The Effect Of MCC950 On Canine Staphylococcus Pseudintermedius Keratitis

The corneal surface is moist and suitable for the growth of a variety of microorganisms that together constitute the normal flora of the ocular surface.In most cases,there are interactions between microorganisms and hosts to maintain the relative balance of the flora.When the body resistance is reduced or the cornea is damaged,the balance is disrupted and opportunistic pathogens infect the cornea via damage the defence barrier of corneal epithelial cell.Staphylococcus pseudintermedius is a resident flora in the conjunctival sac of dogs.It is one of the main opportunistic pathogens causing canine keratitis,which has a high isolated rate in the conjunctival sac of both healthy dogs and dogs with keratitis.In the last decade,S.pseudintermedius resistance has been gradually increasing,increasing the treatment difficulty.MCC950,a diarylsulfonylurea-containing compound,is a specific inhibitor of the NLRP3 inflammasome and inhibits IL-1β maturation and secretion.At present,its therapeutic effect has been confirmed in a variety of inflammatory diseases.In this study,canine corneal epithelial cells and canine corneal stromal cells were stimulated with S.pseudintermedius to make inflammation models and then treated with MCC950.The effects of MCC950 on cell inflammation-related and proliferation-related signaling pathways and cytokines induced by S.pseudintermedius were assessed by qRT-PCR,Western Blot and flow cytometry.An animal model of keratitis was established by injecting S.pseudintermedius into the canine corneal stromal layer,and the effect of MCC950 on canine Staphylococcus pseudintermedius keratitis was investigated by slit lamp examination,paraffin section,qRT-PCR,Western Blot and immunohistochemistry.This study provides a new target and theoretical basis for the clinical treatment of canine bacterial keratitis.1.The therapeutic effect of MCC950 on canine Staphylococcus pseudintermedius keratitisAn animal model of Staphylococcus pseudintermedius keratitis was established by injecting S.pseudintermedius into the canine corneal stroma.After 12 hours of bacterial injection,the control group did not receive any treatment,levofloxacin control group and levofloxacin treatment group were treated with levofloxacin eye drops,MCC950 control group and MCC950 treatment group were treated with MCC950 based on levofloxacin eye drops.Each group was treated using corresponding eye drops every 2 hours(2 drops/eye),up to 36 hours.Canine corneas were observed using a slit lamp;corneas were collected,partly for paraffin section preparation,and partly for extraction of RNA and protein.The corneal pathological damage was observed by HE staining of paraffin sections,the expression of related proteins was detected by immunohistochemistry and Western Blot,and the mRNA expression of related cytokines was detected by qRT-PCR.The results showed that the S.pseudintermedius infected the canine corneal stroma resulted in corneal opacity,edema,ulceration,neovascularization and a large number of neutrophils infiltration in the stroma.After the treatment of MCC950,the above lesions were improved,and the corneal inflammatory damage was alleviated.The phosphorylation levels of IκBα,p65 and the expression of MyD88 were significantly increased(p<0.01),and the expressions of ASC,caspase-1 p20,cleaved IL-1β and NLRP3 were significantly upregulated(p<0.01).The expressions of IL-1β,IL-18,IL-8,IL-6 and TNF-α were significantly increased(p<0.01).S.pseudintermedius can activate corneal NLRP3 inflammasome and NF-κB signaling pathway.After MCC950 treatment,the expressions of the above proteins and mRNA were significantly decreased(p<0.01).2.Isolation and identification of canine corneal epithelial cells and stromal cellsPrimary canine corneal epithelial cells(CCECs)and canine corneal stromal cells(CCSCs)were isolated and cultured in vitro by different enzymatic digestion combined with tissue block adherence.The epithelial layer and partly stromal layer of the beagle cornea were collected aseptically by surgery.The epithelial layer and the stromal layer were separated with 1.2 IU/mL dispase Ⅱ enzyme.The epithelial layer was chopped and seeded into cell flasks for culture.The stromal layer was digested with 0.25%collagenase I to obtain cell suspension.The cells were collected and then seeded into cell flasks.The cell growth curve was determined by CCK-8,and the purity of epithelial and stromal cell was confirmed via respectively detecting cytokeratin 12 and vimentin using immunofluorescence.Both CCECs and CCSCs showed good proliferative viability.The immunofluorescence results showed that CCECs expressed cytokeratin 12,and CCSCs expressed vimentin.All of these demonstrated that canine corneal epithelial cells and stromal cells were successfully isolated and purified.3.The effects of MCC950 on S.pseudintermedius-induced NF-κB signaling pathway and NLRP3 inflammasome in canine corneal epithelial cells and stromal cellsCCECs were pretreated with 50 μmol/L PDTC,10 μmol/L MCC950 and 20μmol/L PDTC+10 μmol/L MCC950 for 1 h,respectively.CCSCs were pretreated with 20 μmol/L PDTC,10 μmol/L MCC950 and 20 μmol/L PDTC+10 μmol/L MCC950 for 1 h,respectively.S.pseudintermedius in logarithmic growth phase was added into cells according to MOI=1:1,and cells were collected in different time points.The inflammatory factors were detected by qRT-PCR,NF-κB signaling pathway and NLRP3 inflammasome-related protein expression were detected by Western Blot,and the lactate dehydrogenase(LDH)release and malondialdehyde(MDA)content were detected using commercial kits.The results showed that compared with the control group,the phosphorylation levels of p65 and IκBα in the S.pseudintermedius group were significantly increased(p<0.01);the protein expressions of NLRP3,ASC,caspase-1 p20 and cleaved IL-1β were significantly increased(p<0.01);the mRNA expressions of IL-1β,IL-6,IL-8,IL-18,TNF-α,NLRP3,ASC and caspase-1 were significantly increased(p<0.01);cellular MDA content and LDH release significantly increased(p<0.01).After PDTC and MCC950 treatment,the phosphorylation levels of p65 and IκBα were significantly decreased(p<0.01);the protein expressions of NLRP3,ASC,caspase-1 p20 and cleaved IL-1β were significantly decreased(p<0.01);the mRNA expressions of IL-1β,IL-6,IL-8,IL-18,TNF-α,NLRP3,ASC and caspase-1 were significantly decreased(p<0.01);the cellular MDA contentand LDH release were significantly decreased(p<0.01).It indicated that MCC950 could inhibit the activation of NF-κB signaling pathway and NLRP3 inflammasome induced by S.pseudintermedius,and reduce the inflammatory response.4.The effects of MCC950 on the proliferation of canine corneal epithelial cells and stromal cells induced by S.pseudintermediusCCECs and CCSCs were pretreated with 10 μmol/L MCC950 for 1 h,S.pseudintermedius in logarithmic growth phase was added into cells(MOI=1:1),and cells were collected in different time points.The growth factors were detected by qRT-PCR,PI3K/AKT and Wnt/pcatenin signaling pathway-related protein expression was detected by Western Blot,and the cell cycle was analyzed by flow cytometry.The results showed that compared with the control group,the phosphorylation levels of PI3K and AKT in the S.pseudintermedius group were significantly increased(p<0.01);the protein expression levels of β-catenin,c-Myc and CyclinD1 were significantly increased(p<0.01).The mRNA expressions of EGF,FGF,TGFβ1,VEGF and CTGF were significantly upregulated(p<0.01).Cells in S phase were significantly increased(p<0.01),and cells in G2 phase were significantly decreased(p<0.05).Compared with the S.pseudintermedius group,MCC950 significantly decreased the phosphorylation levels of PI3K and AKT and the protein expressions of β-catenin,c-Myc and CyclinDl(p<0.01).The mRNA expressions of EGF,FGF,TGF-β1,VEGF and CTGF were significantly down-regulated(p<0.01).Cells in S phase were significantly reduced(p<0.01).In conclusion,MCC950 can inhibit the activation of NF-κB signaling pathway and NLRP3 inflammasome in CCECs,CCSCs and corneal tissue infected by S.pseudintermedius,reduce the expression of inflammatory factors,and ameliorate inflammatory damage.At the same time,MCC950 can inhibit the activation of PI3K/AKT and Wnt/β-catenin signaling pathways in CCECs and CCSCs infected by S.pseudintermedius,reduce the expression of growth factors,and avoid corneal fibrosis healing and scarring.MCC950 can reduce corneal inflammatory response and facilitate corneal healing.