Therapeutic Effect Of Thymol In Mice With Aspergillus Fumigatus Keratitis

Purpose: This study explores the anti-inflammatory effects and mechanisms of thymol in Aspergillus fumigatus keratitis.Methods: 1.The minimum inhibitory concentration(MIC)test,mycelial cell wall fluorescent staining test,cytotoxicity(CCK-8)test,cell scratch test and corneal fluorescein staining test were used to determine the safe and effective concentration of thymol in the treatment of Aspergillus fumigatus keratitis.2.The experimental mice were equally divided into four groups.The method of intra-matrix injection was used to establish fungal keratitis models,then thymol or dimethyl sulfoxide(DMSO)were injected under the subconjunctiva before and after infection.Mouse corneas were observed under a slit-lamp microscope and clinical scores were recorded.Mouse corneas were collected for colony-plate counting test.3.The recruitment of inflammatory cells within the corneal stroma was determined by immunofluorescence,hematoxylin-eosin(HE)staining,and myeloperoxidase(MPO)methods.4.Western blot and qRT-PCR experiments were used to detect the mRNA and protein expression of TLR4,MyD88,NF-kB,IL-1,NLRP3,caspase-1,caspase-8,GSDMD,RIPK3,and MLKL.5.The experimental mice were equally divided into three groups: fungal keratitis group,thymol-treated fungal keratitis group,and natamycin-treated fungal keratitis group.Infection of the mouse cornea was observed under a slit-lamp microscope and the clinical score was recorded to compare the therapeutic effects of thymol and natamycin.Then mouse corneas were collected for colony-plate counting test.Results: 1.The concentration of thymol significantly inhibited the mycelium growth of A.fumigatus starting from 50 μg/ml.The cell growth of HCECs cultured at 50 μg/ml thymol was not significantly inhibited.Cell scratches were healed in 50 μg/ml thymol medium.Corneal fluorescein staining was performed after 50 μg/ml thymol drug instillation,and no obvious yellow-green fluorescent staining was found in the cornea.2.It was observed under the slit lamp microscope that thymol-treated fungal keratitis group had milder corneal inflammation and lower clinical scores.Plate counting test after 3 days infection showed a significantly reduced fungal load in corneal tissue after thymol treatment.3.Immunofluorescence staining and HE staining tests demonstrated that macrophages and neutrophils were significantly reduced in mice corneal tissues after 3days of thymol treatment.MPO test also demonstrated that thymol can reduce myeloperoxidase activity in neutrophils.4.The qRT-PCR and Western blot experiments showed that TLR4,MyD88,NF-kB,and IL-1β expression were downregulated in Aspergillus fumigatus keratitis mouse after 1 day of thymol treatment.5.The qRT-PCR and Western blot experiments showed thymol treatment significantly down-regulated the pyroptosis-associated mRNA and protein expressions of NLRP3,caspase-1,caspase-8and GSDMD in Aspergillus fumigatus keratitis mouse,as well as down-regulated p17IL-1β protein.6.The qRT-PCR and Western blot experiments showed thymol treatment also down-regulated the necroptosis-associated mRNA and protein expressions of RIPK3 and MLKL in Aspergillus fumigatus keratitis mouse.7.Under the slit lamp microscope,the thymol-treated fungal keratitis group had milder corneal inflammation and lower clinical scores than the natamycin-treated fungal keratitis group.Colonies were significantly reduced in both groups,but no significant difference was found.Conclusion: Thymol inhibited the growth of Aspergillus fumigatus mycelium.In addition,thymol played a protective role in A.fumigatus keratitis by reducing inflammatory cells recruitment,inhibiting the TLR4/MyD88/NF-kB/IL-1β signaling pathway and the maturation and release of IL-1β by reducing the pyroptosis and necroptosis.