Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Dali, Yunnan, China; CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China; University of Chinese Academy of Sciences, Beijing, China.
School of Medicine and Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China.
J Ethnopharmacol. 2022 Mar 1;285:114851. doi: 10.1016/j.jep.2021.114851. Epub 2021 Nov 19.
Cybister chinensis Motschulsky belongs to the family Dytiscidae. As a traditional Chinese medicine, the insect is called Longshi in the folk and is commonly used to treat enuresis in children and frequent urination in the elderly.
Inflammation is involved in chronic kidney disease. The previous study proved ethanol extract of C. chinensis exhibited anti-inflammation effects in the Doxorubicin-induced kidney disease. However, the material basis and their possible mechanism of the insect were still unclear. Thus, we aimed to separate the active compounds of the ethanol extract from C. chinensis and to investigate their possible mechanism of anti-inflammation by network pharmacology and molecular docking.
The insect was extracted with 75% ethanol to produce ethanol extracts and then were extracted by petroleum ether, ethyl acetate and n-butanol respectively. Silica gel column chromatography and preparative HPLC were applied to separate the compounds of the extract. The compounds were characterized and identified by NMR and mass. The compound associated genes were collected by BATMAN-TCM database and the inflammation associated genes were obtained through DigSee database. The protein-protein interaction (PPI) network was carried out via Search Tool for the Retrieval of Interacting Genes/Protein (STRING) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) target pathway analysis was performed in Database for Annotation, Visualization and Integrated Discovery (DAVID). The possible mechanism of compounds against inflammation was investigated by molecular docking. Finally, the anti-inflammatory effect of the representative compound was verified by the LPS-induced Raw 264.7 cell inflammatory model. TNF-α, IL-1β and IL-6 of the cell supernatants were analyzed via using ELISA kits and the key proteins in JAK2/STAT3 signaling pathway were verified via the Western blot assays.
Among crude extracts from C. chinensis, ethyl acetate extract showed the obvious anti-inflammatory effects. Nine compounds were isolated from ethyl acetate extract of Cybister chinensis for the first time, including benzoic acid (1), hydroxytyrosol (2), protocatechualdehyde (3), N-[2-(4-hydroxyphenyl)ethyl]acetamide (4), (2E)-3-phenylprop-2-enoic acid (5), 3-phenylpropionic acid (6), methyl 3,4-dihydroxybenzoate (7), 1,4-diphenyl butane-2,3-diol (8) and p-N,N-dimethylaminobenzaldehyde (9). After searching in the database, 1079 compound associated genes and 467 inflammation associated genes were found. The 137 common targets covered 77 signaling pathways, in which HIF-1 signaling pathway, TNF signaling pathway, influenza A, PI3K/Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway and Jak-STAT signaling pathway were important for inflammation. Molecular docking studies showed compound 1, 4, 5, 6, 7 and 8 were the potential inhibitors of JAK2 protein. In addition, the in vitro test showed compound 5 reduced the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner. Furthermore, it was found that compound 5 inhibited the expression of p-JAK2 and p-STAT3 in LPS-induced RAW264.7 cells in a dose-dependent manner.
Based on the network pharmacology and molecular docking, the study suggested that C. chinensis could relieve the inflammation based on the multi-compounds and multi-pathways, which provided the foundation for the medicinal application of C. chinensis.
Cybister chinensis Motschulsky 属于水龟甲科。作为一种传统中药，这种昆虫在民间被称为龙虱，常用于治疗儿童遗尿和老年人尿频。
炎症参与慢性肾脏病。先前的研究证明，中华鳖乙醇提取物在多柔比星诱导的肾病中具有抗炎作用。然而，该昆虫的物质基础及其可能的机制仍不清楚。因此，我们旨在从中华鳖乙醇提取物中分离出活性化合物，并通过网络药理学和分子对接研究其抗炎的可能机制。
昆虫用 75%乙醇提取产生乙醇提取物，然后分别用石油醚、乙酸乙酯和正丁醇提取。硅胶柱层析和制备高效液相色谱法用于分离提取物中的化合物。通过 NMR 和质谱对化合物进行鉴定和鉴定。通过 BATMAN-TCM 数据库收集化合物相关基因，通过 DigSee 数据库获得炎症相关基因。通过 Search Tool for the Retrieval of Interacting Genes/Protein (STRING) 进行蛋白质-蛋白质相互作用 (PPI) 网络分析，并在 Database for Annotation, Visualization and Integrated Discovery (DAVID) 中进行京都基因与基因组百科全书 (KEGG) 目标途径分析。通过分子对接研究化合物抗炎的可能机制。最后，通过 LPS 诱导的 Raw 264.7 细胞炎症模型验证了代表性化合物的抗炎作用。通过 ELISA 试剂盒分析细胞上清液中的 TNF-α、IL-1β 和 IL-6，通过 Western blot 检测 JAK2/STAT3 信号通路中的关键蛋白。
在中华鳖的粗提物中，乙酸乙酯提取物表现出明显的抗炎作用。从中华鳖的乙酸乙酯提取物中首次分离出 9 种化合物，包括苯甲酸（1）、羟基酪醇（2）、原儿茶醛（3）、N-[2-（4-羟苯基）乙基]乙酰胺（4）、（2E）-3-苯基-2-丙烯酸（5）、3-苯基丙酸（6）、3,4-二羟基苯甲酸甲酯（7）、1,4-二苯基丁烷-2,3-二醇（8）和对-N,N-二甲氨基苯甲醛（9）。在数据库中搜索后，发现 1079 个化合物相关基因和 467 个炎症相关基因。137 个共同靶点涵盖了 77 个信号通路，其中 HIF-1 信号通路、TNF 信号通路、流感 A、PI3K/Akt 信号通路、NOD 样受体信号通路、MAPK 信号通路、Toll 样受体信号通路和 Jak-STAT 信号通路与炎症有关。分子对接研究表明，化合物 1、4、5、6、7 和 8 可能是 JAK2 蛋白的潜在抑制剂。此外，体外试验表明，化合物 5 可剂量依赖性降低脂多糖（LPS）刺激的 RAW264.7 细胞中肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6 和 IL-1β 的表达。此外，发现化合物 5 可剂量依赖性抑制 LPS 诱导的 RAW264.7 细胞中 p-JAK2 和 p-STAT3 的表达。
基于网络药理学和分子对接，研究表明中华鳖可通过多种化合物和多种途径缓解炎症，为中华鳖的药用应用提供了依据。
