荆防颗粒抗感染性肺炎的靶点发现及分子机制研究

Anti-infectious pneumonia target discovery and molecular mechanism study of Jingfang Granules

基于靶点“钩钓”策略鉴定荆防颗粒发挥抗感染性肺炎的直接药理靶点群,基于靶点相关药理学信号通路探究荆防颗粒治疗感染性肺炎的分子机制。制备荆防颗粒药效成分键合的磁性微球,并与细菌脂多糖(lipopolysaccharide,LPS)诱导的小鼠肺炎组织裂解液进行孵育,通过高分辨质谱对捕获的蛋白进行分析,筛选与荆防颗粒提取物存在特异性结合的作用靶点群。利用京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析靶点蛋白群相关信号通路。在此基础上,进一步构建LPS诱导的小鼠感染性肺炎模型,通过苏木精-伊红(hematoxylin-eosin,HE)染色、免疫组化染色等方法对靶点蛋白群可能的生物学功能进行验证。累计从肺组织中鉴定到186个荆防颗粒特异性结合的蛋白,基于KEGG通路分析发现这些靶点蛋白作用的信号通路主要包括沙门菌感染、血管和肺上皮黏着连接、核糖体相关的病毒复制、病毒的内吞作用和脂肪酸降解相关信号通路;反映荆防颗粒的靶点功能主要与肺部炎症与免疫、肺能量代谢、肺微循环和病毒感染有关。最后,基于体内炎症模型发现,荆防颗粒显著改善LPS所致感染性肺炎模型小鼠的肺泡结构并下调炎症标志蛋白肿瘤坏死因子α(TNF-α)和IL-6表达;上调线粒体功能关键蛋白细胞色素C氧化酶4(COXⅣ)表达量及ATP含量;上调微循环相关蛋白CD31与Occludin表达;上调病毒感染相关蛋白DDX21与DDX3表达,提示荆防颗粒能够抑制肺部炎症、改善肺部能量代谢和肺微循环、抵抗病毒感染,进而发挥肺保护作用。研究结果从靶点-信号通路-药理功效的角度系统诠释了荆防颗粒治疗呼吸系统炎症的分子药理机制,为其临床合理用药提供了数据支撑,并有助于拓展其新的药理学用途。

This study aimed to identify the direct pharmacological targets of Jingfang Granules in treating infectious pneumonia via "target fishing" strategy. Moreover, the molecular mechanism of Jingfang Granules in treating infectious pneumonia was also investigated based on target-related pharmacological signaling pathways. First, the Jingfang Granules extract-bound magnetic nanoparticles were prepared, which were incubated with lipopolysaccharide(LPS)-induced mouse pneumonia tissue lysates. The captured proteins were analyzed by high-resolution mass spectrometry(HRMS), and the target groups with specific binding to the Jingfang Granules extract were screened out. Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis was used to identify the target protein-associated signaling pathways. On this basis, the LPS-induced mouse model of infectious pneumonia was established. The possible biological functions of target proteins were verified by hematoxylin-eosin(HE) staining and immunohistochemical assay. A total of 186 Jingfang Granules-specific binding proteins were identified from lung tissues. KEGG pathway enrichment analysis showed that the target protein-associated signaling pathways mainly included Salmonella infection, vascular and pulmonary epithelial adherens junction, ribosomal viral replication, viral endocytosis, and fatty acid degradation. The target functions of Jingfang Granules were related to pulmonary inflammation and immunity, pulmonary energy metabolism, pulmonary microcirculation, and viral infection. Based on the in vivo inflammation model, Jingfang Granules significantly improved the alveolar structure of the LPS-induced mouse model of infectious pneumonia and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). Meanwhile, Jingfang Gra-nules significantly up-regulated the expressions of key proteins of mitochondrial function COX Ⅳ and ATP, microcirculation-related proteins CD31 and Occludin, and proteins associated with viral infection DDX21 and DDX3. These results suggest that Jingfang Gra-nules can inhibit lung inflammation, improve lung energy metabolism and pulmonary microcirculation, resist virus infection, thus playing a protective role in the lung. This study systematically explains the molecular mechanism of Jingfang Granules in the treatment of respiratory inflammation from the perspective of target-signaling pathway-pharmacological efficacy, thereby providing key information for clinical rational use of Jingfang Granules and expanding potential pharmacological application.