Exploring The Antifungal Activity And Mechanism Of Hemiprotonic Phenanthroline-Phenanthroline~+ Against Trichophyton Rubrum

Trichophyton rubrum（T.rubrum）is the main pathogenic fungus causing skin fungal diseases.It can infect human skin,nails,and hair,causing diseases such as tinea pedis,tinea capitis,tinea cruris,and onychomycosis.Skin fungal diseases caused by T.rubrum are difficult to cure and prone to recurrence,making them almost a chronic infectious disease.However,the drugs currently used to treat this disease in clinical practice have side effects or are ineffective,which makes eradicating skin fungal diseases caused by T.rubrum a major challenge.Therefore,finding new anti-T.rubrum drugs is of great significance.A newly synthesized compound,hemiprotonic phenanthroline-phenanthroline⁺（ph-ph⁺）,has broad-spectrum antibacterial effects.Previous studies have shown that ph-ph⁺has a good killing effect on Candida albicans and has no significant toxic side effects on animals or irritating effects on the skin.It is expected to be developed as a potential drug for the treatment of skin fungal diseases.Based on this,we intends to investigate the mechanism of ph-ph⁺inhibiting T.rubrum,with the aim of developing a new potential drug that can completely eradicate skin diseases caused by T.rubrum and elucidating the signaling pathway through which ph-ph⁺inhibits T.rubrum.Firstly,the in vitro antibacterial effect of ph-ph⁺on T.rubrum was preliminarily explored using the microbroth dilution method,paper disk method,and modified agar diffusion method.The results showed that the MIC of ph-ph⁺against T.rubrum was 2μg/m L,and the MFC was 8μg/m L.The inhibition rates of ph-ph⁺on T.rubrum conidia were 27.45%and 41.66%,respectively,while the inhibition rates on T.rubrum mycelia were 21.18%and 79.05%,respectively,indicating that ph-ph⁺has different inhibitory effects on T.rubrum mycelia and conidia.Ph-ph⁺can inhibit the germination of T.rubrum conidia into mycelia.The germination rates of T.rubrum conidia at MIC and MFC were45.07%and 11.46%,respectively.The effects of ph-ph⁺on the growth of conidia and mycelia at low and human body temperatures were evaluated,and the results showed that ph-ph⁺can inhibit the growth of mycelia and conidia in a dose-dependent manner at both4℃and 37℃.Subsequently,the morphology of fungal cells,changes in transcription factors,and changes in biochemical indicators were analyzed by transmission electron microscopy,transcriptomics,nail modeling,and detection of biochemical indicators to elucidate the signaling pathways induced by these changes.Ph-ph⁺can effectively treat T.rubrum infection of nails.The mycelia of T.rubrum appeared to be broken and swollen after ph-ph⁺treatment.Ph-ph⁺induced cell death through ferroptosis,during which the cell wall and contents of the cell were separated,and the cell membrane ruptured.The transcriptomic results showed that a total of 97 genes were affected by ph-ph⁺,including65 downregulated genes and 32 upregulated genes.Among the upregulated genes,the Fre B gene encoding a transmembrane component of iron/copper reductase was the most prominent.Among the downregulated genes,the transcription of genes related to GSH synthesis was significantly reduced.After ph-ph⁺treatment,the Fe²⁺and Fe³⁺ions,reactive oxygen species（ROS）,and malondialdehyde（MDA）content in T.rubrum increased significantly,indicating that oxidative stress occurred inside the cells.Glutathione（GSH）,glutathione peroxidase（GSH-Px）,mitochondrial membrane potential（MMP）,mitochondrial respiratory chain activity,and ATP content all decreased,indicating that energy supply inside the cells was insufficient.The results of various experiments proved that ph-ph⁺killed fungi by inducing T.rubrum ferroptosis.As T.rubrum is the pathogen of dermatophytosis,ph-ph⁺was made into a gel formulation for topical application.The orthogonal design method was used to optimize the gel formulation,and the performance of the ph-ph⁺gel was tested.The optimal formulation of ph-ph⁺gel was 0.16%ph-ph⁺,0.80%carbomer 981,15.00%propylene glycol,2.50%lauryl pyrrolidone,and 1.08%triethanolamine.Ph-ph⁺gel could maintain stable viscosity and drug activity at different temperatures,with a drug loading capacity of 98.27%and a skin penetration rate of 55.50%.Finally,guinea pigs were used as experimental animals,and T.rubrum was used to infect the skin on their backs.The success of the T.rubrum-infected guinea pig model was determined by the presence of T.rubrum hyphae in the scales.After successful modeling,the scales were cultured and observed on the 1st,3rd,and 7th days after treatment.The results showed that the therapeutic effect of ph-ph⁺gel was similar to that of the positive control drug terbinafine gel.On the 7th day,the skin of the infected area of the guinea pig was cut and stained with HE and PAS.The results showed that T.rubrum could invade the epidermis and dermis of the guinea pig skin,causing inflammation,thickening of the stratum corneum,and dilation and fusion of hair follicles.After treatment with ph-ph⁺gel,the skin structure was even,the degree of tissue damage was reduced,the degree of keratinization in the epidermis was normal,and there was no dilation and fusion of hair follicles,indicating that the skin was restored.In conclusion,we investigated the antifungal activity and mechanism of ph-ph⁺on T.rubrum,developed a new potential drug for the treatment of dermatophytosis caused by T.rubrum,and elucidated the signal pathway of ph-ph⁺inhibition of T.rubrum.This study provide a new treatment for T.rubrum-induced diseases,making it possible to completely cure skin diseases caused by T.rubrum.At the same time,it provides a different signal pathway for the mechanism of drugs acting on fungi from traditional drugs that cause fungal death,and is expected to verify the iron apoptosis-related mechanism in the fungal field.