基于网络药理学探究白肉灵芝醇提物对矽肺炎症反应改善作用与机制

Network pharmacology reveals the effect and mechanism of Ganoderma leucocontextum ethanol extract on improving inflammatory response in silicosis lungs

目的通过网络药理学、分子对接技术和动物实验探究白肉灵芝醇提物(GLE)对矽肺的改善作用及其潜在分子机制。方法(1)采用超高液相色谱-四极杆静电场轨道阱质谱法(UPLC-QE-MS)分析GLE成分;利用网络药理学和分子对接技术,探究GLE干预矽肺炎症进程中的活性成分及调控的潜在分子通路和靶点。(2)将无特定病原体级雄性C57BL6/J小鼠分为4组,每组10只。采用非暴露式气管滴注法,矽肺模型组和GLE干预组小鼠一次性予质量浓度为50 g/L的二氧化硅混悬液80μL染尘,空白对照组和GLE对照组小鼠均予等体积的无菌0.9%氯化钠溶液;造模后第2天起,GLE对照组和GLE干预组小鼠均予剂量为200 mg/(kg·d)的GLE灌胃,空白对照组和矽肺模型组小鼠均予等体积0.9%氯化钠溶液灌胃,每日1次,连续35 d。实验结束后,观察各组小鼠肺组织病理学改变情况,计算肺质量系数、炎症评分和胶原沉积面积占比,采用酶联免疫吸附实验检测肺组织中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和IL-6水平。结果(1)UPLC-QE-MS技术检测出GLE的76种活性成分;其中,36种成分满足类药性五原则筛选条件。基于网络药理学理论,该36种GLE活性成分中共筛选出改善矽肺炎性反应的潜在作用靶点67个。京都基因与基因组百科全书富集分析和基因本体论功能分析结果显示,IL信号和免疫细胞的细胞因子信号在GLE改善矽肺的过程中发挥关键作用。分子对接结果显示,67个交集靶点中,排名基于前10的依次是TNF、IL6、B淋巴细胞瘤2、细胞肿瘤抗原p53、半胱氨酸蛋白酶-3亚基p12、JUN、表皮生长因子受体、IL1B、67 kDa基质金属蛋白酶-9和前列腺素G/H合酶2。蛋白质-蛋白质相互作用分析结果显示,与关键靶点TNF-α、IL-1β、IL-6亲和力最强的成分是甘草次酸;其次为灵芝酸DM、泽泻醇C、灵芝酸β和红光皂苷元。(2)组织病理学检查结果显示,GLE可缓解矽肺小鼠模型的肺部炎症反应和胶原沉积。GLE干预组小鼠肺质量系数、炎症评分、胶原面积占比和IL-6水平均低于矽肺模型组(P值均<0.05);但该2组小鼠肺组织中TNF-α和IL-1β水平分别比较,差异均无统计学意义(P值均>0.05)。结论GLE可能通过抑制小鼠肺组织中IL-6水平而改善二氧化硅所致肺组织炎症和纤维化;其机制具有多成分、多靶点、多通路协同作用的特点。

Objective To explore the effect of Ganoderma leucocontextum ethanol extract(GLE)on silicosis and its potential molecular mechanism using network pharmacology,molecular docking technology and animal experiments.Methods i)The components of GLE were analyzed using ultra-performance liquid chromatography-Q Exactive-mass spectrometry(UPLC-QEMS)method.The active components,potential molecular pathways and targets of GLE in the intervention of inflammation process of silicosis was explored using network pharmacology and molecular docking technology.ii)Specific pathogen free male C57BL6/J mice were divided into four groups with 10 mice in each group.The mice in the silicosis model group and GLE intervention group were given a dose of 80μL silica suspension with a mass concentration of 50 g/L once by non-exposed tracheal instillation,and the mice in the blank control group and GLE control group were given an equal volume of sterile 0.9%sodium chloride solution.From the second day after modeling,GLE control group and GLE intervention group were given GLE at a dose of 200 mg/(kg·d)by gavage,while blank control group and silicosis model group were given the same volume of 0.9%sodium chloride solution by gavage,once per day for 35 days.After that,the histopathological changes of lung tissues of mice were observed,the lung mass coefficient,inflammation score and the ratio of collagen deposition area were calculated,and the levels of tumor necrosis factor(TNF)-α,interleukin(IL)-1βand IL-6 in the lung tissues of mice were detected by enzymelinked immunosorbent assay.Results i)A total of 76 active components of GLE were detected by UPLC-QE-MS.Among them,36 ingredients met the screening criteria of the five principles of drug-like components.A total of 67 potential targets of the 36 GLE active ingredients to improve the inflammatory response of silicosis were screened based on the network pharmacology theory.The result of Kyoto Encyclopedia of Genes and Genomes enrichment analysis and Gene Ontology functional analysis showed that IL signaling and cytokine signaling of immune cells played a key role in the process of antisilicosis of GLE.The results of molecular docking showed that the top 10 targets based on the 67 intersection targets were TNF,IL6,B-cell lymphoma 2,cellular tumor antigen p53,Caspase-3 subunit p12,JUN,epidermal growth factor receptor,IL1B,67 kDa matrix metalloproteinase-9 and prostaglandin G/H synthase 2.The result of protein-protein interaction analysis showed that glycyrrhetinic acid had the strongest affinity with the key targets TNF-α,IL-1βand IL-6,followed by ganoderma acid DM,alismatol C,ganoderma acidβand red sapogenin.ii)The results of histopathological examination showed that the inflammatory response and collagen deposition were alleviated in the lungs of mice with silicosis.The lung mass coefficient,inflammation score,ratio of collagen deposition area and IL-6 expression in lung were lower in mice of the GLE intervention group(all P<0.05),compared with the silicosis model group.However,there was no significant difference in the levels of TNF-αand IL-1βin lung tissues between the two groups(all P>0.05).Conclusion GLE may reduce silica-induced lung inflammation and fibrosis by inhibiting the IL-6 level in lung tissues of mice.Its mechanism is associated with the synergistic action of multicomponents,multi-targets and multi-pathways.