Experimental study of Toll like receptor 4 on inflammatory response of keratocytes induced by Fusarium Solani in vitro

Abstract

Objective To investigate the expression of Toll like receptor 4 (TLR4) in the cultured mouse keratocytes in vitro stimulated by Fusarium Solani suspension,researchthe relationship between TLR4 expression and inflammatory factors secrection.Methods Mouse keratocytes were cultured in vitro by enzyme digestion method.The cells in generation 3 were identified by immunocytochemistry staining using anti-vimentin antibodies.Fusarium Solani suspension (Spore density was 1 ×106CFU/ml) was self-made.Cultured cells were stimulated with Fusarium Solani suspension.MTT colorimetry was used for assaying the proliferation of the cells induced by Fusarium Solani suspension within 48 hours.The techniques of immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to examine the expression of TLR4 protein and mRNA in the cultured mouse keratocytes at 0,3,6 and 12 hours post stimulation.The release of tumor necrosis factor α (TNF-α) and interleukin-8 (IL-8) was also measured using enzyme linked immunosorbent assays (ELISA) in the presence and absence of specific blocking antibodies to TLR4 at 1,3,6 and 12 hours post stimulation,Results Mouse keratocytes were cultured by enzyme digestion after 1-2 days.MTT colorimetry showed that Fusarium Solani could inhibit growth of the cells.The results of immunohistochemical technique and RT-PCR showed that TLR4 protein and mRNA were weakly expressed on cultured mouse keratocytes at 0 hour,reached the peak at 6 hours,and decreased at 12 hours.Secretion of TNF-α and IL-8 were gradually increased,reached the peak at 6 hours,and then decreased gradually.The effects could be inhibited by treatment with TLR4 antibodies.There was positive correlation between TLR4 mRNA expression and inflammatory factors secretion at 3,6 and 12 hours (P＜0.05).The difference of interclass had statistical significant (P ＜0.05).Conclusions TLR4 may play a critical role in recognizing fungi and mediating immune inflammatory response of cornea. Key words: Keratocytes; Fusarium Solani; Toll like receptor 4; Fungal keratitis; Inflammatory response