热毒宁注射液雾化吸入改善烟熏诱导的慢性阻塞性肺疾病小鼠肺部炎症的作用及机制

Effect and Mechanism of Nebulized Inhalation of Reduning Injection on Improving Pulmonary Inflammation in Mice with Smoke-induced Chronic Obstructive Pulmonary Disease

目的:探究热毒宁注射液(RDN)对烟熏诱导的慢性阻塞性肺疾病(COPD)模型小鼠肺部炎症的治疗作用及其机制。方法:采用高效液相色谱法(HPLC)测定RDN中绿原酸和栀子苷的含量。制备烟熏诱导的COPD小鼠模型,并雾化吸入RDN进行治疗。采用瑞氏染色法检测肺泡灌洗液(BALF)中炎症细胞种类及数量;采用苏木素-伊红(HE)染色法观察肺组织病理学改变;采用免疫组织化学染色法(IHC)检测肺组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和IL-6的表达;采用转录组学技术筛选对照组、模型组及RDN高剂量组小鼠肺组织的差异表达基因,并对其进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析;采用蛋白质免疫印迹法(Western blot)检测Toll样受体4(TLR4)信号通路中相关蛋白的表达水平。结果:RDN中绿原酸和栀子苷的质量浓度分别为4.75、7.60 mg·mL^(–1);RDN能降低BALF中巨噬细胞和中性粒细胞的数量,并能改善小鼠肺间质与肺泡腔出血、肺泡壁水肿、炎性浸润等病理学变化;RDN能抑制肺组织炎症因子TNF-α、IL-1β和IL-6的表达;转录组学测序结果显示,3组小鼠存在149个共有差异表达基因,KEGG富集结果显示其主要涉及IL-17信号通路、TLR信号通路、TNF-α信号通路和核转录因子-κB(NF-κB)信号通路;RDN能降低肺组织TLR4的表达,并能抑制下游相关蛋白p38、c-Jun N末端激酶(JNK)、细胞外蛋白调节激酶(ERK)和p65的磷酸化水平。结论:RDN对烟熏诱导的COPD模型小鼠肺组织炎症反应具有改善作用,机制与其抑制TLR4信号通路有关。

Objective:To investigate the therapeutic effect and mechanism of Reduning Injection(RDN)on the pulmonary inflammation in a cigarette smoke-induced chronic obstructive pulmonary disease(COPD)mouse model.Methods:The contents of chlorogenic acid and geniposide in RDN were determined using high-performance liquid chromatography(HPLC).A cigarette smoke-induced COPD mouse model was established and treated with nebulized inhalation of RDN.Wright's staining was used to detect the types and numbers of inflammatory cells in bronchoalveolar lavage fluid(BALF).The pathological changes in lung tissues were observed using hematoxylin-eosin(HE)staining.The expression of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and IL-6 in lung tissues was detected using immunohistochemical(IHC)staining.Transcriptomic analysis was used to screen differentially expressed genes in the lung tissues of the control group,model group,and RDN high-dose group,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.The expression levels of related proteins in the Toll-like receptor 4(TLR4)signaling pathway were determined by Western blot.Results:The concentrations of chlorogenic acid and geniposide in RDN were 4.75 and 7.60 mg·mL^(–1),respectively.RDN reduced the number of macrophages and neutrophils in BALF and improved pathological changes such as interstitial and alveolar hemorrhage,alveolar wall edema,and inflammatory infiltration in the lung tissues of mice.RDN inhibited the expression of inflammatory factorsTNF-α,IL-1β,and IL-6 in lung tissues.Transcriptome sequencing results showed that there were 149 common differentially expressed genes among the three groups.KEGG enrichment analysis indicated that these genes were mainly involved in the IL-17 signaling pathway,Toll-like receptor signaling pathway,TNF-αsignaling pathway,and nuclear factor-κB(NF-κB)signaling pathway.RDN reduced the expression of TLR4 in lung tissues and inhibited the phosphorylation levels of downstream related proteins p38,JNK,extracellular signal-regulated kinase(ERK),and p65.Conclusion:RDN can improve the inflammatory response in the lung tissues of cigarette smoke-induced COPD model mice,and its mechanism is related to the inhibition of the TLR4 signaling pathway.