Mitochondrial Pathway Is Involved in Advanced Glycation End Products-Induced Apoptosis of Rabbit Annulus Fibrosus Cells.

Abstract

Experimental study. The purposes of this study were to evaluate whether advanced glycation end-products (AGEs) induce annulus fibrosus (AF) cell apoptosis and further to explore the mechanism by which this process occurs. Recent studies revealed that AGEs accumulation is considered an important factor in diabetic intervertebral disc (IVD) degeneration. However, the effect of AGEs on intervertebral disc remains unclear. AF cells were treated with various concentrations of AGEs for 3 days. Cell viability and cell proliferation were measured by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, respectively. Cell apoptosis was examined by Annexin V/PI apoptosis detection kit and Hoechst 33342. The expression of apoptosis-related proteins, including Bax, Bcl-2, cytochrome c, caspase-3, and caspase-9, was detected by western blotting. In addition, Bax and Bcl-2 mRNA expression levels were detected by real-time PCR (RT-PCR). Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production of AF cell were examined by 5,5',6,6' -Tetrachloro-1,1',3,3'- tetraethyl-imidacarbocyanine iodide (JC-1) staining and 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probes, respectively. Our results indicated that AGEs had inhibitory effects on AF cell proliferation and induced AF cell apoptosis. The molecular data showed that AGEs significantly up-regulated Bax expression and inhibited Bcl-2 expression. In addition, AGEs increased the release of cytochrome c into the cytosol and enhanced caspase-9 and caspase-3 activation. Moreover, treatment with AGEs resulted in a decrease in MMP and the accumulation of intracellular ROS in AF cells. The antioxidant N-acetyl-L-cysteine (NAC) significantly reversed AGE-induced MMP decrease and AF cell apoptosis. These results suggested that AGEs induce rabbit AF cell apoptosis and mitochondrial pathway may be involved in AGEs-mediated cell apoptosis, which may provide a theoretical basis for diabetic IVD degeneration. N/A.