The Function And Mechanism Of Sanguinarine Against Fungal Pathogens

ObjectivesInvasive fungal infections?IFIs?are life-threatening to immunocompromised individuals.Though the medical technology is improving,the morbidity and mortality of IFIs remain high.Common pathogens that cause IFIs include Candida spp.,Aspergillus spp.,and Cryptococcus spp.,among which Candida albicans is the predominant one.In addition to disseminated systemic infections,biofilms adhered to the surface of mucosa,solid organs,and medical devices are also common styles of candidiasis.Of particular concern is that biofilms display increased resistance to antifungal therapy,causing great difficulty in clinical treatments.There are still many other concerns for the treatment of IFIs in clinic.On the one hand,available treatment options are limited,and can cause severe side effects and high expenses.On the other hand,the long-term use of existing antifungal drugs has made drug resistance an unavoidable issue in IFIs treatments.Therefore,it's urgent to develop novel antifungal agents with high efficiency and low toxicity.Natural products are abundant resources for drug development.Our team have previously screened in natural products for small molecules with antifungal activities and found that sanguinarine?SAN?had potent antifungal effects.In further research a good antibiofilm effect of SAN was also revealed.The antibiofilm effective concentration is lower than the minimal inhibitory concentration?MIC?against planktonic C.albicans.In this study,we conducted a comprehensive investigation of SAN about its antifungal effects and revealed its dose-dependent mechanism.Methods and resultsWe evaluated the antifungal spectrum of SAN by broth microdilution test and found that SAN exhibited strong activity against common fungal pathogens including C.albicans,C.tropicalis,C.parapsilosis,C.krusei,C.glabrata,Cryptococcus neoformans,Aspergillus fumigatus,and Trichophyton Rubrum,with MIC₅₀ ranging from 0.4 to 6.4 mg/L.Particularly,MIC₅₀ of SAN against C.albicans is 3.2 mg/L.SAN also showed effect against fluconazole-resistant strains with similar MICs to susceptible strains.To validate the fungicidal effect of SAN,agar diffusion test and time-kill curve assay were conducted using C.albicans SC5314and C.neoformans H99.And the results indicated that SAN exhibited fungicidal effect in a dose-dependent manner,3.2 mg/L SAN exhibited strong fungicidal effect against C.albicans.Then we determined the post-antifungal effect?PAFE?of SAN and found it had prolonged PAFE with the increase of SAN concentration.We also evaluated the antibiofilm effect of SAN against C.albicans using XTT method.The results showed that SAN exhibited antibiofilm activity at lower concentrations than MIC₅₀?0.4-1.6 mg/L?.In addition,SAN showed dose-dependent effects against biofilms at both initial and mature stages.Further we examined the in vivo activity of SAN using a murine C.albicans infection model and the results showed that SAN protected the mice in a dose-dependent manner.1 mg/kg SAN significantly extended survival time and improved survival rate of infected mice.The toxicity of SAN against human umbilical vein endothelial cells was also assessed with MTT method.The data showed that SAN exhibited a relative low toxicity to mammal cells with IC₅₀ 7.8 mg/L,which was much higher than MIC₅₀ against C.albicans,indicating the specific activity of SAN against fungal cells.We explored the antibiofilm mechanism of SAN under low dosage??1.6 mg/L?and fungicidal mechanism under high dosage??3.2 mg/L?.In terms of antibiofilm mechanism,firstly,we test the influence of SAN on cell adhesion ability by measuring the cell surface hydrophobicity?CSH?of C.albicans after SAN treatment and on hyphal formation ability by observing hyphal growth in different hyphal-inducing media containing SAN.And the results indicated that SAN decreased CSH and blocked the hyphal growth of C.albicans in a dose-dependent manner.Secondly,real-time RT-PCR was performed to detect the expression of some well-known genes involved in adhesion and filamentation and found that genes involved in cAMP pathway such as ECE1,HWP1,HGC1,ALS3,and CYR1 were down-regulated after SAN treatment in C.albicans biofilms.Finally,we determined the endogenous cAMP level of C.albicans biofilms.The results showed the endogenous cAMP level of C.albicans treated with 1.6 mg/L SAN was significantly lower than that in control group,and exogenous cAMP rescued the morphogenesis defect caused by SAN.Those results indicated that the antibiofilm activity of SAN was associated with the down-regulation of cAMP pathway in C.albicans.As for fungicidal mechanism under high dosage of SAN,changes in gene expression profiles and protein profiles after SAN treatment was investigated using gene expression microarrays and isobaric tags for relative and absolute quantitation?iTRAQ?based proteomics analyses.The results showed that genes and proteins involved in oxidative stress were up-regulated significantly,indicating that SAN might cause oxidative stress in C.albicans cells.Therefore,DCFH-DA fluorescent probes were applied to detect the reactive oxygen species?ROS?level in C.albicans cells after SAN treatment.The results showed that SAN increased the intracellular ROS level significantly.And antioxidants rescued the growth defects caused by SAN in a dose-dependent manner.DNA damage in C.albicans was observed after SAN treatment using DAPI and TUNEL reagents,and the antioxidants mitigated those damage.We also investigated the apoptosis induction effect of SAN by Annexin-V-FITC and PI reagents.The results showed that SAN induced apoptosis in C.albicans and the antioxidants attenuated these effects.ConclusionsSAN had potent antifungal activities with a broad spectrum,and exhibited strong fungicidal and antibiofilm effects.SAN also remained effective in vivo and showed low toxicity to mammal cells.Collectively,SAN can be a good candidate in antifungal drug development to kill both resistant and non-resistant fungal pathogens.The antibiofilm activity of SAN under low dosage is attributed to the down-regulation of cAMP pathway and the fungicidal activity under high dosage might be associated with DNA damage and apoptosis induction via accumulation of intracellular ROS.