Therapeutic Applications Of Honokiol On Aspergillus Fumigatus Keratitis

PURPOSEWe characterized the effects of Honokiol?HNK?on keratomycosis caused by Aspergillus fumigatus and the underlying mechanisms.HNK is known to have anti-inflammatory and anti-fungal properties,but the influence on fungal keratitis remains unknown.METHODS1.Cell Count Kit-8?CCK-8?assay was carried out spectrophotometrically to detect the effect of HNK on the proliferation capacity of human corneal epithelial cells?HCECs?to provide reliable concentration without cytotoxicity,and Cell Scratch Test was used to illustrated further the re-epithelization potentiality of HNK on HCECs.2.Minimum inhibitory concentration?MIC?was determined for A.fumigatus,and Time kill assay pointed that HNK was fungicidal and fungistatic in a concentration-or time-dependent manner.3.Adherence assay was stained by HE staining to demonstrate the effect of HNK on the adherence stage of A.fumigatus,and crystal violet staining was to determined the capacity of inhibating biofilm formation.4.Membrane permeability assay was tested the HNK effects on the cell membrane integrity.5.C57BL/6 mice were infected with A.fumigatus to establish animal models of fungal keratitis.Clinical scores reflected the improvement degree of keratitis outcome after treatment.6.Plate count was used to evaluate the effect of HNK on the residual amount of fungi in Aspergillus fumigatus keratitis in mice.7.RT-PCR?Western Blot?ELISA were used to detect the expression of related pattern recognition receptors?PRRs?and inflammatory factors in fungal keratitis quantitatively.8.Myeloperoxidase assay,flow cytometry and IFS were done to evaluate neutrophil infiltration.RESULTS1.HNK had no significant cytotoxic effect on proliferation at 0-8 ?g/m L for 24 h in CCK-8 test and does not affect cell migration compared with control group in scratch-wound assay.2.HNK MIC₉₀ was 8 ?g/m L and MIC₉₉/MFC was 12?g/m L for A.fumigatus.HNK played the fungistatic and fungicidal roles at 6 h and 24 h respectively.3.HNK?8 ?g/m L?was a safe and effective concentration for A.fumigatus,inhibiting adherence at the beginning,diminishing biofilms formation.4.HNK killed A.fumigatus by increasing membrane permeability.5.HNK improved C57BL/6 mice keratitis outcome by reducing clinical scores including three aspects,opacity density,opacity area and surface regularity after 3 days treatment and improved obviously after 5 days treatment.6.After HNK treatment,the residual amount of A.fumigatus in fungal keratitis in mice was significantly reduced compared with the PBS group.7.Consistent with the disease severity,HNK downregulated m RNA and protein expression levels of TLR-2,HMGB1,IL-1? and TNF-? in keratitis-related molecular mechanisms.8.In terms of neutrophil infiltration?MPO,flow cytometry and IFS?,HNK played a vital role in anti-inflammation.The outcome of flow cytometry showed that HNK significantly reduced the number of neutrophils involved in the inflammatory response and the proportion of neutrophils in the inflammatory cells in the mouse keratitis models.We could see from the pictures of immunofluorescence staining that HNK obviously reduced the neutrophils accumulation,corneal edema and structural disorder.MPO was significantly inhibited in HNK-treated group.CONCLUSIONOur study suggested HNK played anti-fungal and anti-inflammatory roles on keratomycosis by reducing survival of fungi,infiltration of neutrophils and expression of TLR-2,HMGB1 and proinflammatory cytokines,providing a potential therapeutic treatment for fungal keratitis.