Autophagy in rat annulus fibrosus cells: evidence and possible implicationsReport as inadecuate

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Arthritis Research and Therapy

, 13:R132

First Online: 16 August 2011Received: 22 February 2011Revised: 13 April 2011Accepted: 16 August 2011


IntroductionProgrammed cell death of intervertebral disc IVD cells plays an important role in IVD degeneration, but the role of autophagy, a closely related cell death event, in IVD cells has not been documented. The current study was designed to investigate the effect of interleukin IL-1β on the occurrence of autophagy of rat annulus fibrosus AF cells and the interrelationship between autophagy and apoptosis.

MethodsRat AF cells were isolated and exposed, in tissue cultures with or without serum, to IL-1β in different concentrations for 24 hours. Ultrastructural analysis, flow cytometry and lysosomal activity assessment were performed after the in vitro treatment to determine the presence and levels of autophagy. The mRNA expression of autophagy-related proteins Beclin-1, Bcl-2 and microtubule associated protein 1 light chain 3 LC3 were evaluated using real-time PCR. 3-methyladenine 3-MA, a PI3K inhibitor, was used to determine the interaction between autophagy and apoptosis via the suppression of autophagy.

ResultsAutophagy was detected in rat AF cells under serum starvation condition by transmission electron microscopy. PCR and flow cytometry results showed that IL-1β enhanced the autophagy-induction effect of serum deprivation in a dose-dependent manner. However, IL-1β alone failed to induce autophagy in AF cells cultured without serum starvation. When autophagy was suppressed by 3-MA, the apoptosis incidence was increased. Serum supplement also partly reversed the autophagy incidence without affecting the apoptosis incidence in the same cells.

ConclusionsIL-1β up-regulates serum deprivation-induced autophagy of AF cells in a dose-dependent manner. Autophagy may represent a protective mechanism against apoptosis in AF cells and IVD degeneration.

KeywordsIntervertebral disc Autophagy Apoptosis Interleukin-1β Serum deprivation Abbreviations3-MA3-methyladenine

AFannulus fibrosus

ANOVAanalysis of variance

Atgautophagy-related genes

DMEMDulbecco-s modified Eagle medium

FBSfetal bovine serum


IVDintervertebral disc degeneration

LC3microtubule-associated protein 1 light chain 3


MMPsmatrix metalloproteinases

NPnucleus pulposus


PBSphosphate buffered saline.

Electronic supplementary materialThe online version of this article doi:10.1186-ar3443 contains supplementary material, which is available to authorized users.

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Author: Chao Shen - Jun Yan - Lei-Sheng Jiang - Li-Yang Dai


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