基于MEK/ERK通路探讨清热解毒扶正方调控中性粒细胞胞外诱捕网治疗肺炎的机制OA

Objective The mechanism by which the Heat-Clearing,Detoxifying,and Supporting Formula-Toxin-Clearing Formula inhibits mitogenactivated proteinkinase kinase(MEK)/extracellular signal-regulated kinase(ERK)signaling to regulate neutrophil extracellular traps(NET)formation and alleviate pneumonia.Methods Tracheal drip injection of Klebsiella pneumoniae was used to establish a mouse model of pneumonia.Mice were divided into blank control,model,ceftriaxone,and low-and high-dose DSQ groups.The pharmacodynamic effects of Toxin-Clearing Formula on mice with pneumonia were evaluated by assessing general condition,alveolar lavage fluid cell count,and lung pathology.Myeloperoxidase(MPO)activityin lung tissues was detected with a colorimetric assay kit.The protein levels of tumor necrosis factor(TNF)-α and interleukin(IL)-6 in lung homogenates were quantified using ELISA and quantitative reverse-transcription PCR was used to detect TNF-α,IL-6,IL-1β,lymphocyte antigen 6 complex,locus G(Ly6G),MPO,neutrophil elastase(NE),citrullinated histone H3(Cit-H3),and peptide arginine deaminase 4(PAD4)mRNA expression in lung tissues.Immunohistochemistry(IHC)was used to observe Ly6G,MPO,NE,and Cit-H3 expression in lung tissue sections,while immunofluorescence(IF)was used to observe NET expression.Western blot analysis was used to detect MEK,P-MEK,ERK,P-ERK,PAD4,and Cit-H3 protein expression in lung tissues.Results Toxin-Clearing Formula significantly improved symptoms such as shortness of breath and weight loss in mice with pneumonia,reduced inflammatory cell infiltration in lung tissue,and decreased MPO activity and TNF-α and IL-6 levels(P＜0.05,P＜0.01).mRNA and protein expression of Ly6G,MPO,NE,Cit-H3,and PAD4 were reduced(P＜0.05,P＜0.01),NET formation was inhibited,and the phosphorylation level of the MEK/ERK pathway was decreased(P＜0.05,P＜0.01).Conclusions Toxin-Clearing Formula may alleviate pneumonia by inhibiting the MEK/ERK pathway and reducing NET formation.