| As a common endocrine metabolic disease, Diabetes mellitus(DM) is one of the five major diseases of high morbidity and mortality around the world. Along with fast development of economy and improvement of living standards, the incidence of DM presents a rapid upward trend. As a result, it’s predicted that up to 350 million people will be diagnosed with DM by 2025. As one of the most common complications of DM, Diabetic retinopathy(DR) has become an important reason for visually impaired DM patients and even blindness. Therefore, it’s of great significance to explore the underlying mechanisms of DR and search for its clinical treatment.Literature reports that there were swelling of body and a decreased number of RGCs in diabetic rat induced by streptozotocin(STZ). At the same time, more and more research confirmed that retinal ganglion cell lesions occurred in the early stage of DR accompanied with RGCs injury, apoptosis, structural changes and impaired functional status. It’s well known that RGCs is the only visual output cell and RGCs axon is an important part of the optic nerve. Hence the injury, apoptosis and regenerative repair of RGCs is one of the important mechanisms of DR onset and vision loss. Currently, although extensive research on diabetes RGCs had been carried out at home and abroad, the molecular mechanism of injury, apoptosis and regeneration of RGCs is not completely understood and still needs further exploration.Recent research found that Nogo receptor(Ng R) played a key role in the injury, repair and regeneration of RGC. After the interference, it could effectively restore cell vitality, promot cell proliferation and inhibit cell apoptosis of rat RGCs. Our preliminary work suggested that the protein expression of Ng R and Rock1 was increased in DR, which implied that the Ng R-Rho A-Rock1 signal pathway was activated. Furthermore, the Rho A/Rock signal pathway could regulate the expression of F-actin and inhibit the extension of terminal axon growth cones and ganglion regeneration. However, the inhibition of upstream signal protein Ng R could restore retina axon regeneration and inhibit the apoptosis of RGCs. These studies indicated that Ng R-Rho ARock1 signal pathway may be involved in regulation of apoptosis, injury and regeneration process of diabetic retinal ganglion cell.Ciliary neurotrophic factor(CNTF) is of multiple effect, which can nourish the ganglion cells and promote axon growth. Lacking of neurotrophic factors is another important factor leading to impaired vision of DR patients through promoting RGCs apoptosis. Studies have proved that CNTF had obviously protective effect on RGCs in traumatic optic nerve injury, and exogenous CNTF could obviously protect the vision of hereditary diabetes mice and type Ⅰ diabetes rats. These data indicate that CNTF plays an important role in the process of optic nerve repair, axonal regeneration and ganglion cell apoptosis. But little is known about the effect and mechanism of CNTF on diabetic RGCs at home and abroad.To sum up, our study intends to research the effect and molecular mechanisms of si Ng R and CNTF on the survival and regeneration of diabetic retinal ganglion cells in vivo and vitro, and investigated whether there is a synergistic effect between them. It’s expected that what we’ve done here will contribute to a better understanding of the mechanisms of DR, and also provide reliable experimental basis for clinical treatment of DR.Part1 Effect of Ng R interference and CNTF pretreatment on apoptosis of RGCs induced by high glucoseObjective: To clarify the effect of si Ng R and CNTF on the proliferation, injury and apoptosis of RGC-5 induced by high glucose.Method: RGC-5 was transfected with si Ng R or given CNTF, and then induced cell injury by high glucose. The experiment was divided into six groups: control group, high glucose group, high glucose-NC group, high glucose-si Ng R group, high glucose-CNTF group and high glucose-CNTF-si Ng R group. After 48 h, cell proliferation was detected by MTT assay, cell apoptotic was tested by flow cytometry, the m RNA and protein expression was determined by Real-time PCR and Western blot, the neurite length and expression of specific markers of RGCs were detected by immunofluorescence.Result: The results of MTT and flow cytometry showed that high glucose significantly inhibited cell proliferation and promoted cell apoptotic of RGC-5(P<0.01). However, there was a substantial increase for the proliferative capacity and an obvious reduce for apoptotic rate after transfection with si Ng R or pretreatment with CNTF(P<0.01), and the combination of them was synergistic. The Real-time PCR and Western blot indicated that the m RNA and protein expression of Ng R, Rho A and Rock1 was increased in cell model(P<0.01), which was significantly reduced after si Ng R transfection or CNTF pretreament(P<0.01) and the combination of them was synergistic. Immunofluorescence tests showed that high glucose reduced the expression of specific markers and the neurite length of RGCs(P<0.01), which was obviously increased by si Ng R or CNTF.Conclusion: Si Ng R and CNTF could collaborate to promote the survival of injury cells and inhibit apoptosis, and the protective effect of them on RGC-5 cells was closely associated with Ng R-Rho A-Rock1 signal pathway.Part 2 Effect of si Ng R and CNTF on apoptosis and regeneration of RGCs in diabetic ratsObjective: To clarify the effect of intravitreal injection of si Ng R and CNTF on apoptosis and regeneration of RGCs in diabetic rats.Method: Diabetic rats model was established by intraperitoneal injection of STZ, and then was intervened by intravitreal injection of si Ng R and CNTF. The experiment was divided into six groups: control group, diabetic group, diabeticNC group, diabetic-si Ng R group, diabetic-CNTF group and diabetic-CNTFsi Ng R group. After 12 weeks, rat retina tissue was collected from each group. The number and form of ganglion cells was detected by HE staining. Apoptosis rate of RGCs was examined by TUNEL. The expression of apoptosis related proteins was determined by Western blot. The m RNA and protein expression of axon regeneration markers was detected by Real-time PCR, Western blot and immunohistochemistry.Result: The results of HE staining and TUNEL assay showed that the number of RGCs cells reduced significantly, RGCs layer cavitated and cell apoptosis rate increased substantially in diabetic rats(P<0.01). However, intravitreal injection si Ng R or CNTF significantly decreased apoptotic rate and promoted cell survival(P<0.01), and the combination of them was synergistic. The results of Western blot indicated an evident increase for the expression of Bax and caspase-3 and a sharp decrease for F-actin and GAP-43 in retinal tissue of diabetic rats(P<0.01). Besides, the combination of them was synergistic. The experiments of Real-time PCR and immunohistochemistry further confirmed that si Ng R or CNTF could significantly increased the m RNA and protein level of axonal regeneration markers F-actin and GAP-43.Conclusion: Si Ng R and CNTF could cooperatively promote RGCs cell survival in diabetic rats, induce axonal regeneration and inhibit cell apoptosis.Part 3 The molecular mechanism of Ng R interference or CNTF treatment on apoptosis and regeneration of diabetes RGCsObjective: To clarify whether the effect of si Ng R or CNTF on RGCs in diabetic rats was associated with Ng R-Rho A-Rock1 signal pathwayMethod: Intraperitoneal injection of STZ induced diabetic rat model. Drug treatment and experimental groups were the same as part 2. Retinal tissue of rats were collected after 16 weeks. The m RNA and protein expression of Ng R, Rho A and Rock1 were detected by Real-time PCR and Western blot technology.Result: The results of Real-time PCR and Western blot showed that the m RNA and protein expression of Ng R, Rho A and Rock1 was remarkably increased in retinal tissue of diabetic rats, while the expression level of them was significantly reduced after intravitreal injection of si Ng R or CNTF(P<0.01), and the combination of them was synergistic.Conclusion: Si Ng R and CNTF could cooperate to inhibit Ng R-Rho ARock1 signal pathway activity, then induced axonal regeneration and inhibited the apoptosis of RGCs. |
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