Authors: Niu Tian, Xie Liping, Xing Xindan, Jiang Yan, Jin Huiyi, Wang Haiyan, Liu Kun
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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.