<b>Combination of isobavachalcone and amphotericin B has antifungal effect against </b><b>Cryptococcus neoformans</b><b>and protects host tissue damage by inhibiting ferroptosis</b>

Despite advancements in the treatment of cryptococcal infections, the availability of antifungal agents remains limited compared to antibiotics used for bacterial infections. The development of novel therapeutic strategies is imperative, albeit challenging. A promising approach involves the synergistic interaction between existing antifungal agents and supplements to enhance efficacy, reduce dosages, and mitigate resistance. This study aims to investigate the effects of Isobavachalcone (IBC) and Amphotericin B (AmB) in novel in vitro and in vivo models, with a focus on ferroptosis regulation.<br>In a novel Caenorhabditis elegans model of cryptococcal infection, we analyzed ferroptosis-related biomarkers, including glutathione (GSH), malondialdehyde (MDA), ferrous ions, and reactive oxygen species (ROS). The results demonstrated that IBC (4 μg/mL) significantly reduced the minimum inhibitory concentration (MIC) of AmB from 1 μg/mL to 0.25 μg/mL, indicating a fourfold increase in potency. The IBC-AmB combination exhibited detrimental effects on membrane permeability and cell wall integrity in Cryptococcus neoformans.<br>Furthermore, this combination elevated host GSH levels while concurrently decreasing ferrous ions, MDA, and ROS. Mechanistically, the treatment upregulated antioxidant/stress response genes (SKN-1, GST-4, GST-5, GPX-1, DAF-16, CNC-11) and antimicrobial peptides (NLP-29), while downregulating PMK-1. These findings suggest a dual mechanism of action involving direct disruption of fungal membranes and modulation of host ferroptosis.<br>This study underscores the potential of combining natural compounds with existing antifungal agents to address the limitations of the current antifungal drug repertoire.尽管隐球菌感染（cryptococcal infections）的治疗手段已取得进展，但相较于用于细菌感染的抗生素（antibiotics），可用抗真菌药物（antifungal agents）仍较为匮乏。开发新型治疗策略虽颇具挑战，但势在必行。其中一种极具前景的思路，是借助现有抗真菌药物与补充剂之间的协同作用，以提升疗效、降低给药剂量并缓解耐药性。本研究旨在探究异补骨脂查尔酮（Isobavachalcone, IBC）与两性霉素B（Amphotericin B, AmB）在新型体外（in vitro）及体内（in vivo）模型中的作用效果，重点关注铁死亡（ferroptosis）调控。<br>我们利用新型隐球菌感染秀丽隐杆线虫（Caenorhabditis elegans）模型，分析了其中与铁死亡相关的生物标志物，包括谷胱甘肽（glutathione, GSH）、丙二醛（malondialdehyde, MDA）、亚铁离子（ferrous ions）与活性氧（reactive oxygen species, ROS）。结果显示，浓度为4 μg/mL的IBC可将AmB的最低抑菌浓度（minimum inhibitory concentration, MIC）从1 μg/mL显著降至0.25 μg/mL，表明其药效提升了四倍。IBC与AmB联合使用可破坏新生隐球菌（Cryptococcus neoformans）的膜通透性与细胞壁完整性。<br>此外，该联合疗法可提升宿主的GSH水平，同时降低亚铁离子、MDA与ROS的水平。从作用机制来看，该治疗方案可上调抗氧化/应激反应基因（SKN-1、GST-4、GST-5、GPX-1、DAF-16、CNC-11）与抗菌肽（antimicrobial peptides, NLP-29），并下调PMK-1的表达。上述研究结果表明，其作用机制兼具双重性：直接破坏真菌细胞膜，以及调控宿主铁死亡。<br>本研究凸显了将天然化合物与现有抗真菌药物联合使用的潜力，可为应对当前抗真菌药物库（antifungal drug repertoire）存在的局限性提供新的途径。