| Objective:To study the therapeutic effect and potential mechanism of chondroitin sulfate in Aspergillus fumigatus keratitis in mice.Methods:1.The non-toxic concentration of CS to human corneal epithelial cells,RAW 264.7cells and mouse cornea was screened by Draize assay and cell counting kit(CCK-8).2.Cell proliferation assay and cell scratch assay were used to evaluate the effect of CS on the proliferation and migration of HCECs.3.In vitro,HE staining of Aspergillus fumigatus spores adhered to HCECs was performed to verify the inhibitory effect of CS on spore adhesion during Aspergillus fumigatus infection.RT-PCR,ELISA and Western Blot were used to detect the expression of TLR-4,TNF-α,IL-1β,IL-6,COX-2 and MIP-2at gene and protein levels,and immunofluorescence staining was used to evaluate TLR-4expression,and the anti-inflammatory effect of chondroitin sulfate in vitro was studied.4.Eyes of a mouse model of Aspergillus fumigatus keratitis was observed by taking pictures under a slit lamp and scored clinically.Assess the therapeutic effect of CS in vivo by plate count assay,myeloperoxidase(MPO).5.The mouse corneas were harvested 3 days after infection for immunofluorescence staining and hematoxylin-eosin(HE)staining to evaluate the effect of CS on neutrophil recruitment and TLR-4 expression.Quantification of mouse corneal neutrophils by flow cytometry.6.In vitro,RAW 264.7 cells were pretreated with TLR-4 si RNA and scrambled si RNA for 12 h and 48 h,respectively,followed by 1 h stimulation with Aspergillus fumigatus,and finally treated with CS for 3h or 23 h.PCR,ELISA or Western blot were used to detect the expression of TLR-4 and downstream inflammatory factors at gene and protein levels in cells of each group,and to evaluate the potential anti-inflammatory mechanism of CS in Aspergillus fumigatus keratitis.Results:1.In vitro,CCK-8 experiments showed that CS at a concentration of 400 μg/m L was not cytotoxic to HCECs and RAW264.7 cells.Cell proliferation experiments and cell scratch experiments showed that 400 μg/m L CS could significantly promote the proliferation and migration of HCECs.In vivo,the results of the Draize assay showed that CS at a concentration of 0-400 μg/m L was not toxic to mouse cornea.2.In the mouse keratitis model,the inflammatory response of the mouse cornea on the 1,3,and 5 days after infection was significantly alleviated in the 400 μg/m L CS-treated group compared with the PBS control group,and the clinical score was significantly reduced.Plate count experiments and adhesion experiments demonstrated that CS reduced the fungal load.The results of the MPO assay showed that CS could attenuate neutrophil activity.3.In a mouse model of Aspergillus fumigatus keratitis,immunofluorescence staining and HE staining of the corneas of the CS-treated mice 3 days after Aspergillus fumigatus infection showed reduced neutrophil recruitment in the stroma and reduced corneal edema.The results of flow cytometry showed that the absolute number of neutrophils and the proportion of inflammatory cells in the cornea of CS-treated mice were significantly reduced after infection.4.The results of RT-PCR and ELISA experiments showed that CS reduced the expressions of inflammatory factors TNF-α,IL-1β,IL-6,COX-2 and MIP-2in the mouse model of Aspergillus fumigatus infection and the RAW264.7 cell model.5.The results of RT-PCR,Western blot and immunofluorescence experiments showed that CS could inhibit the expression of pattern recognition receptor TLR-4 in mouse model of Aspergillus fumigatus infection and RAW264.7 cell model.6.In Aspergillus fumigatus-infected RAW264.7 cells,TLR-4 si RNA treatment group inhibited the expression of TLR-4,p-NF-κB,TNF-α,IL-1β,IL-6,COX-2 and MIP-2.Conclusion:Chondroitin sulfate may ameliorate the prognosis of Aspergillus fumigatus keratitis by promoting corneal epithelial proliferation,inhibiting the recruitment and activity of neutrophils,and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling. |
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