The Role Of P38 And JNK In Apoptosis Of The Cultured Human Retinal Pigment Epithelial Cells Induced By Hypoxia

Backgruoud: Hypoxia and ischemia are etiological factors in fundus oculi diseases. A great quantity of research prove that hypoxia induces the proliferation of retinal pigment epithelial cells(RPE) and plays a important part in the pathogenesis of fundus neovascular diseases. More and more evidence support that the apoptosis of RPE is a key facor in many funds diseases (for example: age related macular degeneration, hereditary malnutrition in outer layer of retina and proliferative vitreoretinopathy). However, there has been few study about the character of apoptosis RPE cells induced by hypoxia so far. Accordingly, it's important for us to investigate the information about apoptosis of RPE induced by hypoxia and its relative signal paths.AIM:(1) To establish the model of RPE cells in hypoxia status, and observe the gowth situation of them; (2) Study the role of p38 in the process of apoptosis of RPE cells induced by hypoxia; (3) Block the p38 and JNK pathway, and observe the influence in apoptosis of RPE cells in the hypoxia status.METHODS:(1) To set up the hypoxia model, human REP cells were cultured in a special incubator containing volume fraction of 1%O₂, 5% CO₂ and 94% N2 for 0h, 1h, 3h, 6h, 12h and 24h. The level of apoptosis was individually measured with scanning and transmission electron microscopy, terminal deoxynucleotidyl transferase mediated nick end labeling(TUNEL) and flow cytometry(FCM).Meanwhile, the proliferation of RPE cells were also measured with MTT. The contrast was RPE cells cultured in a normoxic incubator(5% CO₂, 95% O₂). (2) After hypoxia 1, 3, 6, 12 and 24 h, western blot analysis was performed to detect the expression of phosphorylated p38. The methods of immunohistochemisty and immunofluorescence were also used to fix the position of p38 and p-p38 protein in the RPE cell. blocking the p38 path with SB203580, detect the expression of phosphorylated p38 by western blot (3) After blocking the p38 and JNK path with SB203580 and SP600125, the level of apoptosis of RPE cultured in hypoxia incubator for 3 hours was individually measured with scanning and transmission electron microscopy, TUNEL and FCM.RESULTS: (1) Hypoxia can induce proliferation and apoptosis in the cultured RPE cells. Compared to the control group, the apoptosis of RPE cells was significantly activated and peaked at 3h in the cultured RPE cells under hypoxia. (2) Western blot showed that the expression of phosphorylated p38 increased gradually and peaked at 3h in the cultured RPE cells treated by hypoxia.The level of phospho-p38 of hRPE in the group treated with SB203580 was decreased. Immunohistochemistry staining proved that p38 protein located in the cytoplasm and nucleus in the cultured RPE cells under normoxic conditions. Under hypoxic conditions, the brown intensity decreased within the cytoplasm and increased in the nucleus. In normoxic surrounding, there was no expression of p-p38 in nucelus. However, the fluorescence intensity in the nucleus increased under hypoxic conditions. (3) After blocking the p38 and JNK path, the level of apoptosis of RPE induced by hypoxia decreased.CONCLUSION: Hypoxia can induce proliferation and apoptosis in the cultured RPE cells; According to this study, it was suggested that p38 and JNK path were involved in RPE apoptosis induced by hypoxia. And it's maybe the basis of possible new therapy for intraocular diseases.