结合代谢组学和网络药理学分析探索葛根抗肺纤维化的机制:精氨酸代谢途径的参与

IF 4.8 2区 医学 Q1 CHEMISTRY, MEDICINAL
Hong Du , Meijuan Shao , Shangcheng Xu , Qian Yang , Jingping Xu , Hong Ke , Li Zou , Liping Huang , Yanru Cui , Fei Qu
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引用次数: 0

摘要

民族药理学意义:葛根(Pueraria lobata (Willd.) Ohwi)是一种典型的药用和食用植物,在中国和东南亚有着悠久的应用历史。作为一种广泛使用的传统药物,葛根具有消炎、解热、抗氧化、止咳平喘等功效。特别是,越来越多的证据表明,小叶女贞子在调节代谢方面对纤维化相关疾病有治疗作用。然而,龙须菜对肺纤维化(PF)的作用机制尚未得到深入探讨:本研究旨在通过代谢组学与网络药理学分析相结合的方法,探讨小叶拟南星精氨酸代谢物对肺纤维化模型的影响。材料与方法:本研究将Sprague Dawley(SD)大鼠随机分为5个实验组:生理盐水对照组、博来霉素诱导肺纤维化组、醋酸泼尼松龙组、3.2 g/kg P.lobata组和6.4 g/kg P.lobata组。通过肺功能、体重、苏木精伊红染色(HE)、Masson染色和羟脯氨酸检测等临床症状来评估小叶紫檀对博莱霉素诱导的大鼠肺纤维化的治疗效果。然后,利用 LC-MS 进行血浆代谢组学分析,探讨对照组、PF 组和 P. lobata 治疗组大鼠的病理差异。然后,结合实验验证进行网络药理学研究,分析代谢研究的结果。我们构建了小叶紫檀治疗PF的 "成分-靶点-疾病 "网络。此外,我们还采用分子对接法验证了潜在活性成分与龙脑香茶菜核心靶点之间的相互作用。最后,我们通过酶联免疫吸附试验(ELISA)检测了大鼠血浆中精氨酸相关代谢途径中的 NOS2 和 L-OT。实时 PCR 检测 TNF-α mRNA 和 MMP9 mRNA 的水平。我们还通过 Western 印迹分析检测了 TNF-α 和 MMP9 的表达:结果:我们的研究结果表明,P. lobata能改善PF大鼠的肺功能,并改善其病理损伤、胶原沉积和体重减轻等病理症状。此外,我们还采用血浆代谢组学方法筛查了氨基酸、脂类、黄酮类、花生四烯酸代谢物、苷类等差异代谢物。最后,我们发现精氨酸代谢信号传导主要参与了小叶紫檀对 PF 大鼠治疗的调控。此外,网络药理学预测精氨酸代谢通路位于前 20 个通路中。接下来,我们整合了代谢组学和网络药理学,通过分子对接确定了NOS2、MMP9和TNF-α为小叶豚鼠调控的枢纽基因。重要的是,研究表明葛根素与 NOS2 有很强的亲和力。葛根素能降低PF大鼠体内TNF-α、MMP-9和NOS2的水平,抑制TNF-α和MMP-9蛋白的表达,降低L-OT和NOS2的含量。这些结果表明,龙须菜可通过精氨酸代谢途径改善大鼠的 PF 状况。因此,龙脑香可能是改善 PF 的潜在治疗药物:在这项工作中,我们利用代谢组学和网络药理学探索了龙脑香叶在治疗 PF 中的作用机制。最后,我们证实了小叶紫檀通过调节精氨酸代谢途径,在减少 L-OT 和 NOS2 相关信号分子的基础上,缓解了 BLM 诱导的大鼠 PF。通过寻找精氨酸代谢途径的生物标志物,我们发现了龙脑香附治疗 PF 的新策略。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Integrating metabolomics and network pharmacology analysis to explore mechanism of Pueraria lobata against pulmonary fibrosis: Involvement of arginine metabolism pathway

Integrating metabolomics and network pharmacology analysis to explore mechanism of Pueraria lobata against pulmonary fibrosis: Involvement of arginine metabolism pathway

Ethnopharmacological relevance

Pueraria lobata (Willd.) Ohwi is a typical medicinal and edible plant with a long application history in China and Southeast Asia. As a widely used traditional medicine, P. lobata exhibits the properties of anti-inflammatory, antipyretic, antioxidant, relieving cough and asthma. Particularly, the increasing evidence indicates that the P. lobata has the therapeutic effect on fibrotic-related diseases in terms of metabolic regulation. However, the mechanisms of P. lobata on pulmonary fibrosis (PF) has not been thoroughly explored.

Aim of the study

This study aimed to explore the effect of arginine metabolites of P. lobata against PF model by integrating metabolomics and network pharmacology analysis. It might provide a new idea for the target finding of P. lobata anti-pulmonary fibrosis.

Materials and methods

In this study, the Sprague Dawley (SD) rats were randomly divided into five experimental groups: saline-treated control group, bleomycin-induced fibrosis group, prednisolone acetate group, P. lobata 3.2 g/kg group and P. lobata 6.4 g/kg group. The therapeutic effect of P. lobata on bleomycin-induced PF in rats was evaluated by clinical symptoms such as lung function, body weight, hematoxylin eosin staining (HE), Masson staining and hydroxyproline assay. Next, the plasma metabolomics analysis was carried out by LC-MS to explore the pathological differences between the group of control, PF and P. lobata-treated rats. Then, the network pharmacology study coupled with experimental validation was conducted to analysis the results of metabolic research. We constructed the “component-target-disease” network of P. lobata in the treatment of PF. In addition, the molecular docking method was used to verify the interaction between potential active ingredients and core targets of P. lobata. Finally, we tested NOS2 and L-OT in arginine-related metabolic pathway in plasma of the rats by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was performed to observe the level of TNF-α mRNA and MMP9 mRNA. And we tested the expression of TNF-α and MMP9 by Western blot analysis.

Results

Our findings revealed that P. lobata improved lung function and ameliorated the pathological symptoms, such as pathological damage, collagen deposition, and body weight loss in PF rats. Otherwise, the plasma metabolomics were employed to screen the differential metabolites of amino acids, lipids, flavonoids, arachidonic acid metabolites, glycoside, etc. Finally, we found that the arginine metabolism signaling mainly involved in the regulating of P. lobata on the treatment of PF rats. Furtherly, the network pharmacology predicted that the arginine metabolism pathway was contained in the top 20 pathways. Next, we integrated metabolomics and network pharmacology that identified NOS2, MMP9 and TNF-α as the P. lobata regulated hub genes by molecular docking. Importantly, it indicated a strong affinity between the puerarin and the NOS2. P. lobata attenuated TNF-α, MMP-9 and NOS2 levels, suppressed TNF-α and MMP-9 protein expression, and decreased L-OT and NOS2 content in PF rats. These results indicated that the effects of P. lobata may ameliorated PF via the arginine metabolism pathway in rats. Therefore, P. lobata may be a potential therapeutic agent to ameliorated PF.

Conclusion

In this work, we used metabolomics and network pharmacology to explore the mechanisms of P. lobata in the treatment of PF. Finally, we confirmed that P. lobata alleviated BLM-induced PF in rats by regulating arginine metabolism pathway based on reducing the L-OT and NOS2-related signal molecular. The search for the biomarkers finding of arginine metabolism pathway revealed a new strategy for P. lobata in the treatment of PF.

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来源期刊
Journal of ethnopharmacology
Journal of ethnopharmacology 医学-全科医学与补充医学
CiteScore
10.30
自引率
5.60%
发文量
967
审稿时长
77 days
期刊介绍: The Journal of Ethnopharmacology is dedicated to the exchange of information and understandings about people''s use of plants, fungi, animals, microorganisms and minerals and their biological and pharmacological effects based on the principles established through international conventions. Early people confronted with illness and disease, discovered a wealth of useful therapeutic agents in the plant and animal kingdoms. The empirical knowledge of these medicinal substances and their toxic potential was passed on by oral tradition and sometimes recorded in herbals and other texts on materia medica. Many valuable drugs of today (e.g., atropine, ephedrine, tubocurarine, digoxin, reserpine) came into use through the study of indigenous remedies. Chemists continue to use plant-derived drugs (e.g., morphine, taxol, physostigmine, quinidine, emetine) as prototypes in their attempts to develop more effective and less toxic medicinals.
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