Study On The Therapeutic Effects And Mechanism Of Eugenol In Fungal Keratitis

Objective:The purpose of this study is to determine the therapeutic effects and mechanism of eugenol(EUG)in fungal keratitis(FK)caused by Aspergillus fumigatus(A.fumigatus)infection.Methods:1.The cytotoxicity of EUG on human corneal epithelial cells(HCECs)was evaluated by intracellular lactate dehydrogenase(LDH)assay experiments.The safety of EUG at therapeutic concentrations for fungal keratitis in C57BL/6 mice was evaluated by Draize experiments in vivo.2.The corneas of C57BL/6 mice were infected by A.fumigatus,and were given 160 μg/m L EUG solution or 0.05% DMSO solution respectively.The clinical manifestations of mice corneas after infection were observed,and the corneal inflammation scores were recorded.HE staining was used to detect the infiltration of inflammatory cells in the corneas treated with EUG or DMSO.The fungal load in mouse cornea was measured by corneal colony counting.The m RNA expressions of IL-1β,TNF-α,i NOS,Nrf2 and HO-1 in mouse cornea were detected by real-time fluorescence quantitative PCR.The protein expressions of IL-1β and TNF-α in mouse cornea were determined by ELISA.Protein expressions of Nrf2 and HO-1 in mouse cornea were detected by Western blot.3.HCECs were pretreated with EUG(80 μg/m L,160 μg/m L)or 0.05% DMSO solution for 1 hour,and then stimulated with inactivated A.fumigatus mycelium.The m RNA expressions of IL-1β,IL-6,IL-8,Nrf2 and HO-1 in HCECs were detected by real-time fluorescence quantitative PCR.The protein expressions of IL-1β,IL-6,Nrf2 and HO-1 were detected by Western blot.The protein expression of IL-8 was detected by ELISA.4.HCECs were pretreated with 15 n M brusatol(BT,an inhibitor of Nrf2)or 10 μM zinc protoporphyrin(Zn PP,an inhibitor of HO-1)respectively for 2 hours,followed by being pretreated with 160 μg/m L EUG solution for 1 hour.Then the cells were stimulated with inactivated A.fumigatus mycelium for 8 hours or 24 hours.Real-time fluorescence quantitative PCR,Western blot and ELISA were used to detect the expressions of IL-1β,IL-6 and IL-8 in HCECs.5.The inhibitory effect of eugenol on A.fumigatus growth was determined by minimum inhibitory concentration test and Calcofluor white staining.A.fumigatus biofilms were preformed and then incubated with 80,160,320 and 640 μg/m L EUG respectively for 24 hours.Crystal violet staining was used to measure the damage of fungal biofilm.The adhesion of A.fumigatus spores to HCEC surface was observed by HE staining.Sorbitol protection test was used to detect the damage of EUG to the fungal cell wall.Propidium iodide uptaking test was used to verify the damage of EUG to fungal cell membrane.Results:1.EUG solution below 320 μg/m L has no toxic effect on HCECs and mouse cornea.2.On the 3 days post A.fumigatus infection(p.i.),compared with the control group,mouse corneas treated with 160 μg/m L EUG showed decreased severity and reduced clinical inflammation score.The infiltration of inflammatory cells in the cornea treated with EUG was significantly reduced.The number of corneal fungal colonies in EUG treated mice decreased significantly.3.On the 3 days p.i.,the m RNA expressions of IL-1β,TNF-α and i NOS in corneas treated with EUG were decreased significantly,as well as the protein expressions of IL-1β and TNF-α.4.In HCECs stimulated by A.fumigatus,EUG pretreatment significantly reduced the expressions of the inflammatory factors.5.In mouse cornea and HCECs infected with A.fumigatus,EUG enhanced the expression levels of Nrf2 and HO-1.Compared with EUG pretreatment alone,the expressions of IL-1β,IL-6 and IL-8 were increased in HCECs pretreated with BT or Zn PP for 2 hours before EUG pretreatment.6.After 48 hours of co-culture with EUG,compared with the control group,EUG started inhibiting the growth of A.fumigatus from40 μg/m L,and its inhibitory effect was enhanced with the concentration increasing.160μg/m L EUG could inhibit the growth of A.fumigatus by 90%.In the presence of exogenous sorbitol,the concentration required for EUG to inhibit 90% of the fungal growth was 160 μg/m L.Compared with the control group,after 24 hours of treatment with EUG(80,160,320,640 μg/m L),the amount of fungal biofilm decreased significantly.In comparison to the control group,160 μg/m L EUG inhibited the adhesion of A.fumigatus spores to the surface of HCECs.Compared with the control group,EUG(80,160 μg/m L)significantly destroyed the integrity of fungal cell membrane.Conclusions:EUG can exert anti-inflammatory and antifungal activities in A.fumigatus keratitis.EUG can reduce the infiltration of inflammatory cells in mouse cornea,decrease the corneal fungal load,improving the prognosis of mouse fungal keratitis.EUG inhibits the expressions of inflammatory factors in cornea and HCECs infected with A.fumigatus by activating Nrf2/HO-1 signaling pathway.In addition,EUG can significantly inhibit the fungal growth and spore adhesion,and destroy the fungal biofilm.Its inhibitory effect on fungal growth is related to the destruction of cell membrane integrity.