Starvation-induced changes of LC-3 and Beclin-1 expression in annulus fibrosus cells

Abstract

Objective To investigate the starvation-induced changes of LC-3 and Beclin-1 expression in annulus fibrosus cells. Methods Rat annulus fibrosus cells in the logarithmic growth phase were cultured with amino acid free EBSS (Earle' s balanced salt) medium instead of the DMEM-F12 medium.Cells were collected at 1, 2 and 3 h after EBSS culture. Autopagosomes in annulus fibrosus cells were detected with monodansylcadaverine ( MDC) fluorescent staining method. Reverse transcription-polymerase chain reaction ( RT-PCR) and Western blotting analyses were used to detect the expression of microtubuleassociated protein 1 light chain 3 (LC3 ) and Beclin-1 in annulus fibrosus cells. Results Compared with the control group, MDC positive cells and autopagosomes were increased with the prolongation of starvation.LC3-Ⅱ and Beclin-1 mRNA expression levels in annulus fibrosus cells were increased with the prolongation of starvation (LC3-Ⅱ : 0.21 ±0.02, 0.45 +0. 12, 0.60±0.05, 0. 74 ±0. 03,P＜0. 05; Beclin-1: 0.20 ±0.06, 0. 37 ±0. 11, 0.52 ±0.07, 0. 69 ±0. 07,P＜0. 05). The ratio of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 protein expression was also increased with the prolongation of starvation (LC3-Ⅱ/LC3- Ⅰ : 0. 12 ±0.05,0.32 ±0.09, 0.63 ±0.07, 0. 92 ±0. 04,P＜0.05; Beclin-1: 0.61 ±0. 10, 0. 83 ±0. 11, 1.09 ±0.05,1. 35 ± 0. 10, P ＜ 0. 05 ). Conclusion Autophagy of annulus fibrosus cells can be induced by starvation,which may be related to the procession of intervertebral disc degeneration. Key words: Intervertebral disc degeneration; Annulus fibrosus cell; Autophagy