Study On The Mechanism Of QuFengXuanFei Decoction In Reducing The Formation Of Neutrophil Extracellular Traps In The Treatment Of Post-infectious Cough By Inhibiting NF-κB Signal Pathway

Part Ⅰ:Experimental study on QuFengXuanFei decoction in the treatment of post-infectious cough by reducing the Neutrophil extracellular trapsObjective:To explore the role of neutrophil extracellular traps in the pathogenesis of postinfectious cough and the effect of QuFengXuanFei decoction.Methods:Balb/c female mice aged 8-12 weeks were randomly divided into normal control group(NC group),PIC model group(PIC group),QuFengXuanFei decoction group(QFXF group),NE inhibitor group(NEi group)and DNase group(Dnase Ⅰ group).In the PIC group,cigarette and sawdust were ignited in the exposure tank to make the smoke chamber.The mice was placed in the smoke room and intranasal administration of 100ug/ml LPS.On the 12th,13th,15th and 16th day,capsaicin 0.1mmol/L atomization inhalation was used to stimulate cough,and cough count was recorded during inhalation.Sixteen hours after the last intervention,the bronchi and alveolus were lavaged by PBS to detect the expression of KC(CXCL-1),MIP-2(IL-8)and substance P in BALF.The content of dsDNA in BALF was detected by Quant-iT PicoGreen kit.Bilateral lung tissues were taken to evaluate the model according to the frequency of cough and HE staining.The expression of CXCR1 and CXCR2 mRNA in lung tissue was detected by qPCR,the expression of Cit-H3 and MPO protein in lung tissue was observed by immunofluorescence,and the expression of Cit-H3 protein was detected by WB.To evaluate the formation of NETs in lungs,and explore the relationship between NETs and airway inflammation of PIC.Results:(1)After the establishment of the PIC model,the frequency of cough was significantly increased,the infiltration of inflammatory cells in the lung was increased,but there was no change in the lung parenchyma,and the content of substance P in BALF increased,which conformed to the characteristics of PIC,and proved that the model was successful.(2)Compared with NC group,the frequency of cough of mice in PIC group increased significantly after capsaicin stimulation.Using Dnase Ⅰ to degrade DNA and inhibit elastase activity by NEi as well as taking QuFengXuanFei decoction could significantly reduce the frequency of cough and the content of substance P in mice,indicating that these interventions are effective in the treatment of PIC and can reduce airway neurogenic inflammation in PIC.(3)The content of neutrophil-associated chemokine in PIC group was significantly higher than that in NC group,and the expression of chemokine receptor gene was significantly up-regulated.The chemokine content in QFXF group,NEi group and Dnase Igroup was significantly lower than that in PIC group,and chemokine receptor gene was significantly down-regulated compared with PIC group,indicating that these interventions can reduce neutrophil recruitment and reduce neutrophil-related airway inflammation.(4)The content of dsDNA in BALF of PIC group was significantly higher than that of NC group,the expression of Cit-H3 and MPO protein in lung tissue was increased,and CitH3+MPO+NETs complex appeared.The use of Dnase Ⅰ and NE inhibitors and QuFengXuanFei decoction could significantly reduce the content of dsDNA and the expression of Cit-H3 and MPO protein in lung tissue and the formation of Cit-H3+MPO+NETs complex.Conclusion:LPS combined with smoke stimulation can induce airway inflammation,increase neutrophil chemotaxis and substance P,increase the content of NETs in the lung,which is finally characterized by an increase in the frequency of cough.After the use of Dnase land NE inhibitor,neutrophil chemotaxis and NETs content decreased significantly,substance P content decreased,and cough frequency decreased significantly,indicating that the content of NETs is closely related to PIC.After intervention with QuFengXuanFei decoction,NETs formation,neutrophil chemotaxis,airway neurogenic inflammation and cough frequency in PIC mice were significantly decreased,which was equivalent to that in Dnase Ⅰ and NEi group,suggesting that QuFengXuanFei decoction may improve airway inflammation in PIC mice by reducing NETs content.Part Ⅱ:Study on the mechanism of QuFengXuanFei decoction in reducing the formation of neutrophil extracellular traps of by inhibiting NF-κB signal pathway.Objective:To screen the possible targets of QuFengXuanFei decoction in reducing NETs formation,and to verify the mechanism of QuFengXuanFei decoction in reducing NETs formation by inhibiting NF-κB signal pathway in vitro.Methods:The mRNA expression of PI3K-Akt,mTOR,Toll-like receptor,MAPK and NFκB signal pathway in lung tissue of mice in NC group,PIC group and QuFengXuanFei group were detected by qPCR.Possible targets of QuFengXuanFei decoction were screened,and the contents of IL-1 β,IL-6 and TNF-α in BALF of mice in each group were detected by Elisa.The peripheral blood of Balb/c mice was collected and the neutrophils were isolated and purified by gradient centrifugation.Neutrophils were co-cultured with 100ng/ml LPS,TC-1 supernatant stimulated by 100ng/ml LPS(LPS+TC-1)and 100nM PMA for 4 hours to induce NETs formation respectively.The formation of NETs was observed by immunofluorescence,the expression of Cit-H3 protein was detected by WB,and the content of NETs was quantified.NF-κB inhibitor PDTC(500uM)and different doses of freeze-dried powder of QuFengXuanFei decoction(1mg/ml,2mg/ml,3 mg/ml)were co-cultured with the cells stimulated by LPS+TC-1 supernatant and PMA,and their effects on the formation of NETs were observed.The expression of phospho-p65 protein and IκB protein in neutrophils with different intervention were detected by WB.The activation of NF-κB signal pathway was observed to explore the relationship between NF-κB signal pathway and NETs formation in neutrophils so as to explore the effect targets of QuFengXuanFei decoction.Results:(1)There was no significant difference in the expression of PI3Kr1,Aktl and mTOR mRNA in lungs of each group.The expression of TLR2,TLR4 and p65 NF-κB mRNA was upregulated in the PIC group,while the expression of p65NF-κB mRNA was down-regulated in the QuFengXuanFei decoction group,and had no significant regulatory effect on the expression of mRNA in other signal pathways.(2)The contents of cytokines IL-1β,IL-6 and TNF-α which are the downstream of NF-κB signal pathway increased significantly in PIC group and decreased significantly after the intervention of QuFengXuanFei decoction,indicating that NF-κB signal pathway may be the target of QuFengXuanFei decoction.(3)After direct stimulation of neutrophils with LPS in vitro,few neutrophils were in NETosis.The supernatant of mouse TC-1 cells with LPS stimulated induced an appreciable number of neutrophils to form NETs.Compared with the control group,the expression of phospho-p65 protein increased and the expression of IκB protein decreased,indicating that the NF-κB pathway was activated.PDTC and QuFengXuanFei decoction can inhibit the activation of NF-κB pathway and reduce the formation of NETs.The effect of QuFengXuanFei decoction is related to the dose.(4)PMA can directly stimulate neutrophils to produce NETs.PDTC and QuFengXuanFei decoction have no effect on NETs formation induced by PMA,indicating that QuFengXuanFei decoction cannot directly eliminate NETs,nor reduce NETs formation through MAPK-PKC pathway.Conclusion:the activation of NF-κB signal pathway in neutrophils mediates the formation of NETs.QuFengXuanFei decoction reduces NETs formation by inhibiting NF-κB signal pathway in neutrophils.QuFengXuanFei decoction has no effect on scavenging NETs in vitro.