Anti-inflammatory Effects Of Astaxanthin Against Fungal Keratitis

Purpose:Fungal keratitis is a major cause of corneal ulcers,resulting in significant visual impairment and blindness.ASX,a lipid-soluble keto-carotenoid,has been shown anti-inflammatory effect in a variety of diseases.In this study,we aimed to characterize the anti-inflammatory effect of ASX in fungal keratitis induced by Aspergillus fumigatus.Method:In order to investigate the effect of ASX against fungal keratitis,we carried out experiments in vivo and in vitro.In vivo,fungal keratitis mouse model was established in C57BL/6 mice using A.fumigatus,C57BL/6 mice in 8 weeks were anesthetized by intraperitoneal injection of 8%chloral hydrate?0.4ml/kg?under the stereomicroscope,A.fumigatus suspension solution was injected into the right cornea intrastromally.Mice were randomly divided into two groups,ASX treated group and DMSO treated group.The corresponding eye drop was performed 3 times a day.The mice were anesthetized at different time points required by different experiment and then the slit lamp was used to take photos of the cornea of the mice and scored the degree of inflammation.The mice were killed by cervical dislocation method after anesthesia at different time points required by each experiment.The expression levels of inflammatory cytokines?IL-1?,TNF-?,HMGB-1?,were detected by Real-Time reverse transcription-PCR?Real-Time RT-PCR?and Enzyme Linked Immunosorbent Assay?ELISA?,Real-Time RT-PCR and Western blot?WB?was used to detect Toll-like Receptors 2?Toll-like Receptors 4?TLR2?TLR4?.Immunofluorescence?IF?was used to detect neutrophils,macrophage and TLR2?TLR4.In vitro,6 groups were divided:Normal group;DMSO group;ASX treatment group;A.fumigatus group;A.fumigatus+DMSO group;A.fumigatus+ASX group.When Raw264.7 cells grow evenly and achieve about 80%fusion,DMSO and ASX were added to the corresponding groups,After the cells were acclimated for 2 hours,the corresponding groups were added with the inactivated stimulation liquid of A.fumigatus?5×10⁶ cfu/ml?.After 8 hours of stimulation,Raw264.7 cells in plate were collected to detect the m RNA of downstream proinflammatory cytokines?IL-1?,TNF-?,HMGB-1?,TLR2,TLR4 by Real-Time RT-PCR.Results:1.On the 3rd day after infection,the clinical manifestations of cornea in ASX treated group were better than that in DMSO group,and the clinical inflammation scores on the 1^st,3^thh and 5^thh after infection were lower than those in DMSO group.The MPO activity in ASX treated group was significantly lower than that in DMSO group on the 3rd day after infection.2.The result of IF showed that compared with DMSO group,the number of neutrophils and macrophages in ASX treated group decreased significantly on the 3^rdd day after infection.The expression of IL-1?,TNF-?,HMGB-1 in ASX treated group was significantly lower than that in DMSO treated group on the 1st,3rd and 5th day.In addition,the results of ELISA showed that the expression level of TNF-?,IL-1?and HMGB-1 in ASX treated group was lower than that in DMSO treated group on the 3rd day after modeling.In vitro,the mRNA and protein expression of IL-1?TNF-?and HMGB-1in ASX pretreated cells was lower than that in DMSO treated cells.3.Compared with DMSO treatment group,the relative mRNA level of TLR2,TLR4in ASX treatment group decreased significantly on the 1^st,3^rdd and 5^thh day after modeling.The result of Western blot showed that the expression of TLR2,TLR4 was up-regulated after A.fumigatus infection on the 3^rdd and 5^thh day,while ASX treatment significantly inhibited the up-regulation.The results of IF showed that the expression of TLR2,TLR4in ASX treated group was significantly lower than that in DMSO treated group.In vitro,the mRNA and protein expression of TLR2,TLR4 in ASX pretreated cells was significantly lower than that in DMSO treated cells.Conclusion:ASX can effectively reduce the disease severity of fungal keratitis induced by A.fumigatus and reduce the clinical inflammatory score.ASX reduced the production and infiltration of neutrophils and macrophages,significantly reduced the expression of various inflammatory factors?TNF-?,IL-1?and HMGB-1?and reduced the inflammatory response.The anti-inflammatory effect of ASX is closely related to TLR2and TLR4.In conclusion,ASX can be used as a potential treatment for fungal keratitis.