Apelin-13通过调节YAP入核抵抗缺氧诱导的视网膜Müller细胞凋亡

Apelin-13 inhibits hypoxia-induced retinal Müller cell apoptosis by regulating YAP entry into the nucleus

摘要:

目的：探讨Apelin-13在缺氧诱导的视网膜Müller细胞凋亡中的作用及机制。

方法：以视网膜Müller细胞为研究对象，实验分为对照组、缺氧组和实验组，对照组细胞培养于正常环境中，缺氧组细胞培养于缺氧环境中，实验组细胞培养于缺氧环境中并以Apelin-13(1μmol/L)处理，MTT法检测细胞活力变化，结晶紫染色法观察细胞形态，免疫荧光染色法观察细胞GFAP和YAP的表达情况，TUNEL染色检测细胞凋亡并计算凋亡指数，蛋白印记实验检测p-LATS1、p-YAP、LATS1及YAP蛋白表达变化。

结果：提取分离的Müller细胞传代后贴壁生长，细胞呈长梭形、多角形、圆形等形态，细胞质丰富，细胞核呈圆形，细胞GFAP表达阳性。0.1、1、10μmol/L Apelin-13处理显著抑制缺氧诱导的Müller细胞活力下降(P<0.05或P<0.01)。与对照组相比，缺氧组细胞凋亡指数明显升高(P<0.01)； 而与缺氧组相比，实验组凋亡指数明显降低(P<0.01)。对照组和缺氧组细胞p-LATS1和p-YAP蛋白表达无明显差异； 与缺氧组相比，实验组细胞p-LATS1和p-YAP蛋白表达明显降低(P<0.01)。对照组和缺氧组细胞核YAP蛋白表达无明显差异； 与缺氧组相比，实验组YAP细胞核表达明显升高(P<0.01)。

结论：Apelin-13能抵抗缺氧诱导的视网膜Müller细胞凋亡，该作用可能与调节YAP入核有关。

Abstract:

AIM: To analyze the function and mechanism of Apelin-13 in preventing the apoptosis of retinal Müller cells induced by hypoxia.

METHODS: In the research, the retinal Müller cells are regarded as research subjects, and the control group, hypoxia group and experiment group are set up. The cells of control group are cultivated in normal environment. The cells of hypoxia group are cultivated in hypoxia environment. The cells of experiment group are cultivated in hypoxia environment and are treated with the Apelin-13(1μmol/L). MTT method is used to monitor the changing of the cell viability, and the crystal violet staining method is adopted to observe the cell morphology. In addition, the immunofluorescence staining method is used to test the expression of GFAP and YAP and the TUNEL staining method is used to monitor the cell apoptosis situation and the apoptosis index is calculated. The protein staining method is used to observe the changing of the expression of p-LATS1, p-YAP, LATS1 and YAP protein.

RESULTS:The separated and extracted Müller cells grow on the wall and show elongation, polygon and circular shapes. The cytoplasm is plentiful and the cell nucleus show circular shape. The GFAP expression of the cell is positive. The treatment with 0.1, 1, 10μmol/L Apelin-13 can obviously prevent the Müller cell viability decreasing induced by hypoxia(P<0.05 or P<0.01). Compared with the control group, the cell apoptosis index of hypoxia group is obviously increased(P<0.01). However, compared with the hypoxia group, the cell apoptosis index of experiment group is obviously decreased(P<0.01). The p-LATS1 and p-YAP protein expression of the control group and hypoxia group does not have big difference. Compared with hypoxia group, the p-LATS1 and p-YAP protein expression of experiment group is obviously decreased(P<0.01). The YAP protein expression of cell nucleus of control group and hypoxia group does not have great difference. Compared with hypoxia group, the cell nucleus expression of YAP cell is gretaly increased(P<0.01).

CONCLUSION: Apelin-13 can be used to prevent the retinal Müller cells apoptosis caused by the hypoxia, which may be related to the regulation of YAP into the nucleus.