Effects Of NGR/NR2B Signaling Pathway On Diabetic Retinal Ganglion Cell

1 Backgroud and ObjectiveDiabetes mellitus is one of the common clinical metabolic disease, with a growing incidence and growing number of patients. It was estimated that patients with diabetes mellitus will projected from 2.8 hundred millions in 2013 to 5.9 hundred millions in 2035. Diabetes mellitus has extensive influence to the organism like retina, resulting in diabetic retinopathy (DR). DR is one of the very common complications of diabetes mellitus, and becomes the leading cause of blindness not only in Africa and American also for 20-74 aged man in China. Therefore, exploring the underlying mechanisms of DR, and investigating strategies for clinical treatment about DR play a vital role. Retinal ganglion cell is the only cell can be stimulated to produce activation potentials in retina, the axon of which composed of optic nerve to transmit visual information to optic center. Damage to retinal ganglion cell, of course, plays an important role in DR. Nogo receptor (NgR) locates extensively in nerve tissue, yet only in ganglion cell in retina. NgR combination with nervous growth inhibiting factor inhibits prolongation of neuronal processes, results in atrophy of somatic body, and induces apoptosis, being an important mechanisms of inhibiting nerve regeneration. NgR is up-regulated in animal models of damaged optic nerve such as glaucoma and crush to inhibit optic nerve regeneration. Therefore, in the present study, we aimed to explore the expression of NgR in diabetic retinal ganglion cell, and to investigate the effects of NgR and the downstream molecular on diabetic retinal ganglion cell with RNAi method. The work we done here will contribute to a better understanding of the mechanisms of DR, and may also provide a new insight to the clinical treatment of DR.2 Methods2.1 Construction of adenovirus vector silence NgR gene expressionThe double-stranded DNA oligo (NgR-shRNA) which can cause RNA interference (RNAi) effect to NgR was made after confirming the site RNAi sequence. The NgR-shRNA sequences were annealed and then linked with pGenesil-1 plasmid that linearized by Hind Ⅲ and BamH Ⅰ. Transformation of escherichia coli DH5a with pGenesil-1-NgR shRNA, and identified by double restriction digestion. Moreover, the adenovirus was harvested from 293 cells after the shuttle plasmid co-transfected with lentiviral packing materials into cells. The virus particles were collected to detect virus titer with dilution method.2.2 The expression and the role of NgR in retinal ganglion cell of diabetic ratsMale SD rats were administered with intraperitoneal injection of streptozotocin to incuce diabetic models. Diabetic rats were intravitreally injected with virus-carried anti-NgR nucleotide (siNgR group), scrambled nucleotide (scRNA group), or virus without nucleotide (diabetic group), and naive rats in control group were administered with virus without nucleotide. Three months after diabetes onset, retinal thickness and density of retinal ganglion cell (RGC) were detected by HE staining, coexistence of NgR/NR2B was revealed by immunofluorescence histochemistry, and expression of NgR and downstream molecular NR2B were observed by Western blot.2.3 NR2B on the apoptosis of retinal ganglion cell in viroWith ifenprodil, the specific antagonist of NR2B, RGC was cultured in 3 groups:control group, high glucose group (with 15 mmol/L glucose) and glucose-ifenprodil group (with 15 mmol/L glucose and 3 μmmol/L ifenprodil).3 days later, RGC morphological alterations were recorded, cell viability were detected by MTT method, apoptosis of RGC were observed by Hoechst 33342 staining, and the expression of NR2B were revealed by Western blot.3 Results3.1 Adenovirus vector silence NgR gene expression was constructed3.1.1 Besides restriction digestion sequence for BamHI and Hind Ⅲ digestion sequence for KpnⅠ is included in Pgenesil-1 plasmid. The sequence was recombined to plasmid with Nogo shRNA. In the present study, two electrophoresis strips were observed, confirming the design in our study.3.1.2 Gene of recombined adenovirus vector was about 900 bp, wihile gene of adenovirus vector itself was about 450 bp. Therefore, we constructed the recombined adenovirus properly.3.1.3 293 cell transfected with recombined adenovirus expressed green GFP gradually. The number of fluorescence dot was increased and the tensity of fluorescence was obviously gradually, presenting cytopathic effects.3.1.4 The virus titre was 2.5 X 109 TU/ml, meeting the requirement for later study in vivo and in vitro.3.2 Up-regulation of NgR contributes to the apoptosis of retinal ganglion cell in diabetic rats3.2.1 Diabetic rats were induced:the glycemia in diabetic group, siNgR group and scRNA group were (20.1±1.6), (22.3±1.9), (21.8±1.4) mmol/L, showing no difference among 3 groups (P>0.05), but higher than that of control group (4.2±0.5) mmol/L (P<0.01).3.2.2 Double immunohistochemistry showed that both NR2B and NgR positive neurons were located in retinal ganglion layer. NR2B and NgR coexist in retinal ganglion cells.3.2.3 The thickness of retina from inner limiting membrane to inner layer of inner nuclear layer in control group, diabetic group, siNgR group and scRNA group were (100.0±0.0)%, (50.3±4.2)%, (55.4±5.1)% and (96.3±6.4)%. Compared with control group, diabetic group and scRNA group were thinner (P<0.01), siNgR group was not altered (P>0.05).3.2.4 The density of retinal ganglion cell in control group, diabetic group, siNgR group and scRNA group were (1238±215)/cm2, (793±153)/cm2, (838±178)/cm2 and (1074±194)/cm2. Compared with control group, the density of retinal ganglion cell in diabetic group and scRNA group were decreased (P<0.01), but siNgR group was not altered (P>0.05).3.2.5 The expression of NgR in retinal ganglion cell in control group, diabetic group, siNgR group and scRNA group were (20.4±2.1)%, (42.6±5.2)%, (39.7±4.7)% and (20.4±2.1)%, of NR2Bwere (29.7±3.5)%, (50.7±7.5)%, (48.6±6.3)% and (28.7±4.9)% respectively. Compared with control group, the expression of retinal NgR and NR2B were increased in diabetic group and scRNA group were decreased (P<0.01), not altered in siNgR group (P>0.05).3.3 NR2B contribute to glucose-induced apoptosis of RGC3.3.1 Morphological study showed that retinal ganglion cells in glucose group were atrophy in somatic body, and lighter cell border compared with control group. However, no morphological difference was observed between control group and glucose-ifenprodil group.3.3.2 Cell viabilities in control group, glucose group and glucose-ifenprodil group were (100.0±0.0)%,(78.6±6.2)% and (95.3±8.1)%. Compared with control group, the cell viability was decreased in glucose group (P<0.01), not altered in glucose-ifenprodil group (P>0.05).3.3.3 Hoechst 33342 staining showed that the apoptotic ratio of RGC in control group, glucose group and glucose-ifenprodil group were (3.2±0.4)%, (16.4±1.5)% and (4.1±0.5)%. Compared with control group, the apoptotic ratio was increased in glucose group (P<0.01), not altered in glucose-ifenprodil group (P>0.05).3.3.4 Western blot revealed that the expression of NR2B in control group, glucose group and glucose-ifenprodil group were (30.3±3.7)%, (42.0±7.1)% and (39.3±6.2)%. Compared with control group, the expression of NR2B was increased in glucose group (P<0.01), but not changed in glucose-ifenprodil group (P>0.05).4 Conclusions4.1 Recombined adenovirus for silencing NgR expression was constructed successfully.4.2 The recombined adenovirus constructed here meeting the requirement for later study in vivo and in vitro.4.3 NgR and glutamate receptor NR2B express and coexist in retinal ganglion cells.4.4 Up-regulation of NgR contributes to the loss of retinal ganglion cell in diabetic rats.4.5 NgR induces the expression of retinal NR2B in diabetic rats, and inhibition of NgRdown-regulats the expression of NR2B in retina.4.6 Glutamate receptor NR2B induce apoptosis of RGC in vitro.4.7 Together, NgR-induced up-regulation of NR2B contributes to the apoptosis of retinal ganglion cell in diabetic rats.