ABSTRACT
Objective To investigate the change of endomucin(EMCN) expression in diabetic retinopathy (DR) and its protective role in neurons apoptosis.Methods Fifty-six clean SD rats were randomly divided into 4 groups,including normal control group with intravitreal injection of normal saline,diabetes mellitus (DM) group with intravitreal injection of normal saline,EMCN transfection group with intravitreal injection of adenovirus associated virus(AAV)-EMCN and mCherry transfection group with intravitreal injection of AAV-mCherry,14 rats for each group.Intravitreal injection was performed 2 weeks before diabetes modeling.Western blot analysis was used to measure the expression of EMCN and phosphorylated Akt (p-Akt)/Akt.Flat-mounted retinas were performed to test the transfection efficiency.Hematoxylin-eosin staining was performed to examine the morphology of retinal tissue.The expression of cleaved caspase-3 in retinas of rats was assayed by immunofluorescence.The retinal apoptotic cells were detected by TUNEL.The use and care of the rats followed the ARVO Statement.Results The levels of fasting plasma glucose were significantly higher in the DM group,EMCN transfection group and mCherry transfection group than those in the normal control group (all at P＜0.001).The expression of EMCN protein at 4 weeks and 8 weeks after modeling in the DM group were significantly lower than that in the normal control group (t=3.71,P＜0.05;t =10.09,P＜0.001).The mCherry transfection group was strongly expressed red fluorescence,the expression of EMCN was significantly lower in retinal tissue of DM group than that in the normal control group (t=13.67,P＜0.001).The expression of EMCN was notably upregulated in retinas of EMCN transfection group,comparing with that of DM group (t =3.18,P＜0.05).The expression of EMCN in mCherry transfection group was similar to that in the DM group (t =2.31,P=0.08).Initial morphologic degenerative changes were found in the DM group and mCherry transfection group,such as inter limiting membrane (ILM) was thicken,the number of RGCs was decreased,and the cells in outer nuclear layer (ONL) and inner nuclear layer (INL) arranged irregularly.The histologic change of retinas in the EMCN transfection group was milder than that in the DM group.The expression of cleaved caspase-3 was upregulated in INL of DM group and mCherry transfection group,compared with that in the normal control group.Compared with the normal control group,the number of TUNEL-positive cells noticeably increased in the ONL of DM group and mCherry transfection group,and the number of TUNEL-positive cells markedly reduced in the EMCN transfection group.The relative expression of p-Akt/Akt was significantly lower in the retinal tissue of DM group than that in the normal control group (t =5.52,P＜0.01).However,the relative expression of p-Akt/Akt was notably upregulated in retinas of EMCN transfection group,compared with that in the DM group (t=3.14,P＜0.05).The relative expression of p-Akt/Akt in mCherry transfection group was similar to that in the DM group (t =0.81,P =0.46).Conclusions The overexpression of EMCN can protect diabetic retinas neurons from apoptosis,and its mechanism maybe associated with activation of Akt signaling pathway.
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.