Study On The Therapeutic Effect And Mechanism Of Baicalein In Aspergillus Fumigatus Keratitis

Objective:To investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus(A.fumigatus)keratitis and the underlying mechanisms.Methods:1.The non-cytotoxic antifungal concentration of baicalein was determined using CCK8,cell scratch assay,minimum inhibitory concentration and Draize test in vitro in vivo.2.Biofilm formation,scanning electron microscopy,propidium iodide uptake test calcofluor white stain and adherence assay were used to evaluate the antifungal effect of baicalein.3.In fungal keratitis(FK)mouse models,clinical score and plate count were used to evaluate FK severity,and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity.PCR,ELISA and Western blot were performed to examine TSLP,TSLPR,IL-1β,IL-6 and TNF-α in the cornea of infected C57BL/6 mice to analyze the anti-inflammatory ability of baicalein.4.Human corneal epithelial cells(HCECs)were first stimulated by A.fumigatus for 1 hour,and then treated with 0.25 m M baicalein or DMSO for 3 hours or 24 hours.PCR,ELISA or Western blot were used to detect the expression of TSLP and inflammatory factors in each group of cells.The secretion and localization of TSLP in HCEC were detected by cell immunofluorescence staining.5.HCECs were pretreated with TSLP small interfering RNA(TSLP si RNA)or human recombinant protein TSLP(rTSLP)and then stimulated with A.fumigatus mycelia for 1h,followed by baicalein for 3 h or 24 h.The expressions of TSLP and inflammatory factors in each group were detected by PCR,ELISA or Western blot.The role of baicalein and TSLP in fungal keratitis was evaluated to explore the antiinflammatory activity of baicalein and underlying mechanisms in vivo and in vitro.Results:1.Baicalein at 0.25 m M(non-cytotoxic)significantly inhibited A.fumigatus growth,biofilm formation and adhesion in vitro.2.In A.fumigatus keratitis mice,compared with DMSO control group,the corneal inflammation response of mice at 1,3,and 5 days after treatment with 0.25 m M baicalein was significantly reduced,and the clinical score was significantly reduced;At 3 days after infection,the number of neutrophils in the stromal layer and epithelial layer of cornea were significantly reduced in the baicalein treatment group.After 5 days of infection,the fungal load of cornea was significantly decreased in the baicalein treatment group.3.Compared with DMSO control group,the m RNA levels of TSLP and TSLR,IL-1β,IL-6 and TNF-α were decreased in baicalein-treated cornea at1,3 and 5 days of infection,and the protein expressions of TSLP,TSLPR,IL-1β,IL-6 and TNF-α were decreased at 3 and 5 days of infection.4.Baicalein could significantly inhibit the m RNA and protein expressions of IL-1β,IL-6 and TNF-α induced by HCEC after stimulation by A.fumigatus.5.The expression of TSLP in HCEC was increased after stimulation by A.fumigatus.Baicalein inhibited the secretion of TSLP in HCEC by inhibiting the expression of TSLP m RNA and protein.6.In HCECs,the m RNA and protein levels of TSLP,IL-1β,IL-6 and TNF-α in TSLP si RNA treated group while higher in rTSLP treated group than in the corresponding control.Baicalein treatment significantly inhibited the expression of TSLP,IL-1β,IL-6 and TNF-α induced by rTSLP.Conclusion:Baicalein plays a protective role in mouse A.fumigatus keratitis by inhibiting fungal growth,biofilm formation and adhesion,and suppressing inflammatory response via downregulation of TSLP/TSLPR pathway.