The Role Of Protein Kinase C? In The Dectin-1 Signaling Pathway Of Human Corneal Epithelial Cells In The Innate Immunity Stage Of Anti-fungal Infection

Objective:To study the function of protein kinase C 8(PKC 8)in innate immune at the early period of exposed to aspergillus fumigatus(AF)hyphae antigens in human corneal epithelial cells,and investigate its effect on the expression of inflammatory factors.Methods:Immortalized cultured human corneal epithelial cells(tHCECs)in vitro were randomly divided into control group,Aspergillus fumigatus infection group,PKC ?inhibition group and Dectin-1 blocking group.tHCECs which Aspergillus fumigatus hyphae suspension stimulated and tHCECs was treated with rottlerin(or laminarin)prior to inactive AF hyphae antigens stimulation 4,8,16 hours post-infection,the expression of inflammatory cytokines mRNA in tHCECs were detected by RT-PCR.Western blot was used to detect the expression of phospho-pkc 8 protein(tyr311)and total PKC ? protein at 5,15,30,45 minutes in fungal infection group.Pretreated cells with Rottlerin(PKC 8 activity pharmacological inhibitor)concentration gradient or laminarin(a dectin-1 activity inhibitor)followed by AF hyphae,the expression of phospho-pkc ? protein(tyr311)and total PKC ? protein were detected by western blot.Results:Both phosphorylation of PKC 8(tyr311)and total PKC 8 protein were expressed in the normal tHCECs,the phosphorylation of PKC 8 is significantly increased after Aspergillus fumigatus infection with a time-dependent manner,and peaked at 30 minutes,There was significant difference(p<0.05),whereas total PKC 8 protein wasnot significant difference(p>0.05).Pretreated with rottlerin or laminarin,phosphorylation of PKC 8 protein was significantly decreased(p<0.05),and the expression of total PKC 8 protein was no significant difference(p>0.05).There were significantly differences between the blank control group and fungal infected group in the expression of inflammatory cytokines IL-6,IL-8,CXCL1(p<0.05),however,the expression of IL-6,IL-8,CXCL1 mRNA were significantly decreased after pretreated with Rottlerin(p<0.05).Conclusions:1.Both phosphorylation of PKC ?(tyr311)and total PKC 8 protein were expressed in the normal tHCECs,and IL-6,IL-8,CXCL1 mRNA were expressed in the tHCECs by PKC ?.2.The expression of IL-6,IL-8,CXCL1 mRNA after AF hyphae stimulated by Dectin-1/PKC 8 sinalling pathway.