Antifungal Activity And Potential Mechanism Of Agrimonolide-6-O-β-D-Glucopyranoside Combined With Fluconazole Against Candida Albicans

Background:The number of systemic fungal infections has increased significantly over the past few decades,with Candida albicans（C.albicans）as the primary pathogen,causing approximately 400,000 systemic fungal infections per year with a mortality rate of 40-60%.Current treatment strategies for fungal infections are hampered by the limited availability of antifungal drugs and the emergence of drug resistance.In recent years,combination drug delivery has been widely studied and applied,and specific drug combinations can overcome existing drug resistance and/or delay or prevent the emergence of further resistance through multiple modalities and are an effective way to address and treat drug-resistant fungal infections.Chinese medicines are commonly used in the treatment of diseases and have the advantages of mild action,multiple routes of action,multiple targets,multiple components,and few adverse effects.In recent years,with the widespread application of Chinese herbal medicine worldwide,research on single herbs,Chinese herbal compound,Chinese herbal preparations,extracts and compounds of Chinese herbal origin used alone or in synergy with commonly used antifungal drugs for the treatment of fungal infections have received increasing attention from researchers and have yielded some results.Objective:The aim of this study was to explore the in vitro and in vivo effects of AG-6 in combination with FLC on drug-resistant C.albicans and to investigate its potential synergistic antifungal mechanism.Methods:1.The antifungal activity of different Agrimoniae Herba extracts alone or in combination with clinically used antifungal drug FLC against clinical FLC-resistant C.albicans isolate CA23 and FLC-susceptible C.albicans strain SC5314 were screened by microdilution method,and the compounds or compound groups with better activity were selected to optimize the optimal concentration or concentration groups,and then the activity was verified against different C.albicans strains.The dynamic antifungal activity of the combined drug groups was evaluated using time-killing curves.2.Label Free quantitative proteomics techniques were used to analyze the effect of AG-6+FLC combination on the differential expression of protein levels in C.albicans and the related pathways.3.OCR was measured by Seahorse XF Cell Mito Stress Test kit（Agilent Technologies）and ECAR was detected using Seahorse XF Glycolysis Stress Test kit（Agilent Technologies）according to XF24 Seahorse Extracellular Flux Analyzer method.Further,to reflect mitochondrial function,intracellular levels of reactive oxygen species（ROS）were detected using a reactive oxygen assay kit,mitochondrial membrane potential changes were detected with a JC-1 mitochondrial membrane potential detection kit,and ATP content was determined using an ATP assay kit.4.The effect of AG-6+FLC combination on the ergosterol content of C.albicans was detected by UV spectral scanning;the effect of AG-6+FLC combination on the cell membrane morphology of C.albicans was observed by transmission electron microscopy;the permeability of cell membrane was analyzed by PI staining;the integrity of the cell membrane of C.albicans after drug treatment was analyzed by AO/EB double staining.5.The effect of AG-6 in combination with FLC on the morphological transformation of C.albicans was observed by an inverted microscopy and scanning electron microscopy,and the effect of AG-6 in combination with FLC on the biofilm formation of C.albicans at different stages was observed by an inverted microscopy,and the expression of related genes was detected by q PCR.6.The in vivo anti-C.albicans effect of AG-6+FLC combination was evaluated by survival rate,fungal load,and histopathology of tissues by using the Galleria mellonella larva infection model and a mouse model of systemic infection.Results:1.MIC₈₀was performed by microdilution method,and the results showed that compounds C070003,C070006,C070017,and C070018 combined with FLC all had good synergistic inhibition activity against CA23 with MIC₈₀of 21.16～89.16μg/m L and FICI of 0.10～0.45μg/m L;and compound C070006（AG-6）combined with FLC had the best combined inhibition effect,so C070006（AG-6）was selected for the follow-up experiments.The concentration of AG-6+FLC combination was selected,and it was found that the ratio of AG-6 to FLC was 1:5,which could reduce the amount of FLC and also reflect the efficacy of AG-6 to the greatest extent,and then the combination of this concentration was used to screen the antifungal activity of different C.albicans,and the results showed that the combination of AG-6+FLC had synergistic antifungal activity against different C.albicans.Considering the follow-up mechanism research,ATCC14053 was selected.The results of time-killing curves also indicated that the combination of AG-6+FLC had dynamic anti-C.albicans activity against ATCC14053.2.Using proteomic techniques,the results showed that the combination of AG-6+FLC treatment of C.albicans resulted in significant changes in important pathways such as fructose and mannose metabolism,glycolysis/gluconeogenesis,and steroid metabolism by analyzing the differential protein KEGG pathway,suggesting that the combination of AG-6+FLC may exert anti-C.albicans effects by affecting these metabolic pathways in C.albicans.3.Using the Agilent XF24 Seahorse Extracellular Flux Analyzer,The results showed that AG-6+FLC combination inhibited glycolysis and increased basal oxygen consumption.And then q PCR was performed to verify the expression levels of three key enzymes in the glycolytic pathway（hexokinase,6-phosphofructokinase,pyruvate kinase）,and the results showed that the expression levels of HXK1,HXK2,FBA1,CDC19,TPI1,and FBP1 genes were significantly downregulated,which also confirmed that AG-6+FLC combination inhibited the genes related to the glycolytic pathway.The results showed that AG-6+FLC combination increased intracellular ROS levels,decreased mitochondrial membrane potential as well as intracellular ATP content in C.albicans,indicating that AG-6+FLC combination impaired mitochondrial function in C.albicans.4.Using UV spectroscopy,the results showed that the treatment of C.albicans with AG-6+FLC reduced the synthesis of ergosterol in the cell membrane of C.albicans.By transmission electron microscopy,it was also observed that after the treatment of C.albicans with AG-6+FLC,the discontinuity of the cell membrane,the damage of membrane structure,the disorder of intracellular material and the partial leakage of material were observed in C.albicans under the lens.When the cell membrane permeability was analyzed by PI staining,it was also observed that almost all of the combined AG-6+FLC group was stained red and the fluorescence intensity was significantly enhanced.The integrity of the cell membrane of C.albicans after drug treatment was also analyzed by AO/EB double staining,and the same results were observed.It was suggested that the combination of AG-6+FLC could cause damage to the cell membrane of C.albicans.5.The results showed that the combination of AG-6+FLC was able to inhibit the morphogenetic switching between the hyphal and yeast forms induced by Spider and SD+10%FBS by inverted microscopy and scanning electron microscopy on the formation of ATCC14053 hyphal;the effect of AG-6+FLC combination on the biofilm formation of C.albicans at different stages was observed by inverted microscopy.The results showed that the combination of AG-6+FLC could inhibit the biofilm formation of C.albicans at different stages.The results showed that AG-6+FLC combination significantly down-regulated the expression of RAS1,CDC24,CDC42,CST20,STE11,HST7,CEK1and CPH1,suggesting that AG-6+FLC combination could inhibit the biofilm formation of C.albicans through cekl-MAPK.6.Using the Galleria mellonella larva C.albicans infection model and a mouse model of systemic C.albicans infection.The in vivo anti-C.albicans effects of AG-6+FLC combination were evaluated by measuring the effects of AG-6+FLC combination on the survival rate of the Galleria mellonella larvae/mice,the fungal load of larvae/mice tissues,and the histopathology of larvae/mice.mice survival time,reduce the number of C.albicans in larval/mouse tissues,and decrease the degree of damage caused by C.albicans to larval/mouse tissues,suggesting that the combination of AG-6+FLC has good synergistic therapeutic effect on C.albicans in vivo in Galleria mellonella larvae/mice,and the results are consistent with in vitro experiments.Conclusion:The combination of AG-6 and FLC has synergistic anti-C.albicans effects in vitro and in vivo,which may exert synergistic inhibition through various molecular mechanisms such as inhibition of glucose catabolic energy production（both glycolysis and mitochondrial function）,thereby affecting the morphological transformation,biofilm formation capacity and cell membrane lipid synthesis of C.albicans.Our study suggests that energy metabolism may be the core molecular mechanism of synergistic bacterial inhibition by multi-target drug combination,providing new perspectives and effective solutions for clinical treatment of drug-resistant strains of infection.