Authors: Wang Yuping, Wang Jing, Yuan Yuanzhi
Researches showed that the mitochondrial-mediated apoptosis and autophagy play an important role in the survival of retinal ganglion cells (RGCs) in glaucoma.However, whether high pressure will lead to mitochondrial-mediated apoptosis and autophagy is not elucidated.
This study was conducted to explore whether elevated pressure can directly induce the mitochondrial-mediated apoptosis and autophagy in cultured RGC-5 cells in vitro.
RGC-5 cells were exposed to self-made pressure device and treated by 0, 20, 40 and 60 mmHg pressure for four hours in pressurized bottles.RGC-5 cells were incubated simultaneously in sealed incubator bottles and served as normal control.The morphological changes of the cells were examined under the inverted phase-contrast microscope.The apoptosis percentages of the cells were detected by flow cytometry.Mitochondrial membrane potentials of the cells were assessed using the JC-1, a fluorescent dye.And the expressions of cytochrome C (Cyt-c), microtubule associated protein light chain 3 (LC3) and Beclin-1 protein in the cells was detected by Western blot.
Cultured cells showed fusiform shape in the normal control group and 0 mmHg group with more dendritic process.The cell density was obviously reduced and the number of dead cells was increased in the 20, 40 and 60 mmHg groups.The apoptotic percentage was (15.69±0.77)%, (15.77±1.14)%, (18.30±1.07)%, (23.28±1.33)% and (34.47±1.17)% in the normal control group and 0, 20, 40 and 60 mmHg groups, respectively, showing a significant intergroup difference (F=150.90, P<0.001), and the apoptotic percentage in the 40 mmHg group and 60 mmHg group was significantly increased in comparison with the normal control group (both at P<0.01). The mitochondrial membrane potential was high in the normal control group and 0 mmHg group, with the reddish fluorescence in the cells, and the reddish fluorescence was weakened in the 20 mmHg group and 40 mmHg group.The lowing of mitochondrial membrane potential was seen in the 60 mmHg group, with the green fluorescence.Compared with the normal control group, the expression of Cyt-c, LC3-Ⅱ and Beclin-1 proteins in pressured groups was correspondingly increased (all at P<0.05).
Elevated pressure induces the morphologic change of RGC-5 cells, results in mitochondrial membrane potential reduction and mitochondrial-mediated apoptosis and autophagy.