Jing Fei, Chen Xi, Xue Jingbo, Huang Kai, Xing Feng, Hu Xudong, Peng Yuan, Liu Chenghai
Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai, China.
Front Pharmacol. 2022 Apr 25;13:805432. doi: 10.3389/fphar.2022.805432. eCollection 2022.
Pro-inflammatory macrophages aggravated progress of pulmonary fibrosis (PF) both in patients and animal models. Fuzheng Huayu (FZHY) formula, a Chinese herbal product, is effective in treating pulmonary fibrosis in our previous study. But its action mechanism against PF relating to macrophage activation was unclear. This study was designed to evaluate the anti-fibrotic and anti-inflammatory roles of FZHY in pulmonary fibrosis and to elucidate the potential mechanisms. Network pharmacology was employed to identify the interrelationships among compounds of FZHY, potential targets and putative pathways on anti-pulmonary fibrosis. According to the data of bioinformatics analysis, the key pharmacological target for FZHY against PF was screened. The network pharmacological prediction was validated by a series of experimental assays, including CCK8, western blot and immunofluorescence staining. Then molecular mechanism of FZHY on relating to the predictive target were studied in bleomycin induced pulmonary fibrosis in mice with methylprednisolone as a positive control, and in lipopolysaccharide (LPS) stimulated cultured macrophages in culture, respectively. The network pharmacology analysis reveal that a total of 12 FZHY-PF crossover proteins were filtered into a protein-protein interaction network complex and designated as the potential targets of FZHY against pulmonary fibrosis, while TNF-α signal pathway ranked at the top. FZHY and methylprednisolone could attenuate the lung fibrosis and decrease pulmonary TNF-α expression in bleomycin induced fibrotic mice, without difference between two treatments. While TNF-α was mainly originated from macrophages identified by double fluorescent staining of TNF-α and F4/80. LPS stimulated cultured macrophage polarization and activation demonstrated by the enhance contents of TNF-α and iNOS but decreased level of Arg-1. FZHY could alleviate the LPS stimulated macrophage polarization and activation demonstrated by decreasing TNF-α and iNOS and increasing Arg-1. In particular, FZHY could significantly reduce the production of p65 and the nuclear translocation of phosphorylated p65. Fuzheng Huayu formula has a good effect against pulmonary fibrosis induced by bleomycin in mice, whose action mechanism was associated with down-regulation of NF-κB/TNF-α signaling pathway in pro-inflammatory macrophages. These findings provided an important strategy for developing new agents against lung fibrosis and accelerated FZHY product application on patients with lung fibrosis.
促炎巨噬细胞在患者和动物模型中均会加剧肺纤维化（PF）的进展。扶正化瘀（FZHY）方是一种中药制剂，在我们之前的研究中对治疗肺纤维化有效。但其针对与巨噬细胞活化相关的PF的作用机制尚不清楚。本研究旨在评估FZHY在肺纤维化中的抗纤维化和抗炎作用，并阐明其潜在机制。采用网络药理学来确定FZHY的化合物、潜在靶点和抗肺纤维化的假定途径之间的相互关系。根据生物信息学分析数据，筛选出FZHY抗PF的关键药理学靶点。通过一系列实验分析，包括CCK8、蛋白质印迹法和免疫荧光染色，对网络药理学预测进行验证。然后，分别以甲基强的松龙作为阳性对照，在博莱霉素诱导的小鼠肺纤维化模型以及脂多糖（LPS）刺激的培养巨噬细胞中，研究FZHY与预测靶点相关的分子机制。网络药理学分析显示，共有12种FZHY-PF交叉蛋白被筛选进入蛋白质-蛋白质相互作用网络复合物，并被指定为FZHY抗肺纤维化的潜在靶点，而肿瘤坏死因子-α（TNF-α）信号通路位居榜首。FZHY和甲基强的松龙均可减轻博莱霉素诱导的纤维化小鼠的肺纤维化并降低肺组织中TNF-α的表达，两种治疗方法之间无差异。而通过TNF-α和F4/80的双重荧光染色确定TNF-α主要来源于巨噬细胞。LPS刺激培养的巨噬细胞极化和活化表现为TNF-α和诱导型一氧化氮合酶（iNOS）含量增加，但精氨酸酶-1（Arg-1）水平降低。FZHY可通过降低TNF-α和iNOS以及增加Arg-1来减轻LPS刺激的巨噬细胞极化和活化。特别是，FZHY可显著降低p65的产生以及磷酸化p65的核转位。扶正化瘀方对博莱霉素诱导的小鼠肺纤维化具有良好的疗效，其作用机制与下调促炎巨噬细胞中核因子-κB（NF-κB）/TNF-α信号通路有关。这些发现为开发抗肺纤维化新药提供了重要策略，并加速了FZHY产品在肺纤维化患者中的应用。
