清肺饮调节TLR4/NF-κB炎症信号通路治疗细菌性肺炎的作用机制

Functional Mechanism of Qingfei Decoction on Regulating TLR4/NF-κB Inflammatory Signaling Pathway in The Treatment of Bacterial Pneumonia

目的:基于Toll样受体4(toll-like receptors,TLR4)/核因子κB(nuclear factor kappa-B,NF-κB)炎症信号通路探讨清肺饮对细菌性肺炎的作用机制。方法:体内实验,取Balb/c小鼠36只随机分为对照组、模型组、清肺饮低剂量组、清肺饮中剂量组、清肺饮高剂量组和头孢曲松(cefatriaxone,CTX)组各6只,除对照组外其余小鼠均给予肺炎链球菌菌液滴鼻并建立肺炎小鼠模型;清肺饮低、中、高剂量组给予清肺饮中药灌胃,头孢曲松组给予头孢曲松液体皮下注射,HE染色观察各组肺组织病理变化并进行炎症损伤评分。体外实验脂多糖(lipopolysaccharide,LPS)刺激人非小细胞肺癌细胞系(A549细胞)诱导体外炎症损伤模型,噻唑蓝(thiazolyl blue tetrazolium bromid,MTT)法检测不同浓度清肺饮对A549细胞存活率的影响,Western Blot法检测TLR4/NF-κB炎症信号通路相关蛋白表达情况,探索清肺饮治疗细菌性肺炎的作用机制和靶点。结果:体内实验各组小鼠HE染色结果显示与对照组比较,模型组小鼠肺间质弥漫性出血,肺泡壁大面积增厚,肺泡、支气管腔内可见大量红细胞渗出和炎症细胞浸润;清肺饮中、高剂量组与头孢曲松组小鼠肺间质及肺泡毛细血管扩张、出血、炎症细胞浸润程度均减轻,白细胞介素-1β(Interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)含量和肺炎症状评分均明显降低(P<0.05,P<0.01);体外实验100μg/mL、200μg/mL清肺饮可明显抑制A549细胞存活率(P<0.01),50μg/mL以内的清肺饮对细胞存活率几乎无影响(P>0.05);与0μg/mL LPS组比较,10μg/mL LPS刺激A549细胞其NF-κB p65蛋白表达明显上调(P<0.01),NF-κB的抑制蛋白(inhibitor of NF-κB,IκBα)明显下调(P<0.01),清肺饮干预后TLR4、髓样分化因子(myeloiddifferentiationfactor88,MyD88)、NF-κB p65表达降低、IκBα蛋白表达上调,尤以清肺饮高剂量组显著(P<0.01)。结论:清肺饮可减轻肺炎链球菌感染的小鼠肺组织损伤,并可通过抑制TLR4/NF-κB炎症通路的活化而发挥抗炎的生物学功效。

Objective:To explore the effect of Qingfeiyin(QFY)on bacterial pneumonia based on TLR4/NF-κB inflammatory signaling pathway.Methods:In vivo,36 Balb/c mice were divided into Control,Model,QFY(L)、QFY(M)、QFY(H)and CTX groups,6 mice in each group.Mice were infected with Streptococcus pneumonia by nose dropping.HE staining were performed to evaluate the effect of QFY on pneumonia mice.In vitro,A549 cells were stimulated with LPS to establish an inflammatory injury model.The cell viability was determined by MTT method.Western Blot were used to evaluate the expression of TLR4/NF-κB inflammatory signal pathway related proteins.Results:In vivo,HE showed that pulmonary interstitial diffuse hemorrhage,large area thickening of alveolar wall,disappearance of alveolar cavity structure,massive red cell exudation and inflammatory cell infiltration can be seen in thealveoli and bronchi of model mice compared with the Control group after S.pn infection,but the levels of IL-1β,TNF-αand the total lung inflammatory score were decreased in the QFY(M),QFY(H)and CTX groups(P<0.05,P<0.01).In vitro,The survival rate of A549 cells were significantly inhibited by 100μg/mL、200μg/mL QFY(P<0.01),but<50 g/mL QFY almost had no impact(P>0.05).The NF-κB p65 protein was up-regulated(P<0.01)and IκBαprotein were down-regulated(P<0.01)in Model group vs.Control group,but the expression of TLR4,MyD88,NF-κB p65 proteins were decreased after treatment of QFY;Furthermore,the expression of IκBαprotein was up-regulated,especially in QFY(H)group(P<0.01).Conclusion:The lung tissue damage in S.pn infected mice can be reduced by QFY and QFY may play the anti-inflammation role by suppressing the activation of TLR4/NF-κB pathway.