Experimental Study On The Suppression Of Inflammation By Celastrol In Fungal Keratitis In Mice Through LOX-1-p38MAPK Pathway

Purpose: To explore the role of Celastrol(CLT)in the suppression of inflammation and mechanism in Aspergillus fumigatus(A.fumigatus)keratitis.Methods: 1.The left eyes of C57BL/6 mice were injected with CLT or DMSO(control)subconjunctivally at 1 day post infection.The right eyes of C57BL/6 mice are the normal group.Eyes of C57BL/6 mice were treated tropically with 3 ?l of CLT or DMSO(control)twice daily from 2 days post infection.Disease severity of A.fumigatus keratitis mice corneas was recorded by clinical score and photographs with a slit lamp at 1day,3 days and 5 days post infection.The infiltration of inflammatory cells in different treatment groups was compared by hematoxylin and eosin(H&E)staining at 3 days after infection.2.The m RNA and protein levels of pro-inflammatory mediators,anti-inflammatory mediators and LOX-1 in the corneas infected with A.fumigatus in CLT or DMSO treatment group were tested by q RT-PCR,ELISA and Western blot.3.Divided into different groups of cell experiments according to the different pretreatment and with or without stimulation of hyphae.PCR and ELISA were used to detect the m RNA and protein levels of IL-1?,TNF-? and MIP-2 in different groups.The m RNA and protein levels of IL-10 and TGF-? in different groups were tested by q RT-PCR,ELISA and Western blot.4.The m RNA levels of LOX-1 in macrophages of different treatment groups were detected by q RT-PCR.The protein levels of LOX-1,p38 MAPK and p-p38 MAPK in macrophages of different treatment groups were detected by Western blot.Results: 1.Compared with the DMSO-treated group,CLT treatment significantly decreased clinical scores of corneas infected with A.fumigatus at 3 and 5 days post infection.CLT treatment markedly restrained the infiltration of inflammatory cells in the cornea infected with A.fumigatus.2.Pro-inflammatory mediators,anti-inflammatory mediators and LOX-1 m RNA was declined in the CLT treatment group compared with DMSO treatment group.3.Compared with DMSO pretreatment,the m RNA expression levels of IL-1?,TNF-? and MIP-2 and protein expression levels of IL-1? and TNF-? were decreased significantly in CLT-treated group of microphages stimulated with A.fumigatus.Anti-inflammatory effect of CLT was concentration-dependent.CLT pretreatment obviously decreased m RNA expression levels of IL-10 and TGF-? and protein expression levels of IL-10 in macrophages stimulated with A.fumigatus compared with DMSO pretreatment.4.CLT pretreatment markedly inhibited m RNA expression levels of LOX-1 and p-p38 MAPK protein expression in macrophages stimulated with A.fumigatus compared with DMSO pretreatment.No differences of protein levels of p38 MAPK were detected between two groups.Conclusion: Compared with DMSO treatment,CLT significantly suppressed inflammation levels of corneas infected with A.fumigatus and reduced the level of inflammation in primary peritoneal macrophages of mice stimulated with A.fumigatus.CLT ameliorates A.fumigatus keratitis via downregulating inflammatory cytokines through LOX-1-p38 MAPK pathway.