMC induced by PDGF-BB.3.Yiqi Guben capsule could reduce the count of IL-6,IL-8,IL-1?,TNF-?,TGF-?1secreted during the proliferation and migration of ASMC induced by PDGF-BB.4.The mechanism of Yiqi Guben capsule inhibited the proliferation and migration of ASMC induced by PDGA-BB was related to the regulation of NF-?B signaling pathway. |
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