| BackgroundThe pathological changes for most ophthalmic diseases are resulted from retinal blood supply insufficiency. And further cause the damage of RGCs and axons, mainly presented as the apoptosis of RGCs. Currently, there is no effective treatment for the diseases. In vitro RGC culture is an effective way to investigate different ophthalmic diseases, which provides methods not only to study the development of the diseases, but also to find a treatment for the diseases. Acteoside is a natural polyphenols containing phenylpropanoid glycosides (PPGs), which is a main component of (2.1%) ilex latifolia in Yunnan. Ilex latifolia grows in northeast of Yunnan, subtropical broad-leaved forest area in Zhaotong with altitude of 1000 to 1500 meters. In recent years, accumulated evidences indicated that Acteoside with antioxidant activity play roles in the protection of acute ischemic central neuronal damage and chronic progressive degeneration of central neurons. Studies using animal models also showed that acteoside could protect RGCs suffered from the optic nerve damage. In our study, we will investigate the protection effects of acteoside in optic nerve and try to find a new treatment method to the diseases.PurposeTo establish in vitro RGC culture model to investigate the effects of acteoside in the protection of RGC damage.1. To establish normal SD rat RGC culture model.2. To establish damaged SD RGC culture model induced by low serum.3. To observe the effects of acteoside on normal RGC model.4. To observe the effects of acteoside on damaged RGC model.Methods1. To establish normal SD rat RGC culture model.16 retinal tissues were taken from 1-3 day old SD rats. Cell suspension was made by the digestion of the tissues with 0.2% trypsin. The cell were seeded in a plate, and cultured in 37 C,5% CO2. One third of cell culture solution was replaced and cytosine arabinoside was added (with final concentration of 10 umol/L) at the 72 hour. The cell morphologies and THY 1.1 specific fluorescent staining were performed at the 96 hour.2. To establish damaged SD RGC culture model induced by low serum. Follow the first step to get the RGC cell suspension and then seed the cells in 6 different culture plates with different serum levels:10%,7.5%,5%,2.5%,1.25%,0%. Cell morphologies were observed at the 12 hour and 24 hours. The cell activities were tested with WST-8.3. To observe the effects of acteoside on normal RGC model:Follow the first step to get the RGC cell suspension and then seed the cells in 6 different culture plates with different acteoside concentrations:A:(0)、B (5mg/ml)、C(3mg/ml)、D(1mg/ml)、 E (0.5mg/ml)、F (PBS). Observation was performed at the 24 and 48 hours: immunohistochemical staining and The cell activities were tested with WST-8.4. To observe the effects of acteoside on damaged RGC model. Retinal cells are from 32 SD rats 1-3 days old. Follow the above step 2 method to establish the damaged RGC model.1 mg/mL of acteoside was added to observe its effects. Observations were performed at 12 and 36 hours:1) light microscope for cell morphology; 2) Electron microscope for fine structure of cells; 3) WST-8 for cell activities; 4) Immunohistochemical staining; 5) Western blot for protein expression of GAP-43、 Bcl-2 and Bax.Result1. The density of cell suspension is 3.0×106/ml. single, spread cells were observed under microscope. From inoculation to 7 days, the cells presented as normal growth with growing axons and formed axon nets; Thy-1.1 specific staining showed that 92.3 % are RGCs.2. (1) Cell cultures with different serum levels were observed at 12 and 24 hours. The normal RGCs were decreased with the decreasing of serum concentration. RGC apoptosis could be observed visually.(2) Cell activities tested with WST-8. Cell activities were significantly decreased for those cell cultures with 2.5%,1.25%,0% of serum concentrations (P<0.05). However, there is no activity difference was observed among those with serum concentration of 10%,7.5%, and 5%(p>0.05).At 12h and 24h, there’s no different in every serum concentrations.3. After adding acteoside:1) Immunohistochemical staining indicated that group B at 24 hour is lighter than others; the cell density of group B at 48 hour is lower than all other groups.2) Cell counting:there is no significant difference observed among groups at 24 hour (p>0.05); however, cells of group B at 48 hour decreased compared with other groups (p<0.05).3) Cell activities:Group B at 24 and 48 hours showed decreased cell activities compared with other groups (p<0.05)4.12h、36h. 1mg/ml acteoside was added into damaged cell culture models with 1:50 volume ratio.(1) Compared with groups without adding acteoside, Groups with acteosides have increased normal RGCs and axons.(2) Observed by transmission electron microscope:Groups with acteosides have more normal structures than groups without acteosides.(3) WST-8 (cck-8) cell activity test:Cell activities increased significantly at 12 and 36 hours (p<0.05),and at 36 hours increased significantly than 12 hours (p<0.05)(4) Immunohistochemical staining indicated that groups with acteosides are deep colour than others;Cell counting:there is significant difference observed among groups with acteosides,36 increased than 12 hours (p<0.05)(5) Western Blot detection Protein expression:a. GAP-43:Groups with acteosides are increase at 12 and 36 hours (p<0.05); and at 36 increased than 12 hours (p<0.05)b. Bcl-2:Groups with acteosides are increase at 12 and 36 hours (p<0.05); and at 36 increased than 12 hours (p<0.05)c. Bax:Groups with acteosides are decreased at 12 and 36 hours (p<0.05); and at 36 decreased than 12 hours (p<0.05)d. Bcl-2/Bax:at 12h, group without acteoside Bcl-2/Bax=0.964979, group with acteoside Bcl-2/Bax=2.061404, acteoside/without acteoside=2.14; at 36h, group without acteoside Bcl-2/Bax=0.977391, group with acteoside Bcl-2/Bax=4.052812, acteoside/without acteoside=4.15; at 12h and 36h, Bcl-2/Bax of the group with acteoside is more than 1, and at 36 increased than 12 hours. This suggests that Apoptosis resistance of RGCs is increasedConclusion1. Thy-1.1 specific staining showed that 92.3% are RGCs. In vitro RGC culture model is established successfully2. Mimic the microenvironment for cell growth by changing the components of cell culture solutions. Morphological and activity testing indicated that we establish the damaged RGC culture model successfully, which is induced with 1.25% fetal bovine serum.3. RGC activities and cell counting showed that 0.5 mg/ml、1mg/ml、mg/ml acteoside are safe.4. Observations of cell morphology, microstructures and activities, results of immunohistochemical staining, and protein expression of GAP-43、Bcl-2、Bax indicated that acteoside plays a role in the protection of RGCs from the damage induced by low serum. |
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