Jiang Tao, Zhu Xiujing, Yin Zixin, Gao Rui, Li Yufen, Li Chenhao, Meng Qianting, Zhu Xiaojuan, Song Wu, Su Xin
Changchun University of Chinese Medicine, Changchun 130117, China.
Phytomedicine. 2025 Apr;139:156477. doi: 10.1016/j.phymed.2025.156477. Epub 2025 Feb 7.
Baimao-Longdan-Congrong-Fang (BLCF), a traditional Chinese herbal formula described in the Taiping Shenghui Fang (998 AD), consists of medicinal plants with heat-clearing and tonifying properties. BLCF has a promise as a treatment for Staphylococcus aureus (S. aureus) pneumonia, according to its historical use and current pharmacological research.
In this study, the inhibitory effects of BLCF on S. aureus virulence factors were evaluated in vitro, and its mechanisms of action were investigated in a methicillin-resistant S. aureus (MRSA) pneumonia mouse model.
The inhibitory effect of BLCF on S. aureus virulence factors, including sortase A (SrtA) and α-hemolysin (Hla), was investigated by fluorescence resonance energy transfer (FRET) and hemolysis assays. A C57BL/6J mouse model of MRSA pneumonia was employed to evaluate its therapeutic efficacy. Accordingly, an integrated strategy of medicinal chemistry, network pharmacology analysis, GEO database analysis, bioinformatics, molecular docking, molecular dynamics simulation, GeneMANIA-based functional association (GMFA), and GSEA was used to identify and illustrate potential therapeutic targets and mechanisms. Subsequently, the mechanistic results were confirmed by Western blot analysis and RT-qPCR.
While BLCF exhibited weak inhibitory activity against S. aureus USA300, Newman, and SA37 strains, it significantly suppressed SrtA-related virulence functions without affecting bacterial growth. FRET and hemolysis assays confirmed that BLCF inhibited SrtA activity (IC = 1.25 mg/mL) while decreasing hemolytic activity. Furthermore, BLCF protected mice from MRSA infection, increasing their survival rates. Bioinformatics analysis identified 26 active compounds and 2 hub genes (Tnf and Mmp9) that were associated with 5 types of immune cell, including activated CD4 T cells, myeloid-derived suppressor cells, activated dendritic cells, macrophages, and mast cells. Molecular docking revealed 3 active compounds (isoacteoside, verbascoside, and echinacoside) that exhibited strong binding affinities to TNF, MMP9, and SrtA. Molecular dynamics simulations validated the stable interactions between isoacteoside and the target proteins, yielding binding energies of -136.76 ± 8.83 kJ/mol, -174.98 ± 14.89 kJ/mol, and -186.34 ± 9.06 kJ/mol, respectively. The therapeutic effect of BLCF was closely linked to the NF-κB signaling pathway, as revealed by GMFA and GSEA analyses. In vivo, BLCF reduced lung bacterial load, improved the wet/dry ratio, and decreased inflammatory cytokines, thereby enhancing lung histopathology through modulation of the TNF-α/TNFR1/NF-κB/MMP9 axis.
BLCF can effectively treat MRSA pneumonia in mice by inhibiting SrtA activity, decreasing hemolytic activity, and regulating the TNF-α/TNFR1/NF-κB/MMP9 axis. These findings suggest BLCF, a traditional herbal formula, as a promising novel therapeutic approach to treat pneumonia.
白毛龙胆苁蓉方（BLCF）是公元998年《太平圣惠方》中记载的一种中药复方，由具有清热和滋补特性的药用植物组成。根据其历史应用和当前药理学研究，BLCF有望用于治疗金黄色葡萄球菌（S. aureus）肺炎。
本研究在体外评估了BLCF对金黄色葡萄球菌毒力因子的抑制作用，并在耐甲氧西林金黄色葡萄球菌（MRSA）肺炎小鼠模型中研究了其作用机制。
通过荧光共振能量转移（FRET）和溶血试验研究了BLCF对金黄色葡萄球菌毒力因子的抑制作用，包括分选酶A（SrtA）和α-溶血素（Hla）。采用C57BL/6J小鼠MRSA肺炎模型评估其治疗效果。因此，采用了药物化学、网络药理学分析、GEO数据库分析、生物信息学、分子对接、分子动力学模拟、基于GeneMANIA的功能关联（GMFA）和基因集富集分析（GSEA）等综合策略来识别和阐明潜在的治疗靶点和机制。随后，通过蛋白质免疫印迹分析和逆转录-定量聚合酶链反应（RT-qPCR）对机制研究结果进行了验证。
虽然BLCF对金黄色葡萄球菌USA300、Newman和SA37菌株表现出较弱的抑制活性，但它能显著抑制与SrtA相关的毒力功能，而不影响细菌生长。FRET和溶血试验证实，BLCF抑制SrtA活性（IC = 1.25 mg/mL），同时降低溶血活性。此外，BLCF保护小鼠免受MRSA感染，提高了它们的存活率。生物信息学分析确定了26种活性化合物和2个枢纽基因（Tnf和Mmp9），它们与5种免疫细胞类型相关，包括活化的CD4 T细胞、髓源性抑制细胞、活化的树突状细胞、巨噬细胞和肥大细胞。分子对接显示3种活性化合物（异麦角甾苷、毛蕊花糖苷和紫锥菊苷）对TNF、MMP9和SrtA表现出强烈的结合亲和力。分子动力学模拟验证了异麦角甾苷与靶蛋白之间的稳定相互作用，其结合能分别为-136.76 ± 8.83 kJ/mol、-174.98 ± 14.89 kJ/mol和-186.34 ± 9.06 kJ/mol。GMFA和GSEA分析表明，BLCF的治疗效果与核因子κB（NF-κB）信号通路密切相关。在体内，BLCF降低了肺部细菌载量，改善了肺组织湿/干比，并减少了炎性细胞因子，从而通过调节TNF-α/TNFR1/NF-κB/MMP9轴增强了肺组织病理学。
BLCF可通过抑制SrtA活性、降低溶血活性和调节TNF-α/TNFR1/NF-κB/MMP9轴有效治疗小鼠MRSA肺炎。这些发现表明，传统中药复方BLCF是一种有前景的新型肺炎治疗方法。
