Table2_Integrative network pharmacology and experimental verification to reveal the anti-inflammatory mechanism of ginsenoside Rh4.docx

Inflammation is an innate immune response to infection, and it is the main factor causing bodily injury and other complications in the pathological process. Ginsenoside Rh4 (G-Rh4), a minor ginsenoside of Panax ginseng C. A. Meyer and Panax notoginseng, has excellent pharmacological properties. However, many of its major pharmacological mechanisms, including anti-inflammatory actions, remain unrevealed. In this study, network pharmacology and an experimental approach were employed to elucidate the drug target and pathways of G-Rh4 in treating inflammation. The potential targets of G-Rh4 were selected from the multi-source databases, and 58 overlapping gene symbols related to G-Rh4 and inflammation were obtained for generating a protein–protein interaction (PPI) network. Molecular docking revealed the high affinities between key proteins and G-Rh4. Gene ontology (GO) and pathway enrichment analyses were used to analyze the screened core targets and explore the target–pathway networks. It was found that the JAK-STAT signaling pathway, TNF signaling pathway, NF-κB signaling pathway, and PI3K-Akt signaling pathway may be the key and main pathways of G-Rh4 to treat inflammation. Additionally, the potential molecular mechanisms of G-Rh4 predicted from network pharmacology analysis were validated in RAW264.7 cells. RT-PCR, Western blot, and ELISA analysis indicated that G-Rh4 significantly inhibited the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, as well as inflammation-related enzymes in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Moreover, in vitro experiments evaluated that Ginsenoside Rh4 exerts anti-inflammatory effects via the NF-κB and STAT3 signaling pathways. It is believed that our study will provide the basic scientific evidence that G-Rh4 has potential anti-inflammatory effects for further clinical studies.

炎症是机体对感染的一种先天免疫反应，它是导致病理过程中机体损伤及其他并发症的主要因素。人参皂苷Rh4（G-Rh4），为人参（Panax ginseng C. A. Meyer）和三七（Panax notoginseng）中的一种次要人参皂苷，具有卓越的药理特性。然而，其中许多主要的药理机制，包括抗炎作用，尚待揭示。本研究采用网络药理学和实验方法，以阐明G-Rh4治疗炎症的药物靶点和作用途径。G-Rh4的潜在靶点从多源数据库中筛选得出，共获得与G-Rh4及炎症相关的58个重叠基因符号，用于构建蛋白质-蛋白质相互作用（PPI）网络。分子对接揭示了关键蛋白质与G-Rh4之间的高亲和力。通过基因本体（GO）和通路富集分析，对筛选出的核心靶点进行了分析，并探索了靶点-通路网络。研究发现，JAK-STAT信号通路、TNF信号通路、NF-κB信号通路和PI3K-Akt信号通路可能是G-Rh4治疗炎症的关键和主要通路。此外，通过网络药理学分析预测的G-Rh4的潜在分子机制在RAW264.7细胞中得到验证。实时荧光定量PCR、Western印迹和ELISA分析表明，G-Rh4显著抑制了由脂多糖（LPS）刺激的RAW264.7细胞中促炎细胞因子（如TNF-α、IL-6和IL-1β）及炎症相关酶的产生。此外，体外实验评估了人参皂苷Rh4通过NF-κB和STAT3信号通路发挥抗炎作用。本研究认为，将为G-Rh4具有潜在抗炎作用提供基本的科学依据，为后续的临床研究奠定基础。