ABSTRACT
Background Most anti-inflammation eyedrops are limited in clinical application owing to multiple adverse effects.A novel peptide GC31 derived from human thrombomodulin has a natural anti-inflammatory activity.Compared with conventional anti-inflammatory eyedrops,GC31 possesses more advantages and potential clinical transforming value.However,relevant study is still lack.Objective The purpose of this study was to evaluate the anti-inflammatory effect of GC31 and the possible mechanisms.Methods Sixty SPF male Wistar rats aged 8-10 weeks were randomized into 6 groups using randomized number table.Non-specific keratitis models were established in 40 rats by intrastromal injection of 10 μl of lipopolysaccharide (LPS) dissolved in PBS.Different doses of GC31 (125 μg or 250 μg) or dexamethason soluble in PBS were sunconjunctically injected in the experimental eyes respectively in the low dose GC31 group,high dose of GC31 group and the dexamethason group,and 10 μl of PBS was used in the same way in the PBS control group.No drug was injected in the model group,and the normal rats were employed as the blank control group.The corneas were examined by slit lamp microscope and were scored based on the criteria of Anand 24 hours after injection.Then the corneas were collected for histopathological examination.Expression of nuclear factor-κB (NF-κB) p65 in the corneas was detected using immunochemistry.Expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) proteins were assayed using ELISA.Real-time PCR was used to detect the expressions of IL-6 mRNA and TNF-α mRNA.The use and care of the experimental animals followed Regulation for the Administration of Affair Concerning Experiment animals by State Science and Techonology Commission.Results A significant difference was seen in the ocular inflammatory scores among the six groups (F =301.238,P =0.000).The inflammatory scores were significantly lower in the high dose of GC31 group than those in the model group (1.85 ± 0.36 versus 2.90± 0.43) (t' =-5.144,P =0.000) ; and the scores in the dexamethason group was lower than those in the high dose of GC31 group(t' =-3.931,P=0.000).Infiltration of inflammatory cells in corneal tissue was milder in the high dose of GC31 and the dexamethason group compared with the model group.The positive response for NF-κB p65 was obviously weaker in the rat corneas in the low and high dose of GC31 groups and the dexamethason group in comparison with the model group.The contents of IL-6 and TNF-α proteins in the corneas were significantly reduced in the low and high dose of GC31 group and the dexamethason group compared with the model group (low dose group:t=-2.626,P=0.009;t'=-2.310,P=0.017.high dose group:t =-3.361,P=0.001 ;t'=-3.151,P=0.002),and the contents of IL-6 and TNF-α proteins in the dexamethason group were lower than those in the high dose of GC31 group (t=-3.361,P=0.001;t'=-3.360,P=0.000).In addition,the expression trend and compared results of IL-6 mRNA and TNF-α mRNA among the groups were similar to those of the IL-6 and TNF-α proteins (all at P＜0.01).Conclusions GC31 suppresses LPS-induced corneal inflammation response by downregulating the expression of inflammatory eytokines.The effect is more dominant in the doses of 250 μg than that in the doses of 125 μg.