Age-dependent expression of the vitamin D receptor and the protective effect of vitamin D receptor activation on H2O2-induced apoptosis in rat intervertebral disc cells

Abstract

Accumulating evidence shows that genetic polymorphism of the vitamin D receptor (VDR) gene is associated with intervertebral disc degeneration (IDD), implying that VDR may be involved in the pathogenesis of IDD. However, the exact relationship between VDR and IDD remains unknown. The aim of this study was to investigate the age-dependent expression of VDR in rat intervertebral discs and to determine the effect of VDR on oxidative stress-induced cell apoptosis of the annulus fibrosus (AF) and the underlying mechanism. Sprague-Dawley rats were subjected to magnetic resonance imaging (MRI) and CT scans at young (2–3 months), adult (6–7 months), and old (14–15 months) ages. The images revealed age-related degeneration of the lumbar intervertebral discs and endplates. Immunohistochemistry demonstrated positive expression of VDR in the AF. The expression level of VDR in aged rats was significantly reduced compared with that in the young and adult animals and exhibited a negative correlation to IDD severity. Western blot analysis further demonstrated that the amount of VDR protein was significantly decreased in severe degenerative discs. AF cells were also isolated from young rat lumbar discs and subjected to different concentrations of hydrogen peroxide (H2O2) for various amounts of time. The results revealed that H2O2 inhibited the viability of AF cells and induced mitochondrial pathway apoptosis. However, pretreatment of AF cells with 10−7 and 10-8 M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] effectively increased cell viability, increased mitochondrial membrane potential, decreased the level of reactive oxygen species, increased mitochondrial ATP content, reserved the activity of key enzymes in the oxidative respiratory chain, and thus protected the mitochondria from H2O2-induced damage. Whereas, siRNA knock-down of VDR abolished the protective effects of 1,25(OH)2D3. Moreover, 1,25(OH)2D3 inhibited H2O2-induced autophagy of AF cells through inhibition of the mTOR/p70S6K signal pathway. Our study demonstrated that decreased expression of VDR may play a role in age-related intervertebral disc degeneration in rats and that activation of VDR ameliorates oxidative stress-induced apoptosis in AF cells by preserving mitochondrial functions.