Experimental Sciences
Mitochondrial pathway of retinal pigment epithelial cell apoptosis induced by blue light in vitro
Hong Li, Jianping Lyu, Shanjun Cai, Gang Su, Zhipeng Wu, Xin Gong
Published 2015-01-10
Cite as Chin J Exp Ophthalmol, 2015, 33(1): 16-20. DOI: 10.3760/cma.j.issn.2095-0160.2015.01.004
Abstract
BackgroundStudies determined that blue light exposure causes apoptosis of human retinal pigment epithelial (RPE) cells, but its mechanism is still below understood.
ObjectiveThe aim of this study was to investigate whether or how mitochondrial apoptotic pathway is involved in blue-light induced apoptosis of human RPE cells in vitro.
MethodsHuman RPE cells were isolated from fresh donor eyes and primarily cultured and passaged.The cells were identified with keratin antibody by immunochemistry.Then the cells were the non-light exposed group, simple light-exposed group, light-exposed+nifedipine group, light-exposed+calphostin C group and the light-exposed+phorbol myristate acetate (PMA) group.Human RPE cells in light-exposed group were consequently cultured for 24 hours following the exposure of (2 000±500)lx blue-light for 6 hours, and then the expression levels of bax, bcl-2, bcl-xl in the cells were detected by Western blot to evaluate the effect of blue light on the apoptosis.The cells in the light-exposed+nifedipine group, light-exposed+calphostin C group and the light-exposed+PMA group were treated with the corresponding drugs 1 hour prior to light irradiation and sequently received 6-hour light irradiation and 48-hour culture.The expression of caspase-9 protein in the cells were assayed with Western blot to assess the influence of Ca2+ channel and protein kinase C (PKC) pathway on mitochondria of RPE cells.
ResultsCultured cells grew well with visible pigment in cytoplasm.The cells showed the positive response for keratin and presented a cobblestone-like appearance.The expression bands of bax, bcl-2 and bcl-xl proteins were clearly visible at the molecular weight of 23 000, 26 000 and 30 000 in both non-light exposed group and the simple light-exposed group, and the absorbance values of the cells to bax were elevated, while the absorbance values to bcl-2 and bcl-xl were declined in the simple light-exposed group compared with the non-light exposed group (t=-4.409, P=0.012;t=7.575, P=0.002;t=6.068, P=0.004).Compared with the non-light exposed group, the absorbance values of caspase-9 were significantly raised in the simple light-exposed group, light-exposed+calphostin C group and the light-exposed+PMA group (P=0.005, 0.002, 0.000), but no significant difference between the non-light exposed group and light-exposed+nifedipine group (P=0.191).Compared with the simple light-exposed group, the expression level was considerably higher in the light-exposed+PMA group (P=0.005);while that in the light-exposed+nifedipine group or light-exposed+calphostin C group was not significantly different (P=0.057, 0.643).
ConclusionsBlue light exposure induces apoptosis of RPE cells by up-regulating the expressions of bax and caspase-9 proteins and down-regulating the expressions of bcl-2 and bcl-xl. The mitochondrial apoptosis pathway and PKC pathway participate in blue-light induced apoptosis of human RPE cells in vitro.
Key words:
Blue light; Light-induced injury; Retinal pigment epithelium; Apoptosis; Mitochondria; Cysteine-aspartic acid proteases; Protein kinase C; Cell culture
Contributor Information
Hong Li
Department of Ophthalmology, Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China
Jianping Lyu
Shanjun Cai
Gang Su
Zhipeng Wu
Xin Gong