本研究旨在通过“靶向捕捞”策略确定井方颗粒治疗感染性肺炎的直接药理靶点。此外,基于靶点相关的药理信号通路,探讨井方颗粒治疗感染性肺炎的分子机制。首先,制备了经芳颗粒提取物结合的磁性纳米颗粒,将其与脂多糖(LPS)诱导的小鼠肺炎组织裂解物孵育。通过高分辨率质谱(HRMS)分析捕获的蛋白质,筛选出与荆方颗粒提取物具有特异性结合的目标组。使用京都基因和基因组百科全书(KEGG)富集分析来鉴定靶蛋白相关的信号通路。在此基础上,建立LPS诱导的小鼠感染性肺炎模型。通过苏木精-伊红(HE)染色和免疫组织化学法验证了靶蛋白的可能生物学功能。从肺组织中鉴定出186个井方颗粒特异性结合蛋白。KEGG通路富集分析表明,靶蛋白相关信号通路主要包括沙门氏菌感染,血管和肺上皮粘附连接,核糖体病毒复制,病毒内吞作用,和脂肪酸降解。荆方颗粒的靶功能与肺部炎症和免疫有关,肺能量代谢,肺微循环,和病毒感染。基于体内炎症模型,经方颗粒可显著改善LPS诱导的小鼠感染性肺炎模型的肺泡结构,下调肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的表达。同时,荆方Gra-nules显著上调线粒体功能关键蛋白COXⅣ和ATP的表达,微循环相关蛋白CD31和Occludin,以及与病毒感染DDX21和DDX3相关的蛋白质。这些结果表明,经方Gra-nules可以抑制肺部炎症,改善肺能量代谢和肺微循环,抵抗病毒感染,从而对肺起到保护作用。本研究从靶点-信号通路-药效的角度,系统地阐述了经方颗粒治疗呼吸道炎症的分子机制,从而为临床合理使用井方颗粒和扩大潜在的药理应用提供关键信息。
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.