The Effect And Mechanism Of Lidocaine On Allergic Airway Inflammation

Asthma is one of the most common airway diseases characterized by airway inflammation.The prevalence of asthma is increasing year by year,and the number of global asthma patients has exceeded 300 million.Emerging evidence shows that TLR2 signaling pathway regulates the release of inflammatory cytokines and is involved in the pathogenesis of asthma.It has been reported that lidocaine can significantly improve airway inflammation in asthmatic patients and reduce the use of glucocorticoid.In the present study,we observe the effect of lidocaine on the TLR2 expression induced by S.aureus in RAW264.7 cells at the cellular level.Then we observe the effect of lidocaine on allergic airway inflammation and TLR2 expression in lung tissues of wild-type(WT)allergic mice.At the same time,we used TLR2-agonist-pretreated WT mice and TLR2 knockout mice to further explore the mechanism of lidocaine inhibiting allergic airway inflammation.Objective 1.Investigate the effect of lidocaine on the TLR2 signaling pathway of macrophages infected with S.aureus.2.Study the effects of inhaled lidocaine on lung inflammation and TLR2 signaling in allergic mice.3.Investigate the effect of lidocaine on the activation of inflammasome in allergic mice.Methods 1.Detect the effect of lidocaine on the activation of TLR2 signaling pathway in S. aureus-stimulated macrophages Mouse mononuclear macrophages RAW264.7 were seeded in 12-well cell culture plates at a density of 2.5 × 10 5 / well.The cells were pretreated with different concentrations of lidocaine for 24 hrs.And then staphylococcus aureus stimulated for 1 h.The expression of proteins of TLR2 signaling was detected by Western blotting.2.Evaluate the effect of lidocaine on allergic airway inflammation in mice and TLR2 signaling activation C57 BL / 6 female mice were randomly divided into control group(Con group),asthma group(OVA group)and lidocaine treatment group(OVA + 1% Lido group).Mouse lung tissue and bronchoalveolar lavage fluid(BALF)were collected.Histological analysis of mice airways were determined by HE staining for airway inflammation manifestations and by PAS staining for goblet cell hyperplasia and mucus production.The number of inflammatory cells in BALF was detected by Wright's staining,and the levels of IL-13?IL-6?TNF-? and IFN-? in BALF were investigated by ELISA.The expression of proteins of TLR2 signaling was detected by Western blotting.3.Evaluate the effect of lidocaine on allergic airway inflammation in mice pretreated with Pam3CSK4 and TLR2 signaling activation The C57 BL / 6 female mice were pretreated with Pam3CSK4,a TLR2 agonist,2h before sensitization and challenge.The bronchoalveolar lavage fluid and lung tissue samples were collected.Histological analysis of mice airways were determined by HE staining for airway inflammation manifestations and by PAS staining for goblet cell hyperplasia and mucus production.The number of inflammatory cells in BALF was detected by Wright's staining,and the levels of IL-13?IL-6?TNF-? and IFN-? in BALF were investigated by ELISA.The expression of proteins of TLR2 signaling was detected by Western blotting.4.Evaluate the effect of lidocaine on allergic airway inflammation in TLR2-/-mice TLR2-/-mice were sensitized and challenged with OVA.Mouse lung tissue and bronchoalveolar lavage fluid(BALF)were collected.Histological analysis of mice airways were determined by HE staining for airway inflammation manifestations and by PAS staining for goblet cell hyperplasia and mucus production.The number of inflammatory cells in BALF was detected by Wright's staining,and the levels of IL-13?IL-6?TNF-? and IFN-? in BALF were investigated by ELISA.The expression of proteins of TLR2 signaling was detected by Western blotting.5.Evaluate the effect of lidocaine on the activation of inflammasome in allergic mice C57 BL / 6 female mice were sensitized and challenged with OVA.Mouse peripheral blood,lung tissue and bronchoalveolar lavage fluid(BALF)were collected.The hemocytometer was used to detect the number of inflammatory cells in blood.The levels of IL-4,IL-18 in BALF and OVA s Ig E in serumwere investigated by ELISA.The expression of proteins of NLRP3 signaling was detected by Western blotting.Results 1.Lidocaine inhibits S.aureus-stimulated activation of TLR2 signaling in macrophages Increased expression of TLR2?NF-?Bp65 and NLRP3 was found in RAW264.7 cells upon S.aureus infection.Lidocaine dose-dependently inhibited the expression of TLR2,NF-?Bp65 and NLRP3.This inhibition was most pronounced at a lidocaine concentration of 200 ?g / ml.2.Inhaled lidocaine significantly inhibits OVA-induced airway inflammation and lung TLR2 expression Compared with the control group,after OVA stimulation,airway inflammatory manifestations including peribronchial inflammatory infiltration,wall thickening,epithelial goblet cell hyperplasia and mucus production,the number of inflammatory cells and the level of IL-13?IL-6?TNF-? and IFN-? were all markedly increased.Compared with the OVA group,inhaled lidocaine significantly reduced bronchial inflammatory cell infiltration,the goblet cells and the concentration of inflammatory cells and inflammatory cytokines in BALF.Compared with the control group,the expression of TLR2,NF-?Bp65 and NLRP3 in the lung tissue of OVA-stimulated mice were significantly increased,while the inhaled lidocaine significantly reduced the expression of TLR2,NF-?Bp65 and NLRP3 expression.3.Lidocaine significantly inhibits airway inflammation in WT allergic mice pretreated with Pam3CSK4 Pam3CSK4 pretreatment significantly aggravated airway inflammation in WT allergic mice,mainly in bronchial inflammatory cell infiltration,the goblet cells and the concentration of inflammatory cells and inflammatory cytokines in BALF.Lidocaine completely inhibits airway inflammation in Pam3CSK4-pretreated WT allergic mice.Pam3CSK4 pretreatment significantly increased the expression of TLR2,NF-?Bp65 and NLRP3 in the lung tissue of WT allergic mice.Inhalation of lidocaine completely inhibited TLR2,NF-?Bp65 and NLRP3 expression in the lung tissue of Pam3CSK4-pretreated WT allergic mice.4.Lidocaine does not further ameliorate lung inflammation in TLR2-/-allergic mice After OVA challenge,in comparison with WT mice,peribronchial inflammatory infiltration,epithelial goblet cell hyperplasia and mucus production,the number of inflammatory cells and the level of IL-13?IL-6?TNF-? and IFN-? were all markedly weakened in TLR2-/-mice.Furthermore,the administration of lidocaine did not further mitigate the histopathological changes and airway inflammation in TLR2-/-allergic mice.TLR2 deficiency diminished OVA-induced expression of phosphorylated NF-?Bp65 and NLRP3 compared to that in OVA-challenged WT mice.However,treatment with lidocaine did not further decrease the OVA-induced upregulation of P-NF-?Bp65 and NLRP3 in TLR2-/-mice.5.Lidocaine only inhibited the expression of NLRP3 and ASC in lung tissue of allergic mice Compared with the control group,after OVA stimulation,the total number of peripheral white blood cells and lymphocytes increased significantly.And and the levels of IL-4 in BALF and OVA s Ig E in serum were significantly increased.Compared with the OVA group,inhaled lidocaine significantly reduced inflammatory cell in blood,OVA s Ig E in serum and IL-4 in BALF.Compared with the control group,the expression of NLRP3 and ASC in the lung tissue of OVA-stimulated mice were significantly increased,but the expression of caspase-1 and IL-1? in lung tissue and the level of IL-18 in BALF were not significant increase.Inhaled lidocaine significantly reduced the expression of NLRP3 and ASC in the lungs of allergic mice.Conclusions 1.Lidocaine inhibits S.aureus-stimulated activation of TLR2 signaling in macrophages 2.Lidocaine reduces allergic airway inflammation by inhibiting activation of TLR2 signaling pathway.3.Lidocaine inhibits Th2 response and NLRP3 expression in allergic mice to improve airway inflammation.