The Mechanism Of MMP-9 Mediates Early Airway Remodeling In TDI Induced Asthma By Influencing RAGE

BackgroundAsthma is one of the most common chronic respiratory diseases.At present,it is believed that inflammation and airway remodeling develop in parallel in asthma.In addition to resisting airway inflammation,the intervention of airway remodeling in the development of asthma has opened a new way for treating severe refractory asthma.It may alleviate the airway remodeling changes in asthmatic patients by inhibiting the pathological proliferation of airway epithelial cells,subepithelial collagen deposition and epithelial-mesenchymal transformation(EMT),which can prevent the occurrence of severe asthma or refractory asthma.Matrix metalloproteinase(MMPs)and tissue inhibitor of metalloproteinase(TIMPs)participate in the degradation and deposition of extracellular matrix,and the balance between them is crucial in the process of airway remodeling in asthma.The level of receptor for advanced glycation end products(RAGE)is related to the severity of asthma.The purpose of this study was to explore the role of MMPs and RAGE in early airway remodeling in TDI induced asthma.MethodsBALB/C male mice aged 6-8 weeks were randomly divided into AOO group,TDI group,TDI+SB-3CT group and TDI+FPS-ZM1 group.On the 1 st and 8th day,the mice were sensitized by applying 0.3%TDI sensitizer on the back of their ears.On the 15th,18th and 21st day,the mice were sensitized by using 3%TDI provocation solution atomization after intraperitoneal injection of the corresponding drugs 2 hours earlier:1)AOO group:5%DMSO;2)TDI group:5%DMSO;3)TDI+SB-3CT group:25mg/kg SB-3CT(MMP-9 inhibitor),DMSO concentration was adjusted to 5%;4)TDI+FPS-ZM1 group:1.5mg/kg FPS-ZM1(RAGE inhibitor),DMSO concentrations was adjusted to 5%.On the 23rd day,serum,alveolar lavage fluid and lung tissue were collected for ELISA,HE staining,Masson staining,immunohistochemistry and other experiments.When the growth density of HBE135 cells reached 80-90%in 60mm cell culture dishes,5mL TDI-HSA and KM mixture with a concentration of 40μg/mL was added after washing with PBS twice.Culture HBE135 cells for 0,12,24,48,72 and 96 hours respectively.The culture dishes were washed with PBS for 2-3 times at corresponding time,marking and storing them in the refrigerator at-80℃.Cell proteins were extracted.Western blot(WB)was used to detect the expression of MMP-9,TIMP-1,E-cadherin and N-cadherin in bronchial epithelial cells of different groups.Results1.Inhibition of MMP-9 or RAGE can reduce the airway inflammation caused by TDI.Compared with the TDI group,the levels of IL-4 and IL-13 in the alveolar lavage fluid in the TDI+SB-3CT group and TDI+FPS-ZM1 group were significantly reduced.2.Inhibition of MMP-9 or RAGE can improve airway epithelial proliferation induced by TDI.HE staining showed significant proliferation of airway epithelial cells in the TDI group,while airway epithelial cells in the AOO group,TDI+SB-3CT group,and TDI+FPS-ZM1 group were uniformly distributed in a single layer.3.MMP-9 or RAGE inhibitor can inhibit the subepithelial deposition of collagen in the airway caused by TDI.The Masson staining showed that compared with the AOO group,the TDI group had significant subepithelial collagen deposition in the airway,which were alleviated in the TDI+SB-3CT group and TDI+FPS-ZM1 group.4.Inhibition of MMP-9 can reduce the expression of cytoplasmic RAGE of airway epithelial cells.RAGE immunohistochemistry showed RAGE was expressed in the cell membrane of airway epithelial cells in the AOO group,whose expression was increased in the TDI group.In the TDI+SB-3CT group,RAGE was mainly uniformly distributed on the cell membrane,and the expression in the cytoplasm was not as significant as in the TDI group.5.TDI causes EMT in bronchial epithelial cells,as well as degradation and deposition of extracellular matrix.As TDI-HSA stimulation of HBE135,the expression of E-cadherin gradually decreased and the expression of N-cadherin increased.In addition,the expression levels of MMP-9 and TIMP-1 first increased and then decreased,and the ratio of the two increased at 24 hours and showed a downward trend after 24 hours.ConclusionsMMP-9 is involved in the airway inflammation and the airway remodeling of TDI induced asthma,such as airway epithelial proliferation,and subepithelial collagen deposition in airway.Besides,MMP-9 may mediate airway remodeling in asthma by influencing RAGE.