基于TLR4/NF-κB信号通路研究清开灵口服液防治肺炎的作用机制

Mechanism of Qingkailing Oral Liquid in preventing and treatment of pneumonia based on TLR4/NF-κB signaling pathway

目的基于Toll样受体4(Toll-like receptor 4,TLR4)/核因子-κB(nuclear factor-κB,NF-κB)信号通路研究清开灵口服液防治肺部炎症的作用及机制。方法SD大鼠随机分为对照组、模型组及清开灵口服液低、中、高剂量(4、8、16 mL/kg)组和头孢氨苄(175 mg/kg)组,各给药组ig相应药物,1次/d,连续6 d。给药同时进行造模,肺部注射肺炎克雷伯菌菌液造模3 d,对照组肺部注射生理盐水。给药结束后采集大鼠血液、肺泡组织灌洗液(bronchoalveolar lavage fluid,BALF),进行菌落培养和计数;采用全自动血液细胞分析仪检测白细胞数量及中性粒细胞、单核细胞比例;采用ELISA法检测血清中白细胞介素-1(interleukin-1,IL-1)、IL-2、IL-6、IL-10、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、缓激肽(bradykinin,BK)、单核细胞趋化因子-1(monocyte chemotactic protein-1,MCP-1)水平及环氧化酶-1(cyclooxygenase-1,COX-1)、COX-2活性;采用苏木素-伊红(HE)染色观察大鼠肺组织病理变化;采用免疫组化法检测肺组织TLR4蛋白表达;采用Western blotting法检测肺组织磷酸化p65(phosphorylated p65,p-p65)、p65、磷酸化NF-κB抑制因子(phosphorylated inhibitor of NF-κB,p-IκB)和IκB表达。结果与模型组比较,清开灵口服液组大鼠全血及BALF中经培养后的菌落数显著降低(P<0.05);全血中白细胞数目及中性粒细胞、单核细胞比例均显著降低(P<0.05、0.01);血清中IL-1、IL-2、IL-6、IL-10、TNF-α、BK、MCP-1水平及COX-1、COX-2活性均显著降低(P<0.05、0.01);肺实质中炎性细胞浸润、红细胞渗出及肺泡上皮细胞脱落等现象明显减少;肺组织中TLR4蛋白阳性表达明显减少,pp65/p65和p-IκB/IκB蛋白表达水平显著下降(P<0.05、0.01)。结论清开灵口服液具有抑制肺炎克雷伯菌所致肺炎的作用,其机制可能与调控TLR4/NF-κB信号通路有关。

Objective To study the effect and mechanism of Qingkailing Oral Liquid(清开灵口服液,QKL)on pulmonary inflammation based on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway.Methods SD rats were randomly divided into control group,model group,QKL low-,medium-and high-dose(4,8,16 mL/kg)groups and cephalexin(175 mg/kg)group.Each administration group was ig corresponding drugs for 6 d.Models were established at the same time of administration,and models were established by pulmonary injection of Klebsiella pneumoniae solution for 3 d,control group was injected with normal saline.After the administration,blood and bronchoalveolar lavage fluid(BALF)of rats were collected for colony culture and counting;Automatic blood cell analyzer was used to detect the number of white blood cells(WBC)and ratio of neutrophils and monocytes;ELISA was used to detect interleukin-1(IL-1),IL-2,IL-6,IL-10,tumor necrosis factor-α(TNF-α),bradykinin(BK),monocyte chemotactic protein-1(MCP-1)levels and cyclooxygenase-1(COX-1),COX-2 activities in serum;Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung tissue in rats;Immunohistochemical staining was used to detect protein expression of TLR4 in lung tissue;Western blotting was used to detect protein expressions of phosphorylated p65(p-p65),p65,phosphorylated inhibitor of NF-κB(p-IκB)and IκB in lung tissue.Results Compared with model group,number of colonies in whole blood and BALF after culture were significantly decreased in QKL group(P<0.05),number of WBCs and percentage of neutrophils and monocytes in whole blood were significantly decreased(P<0.05,0.01),IL-1,IL-2,IL-6,IL-10,TNF-α,BK,MCP-1 levels and COX-1,COX-2 activities in serum were significantly reduced(P<0.05,0.01);Inflammatory cell infiltration,erythrocyte exudation,and alveolar epithelial cell detachment in lung parenchyma were significantly reduced;TLR4 protein positive expression in lung tissue were significantly reduced,p-p65/p65 and p-IκB/IκB protein expressions were significantly decreased(P<0.05,0.01).Conclusion QKL has an inhibitory effect on pneumonia caused by K.pneumoniae,and its mechanism may be related to regulating TLR4/NF-κB signaling pathway.