| Purpose: To study the therapeutic effect of perillaldehyde in corneal antifungal immunity and its possible molecular mechanism.Methods: 1.C57BL/6 mouse cornea was infected by Aspergillus fumigatus,a mouse fungal keratitis animal model was established,and it was randomly divided into a solvent control group and a perillaldehyde-treated group.The mice were injected with 6 m M perillaldehyde 5ul under the conjunctiva and treated with the same concentration of perillaldehyde three times a day.The condition of the cornea in each group at different modeling time points was recorded with a slit lamp.And corneal clinical scores of mice in the drug-treated group.Colony counting was used to detect the number of viable colonies in the cornea of mice treated with perillaldehyde and the solvent control group.MPO and immunofluorescence were used to detect the effect of perillaldehyde on corneal neutrophil recruitment in Aspergillus fumigatus-infected mice.Real-time PCR and Western blot were used to detect the proinflammatory factors IL-1β,IL-6,TNF-α,and pathway proteins Nrf2,HO in the cornea of mice in the injury control group,solvent control group,and perillaldehyde-treated group,respectively.-1 and Dectin-1 expression.2.Pretreatment of immortalized human corneal epithelial cells with different concentrations of perillaldehyde(100u M,200 u M,400 u M,600 u M)for 2 hours,and then stimulated with Aspergillus fumigatus hyphae.Real-time PCR was used to detect human cornea 8h after infection in each group.Gene expressions of pro-inflammatory factors IL-1β,IL-6,IL-8,TNF-α and pathway proteins Nrf2,HO-1,Dectin-1 in epithelial cells.Human corneal epithelium was detected 24 h after infection in each group by ELISA.The protein expressions of proinflammatory factors IL-6,IL-8 and TNF-α were detected in the cells.The protein expressions of Nrf2 and HO-1 at 8 h after infection were detected by Western blot.Nrf2 expression and nuclear transfer were detected by immunofluorescence.3.After pretreatment with immortalized human corneal epithelial cells by pretreatment with perillaldehyde(200u M,400 u M)at different concentrations for 2h,human corneal epithelial cells were stimulated with Dectin-1 ligand curdlan(Curdlan)for 8 h,and detected by Realtime PCR.Gene expression of human corneal epithelial cells proinflammatory factors IL-6,IL-8 and TNF-α in each group,and the proinflammatory factors IL-6,IL-8 and TNF-α protein expression,Western blot was used to detect the protein expression of Dectin-1 in human corneal epithelial cells 24 h after infection.4.Pretreatment of immortalized human corneal epithelial cells with Nrf2,HO-1 inhibitors Brusatol(BT)and Zn PP,followed by treatment with 600 u M perillaldehyde for 2h,and aspergillus fumigatus stimulated human corneal epithelial cells for 8h and 24 h,Real-time PCR and ELISA were used to detect the m RNA and protein expressions of IL-6,IL-8,and TNF-α in human corneal epithelial cells of each group,and Nrf2 and HO of human corneal epithelial cells were detected by Western blot in protein expression.5.Co-cultivate different concentrations of perillaldehyde(0.2m M,0.6m M,0.8m M,1.0m M,1.2m M,1.8m M)with Aspergillus fumigatus spores,solvent control and Aspergillus fumigatus spores in a liquid agar medium for 24 hours After 48 h,72h,96 h,and 120 h,the suspension of each group at each time point was added to a 96-well plate to measure the absorbance at 540 n M,and the mycelial growth of each group was observed by calcium fluorescence staining method.Results: 1.Compared with the solvent control group,the degree of corneal turbidity,the ulcer area,the clinical score of corneal inflammation and the corneal colony count were significantly reduced in the perillaldehyde treated group.The MPO level of infected cornea in the Perilla group was significantly lower than that in the solvent control group,and corneal immunofluorescence showed that perillaldehyde could reduce the recruitment of neutrophils in the corneal matrix caused by Aspergillus fumigatus infection.2.Aspergillus fumigatus increased the m RNA level and protein level of proinflammatory cytokines in human corneal epithelial cells,and the perillaldehyde pretreatment group could significantly inhibit the expression level of these cytokines in a concentration-dependent manner.3.In the cornea of mice after 3 and 5 days of fungal infection,the m RNA and protein levels of IL-1 β,IL-6 and TNF-α in the perillaldehyde treated group were significantly lower than those in the solvent control group.4 in the biochemical,corneal epithelium cells,the pure Perilla aldehyde groups in Nrf2,HO-1 m RNA and protein levels,Aspergillus fumigatus stimulation group corneal epithelium cells Nrf2,HO-1 m RNA and protein levels higher than normal control group,and perillaldehyde pretreatment to further increase the Aspergillus fumigatus induction of Nrf2,HO-1 m RNA and protein levels;In the cornea of mice infected with Aspergillus fumigatus for 5 days,the fungus increased the m RNA and protein levels of Nrf2 and HO-1,and the perillaldehyde treated group further increased the m RNA and protein levels of Nrf2 and HO-1 induced by Aspergillus fumigatus.Immunofluorescence results showed that perillaldehyde promoted the transfer of Nrf2 from cytoplasm to nucleus in human corneal epithelial cells.5.The corneal epithelial cells were pretreated with BT(Nrf2 inhibitor)and Zn PP(ho-1 inhibitor).The levels of Nrf2 and HO-1 proteins were decreased compared with the normal control group,and the expression levels of perillaldehyde-induced Nrf2 and HO-1 were down-regulated.The m RNA and protein levels of proinflammatory factors IL-6,IL-8 and TNF-α in cells were increased by Aspergillus fumigatus stimulation,and the expression of these inflammatory factors was significantly decreased in the perillaldehyde pretreatment group,while the m RNA and protein levels of these inflammatory factors inhibited by perillaldehyde were partially upregulated in the BT and Zn PP pretreatment groups.6.Aspergillus fumigatus infection significantly increased the m RNA and protein levels of Dectin-1 in the cornea of mice compared with the control group,while the expression of Dectin-1 in the cornea of infected mice treated with perillaldehyde significantly decreased compared with the control group.The m RNA level of Dectin-1 increased after the stimulation of human corneal epithelial cells by Aspergillus fumigatus alone,and the m RNA level of Dectin-1 induced by the stimulation of Aspergillus fumigatus was significantly decreased by perillaldehyde pretreatment.The protein level of Dectin-1 in corneal epithelial cells in the curdlanpretreated group was higher than that in the normal control group,while the protein level of Dectin-1 induced by curdlan-pretreated perillaldehyde could be significantly reduced.Curdlan significantly promoted the m RNA and protein levels of proinflammatory cytokines IL-6,IL-8 and TNF-α in human corneal epithelial cells,while the m RNA and protein levels of these cytokines in perillaldehyde pretreated human corneal epithelial cells were significantly lower than those in the curdlan-stimulated group,showing a concentration dependence.7.Perillaldehyde and Aspergillus fumigatus spores in vitro AGAR medium in liquid trained after 48 h,0.6 m M Perillaldehyde treatment group compared with solvent control group of 540 nm absorbance significantly reduced,and with the increase of drug concentration and absorbance value gradually reduce,calcium fluorescence staining showed with the increase of concentration of perillaldehyde,the number of fungal hyphae and spores gradually reduce,when the concentration of perillaldehyde reaches 1.8 m M,not observed in the presence of fungal hyphae and spores.Conclusion: Perillaldehyde have anti-inflammatory and fungicidal effects in fungal keratitis.Perillaldehyde can reduce the inflammatory response of fungal keratitis by activating the Nrf2/HO-1 signaling pathway,inhibiting the inflammatory response mediated by Dectin-1,and reducing the infiltration of neutrophils during fungal infection.In addition,perillaldehyde can reduce the corneal tissue damage caused by Aspergillus fumigatus through inhibiting the growth of Aspergillus fumigatus. |
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