Regulatory effect of nicotinamide on apoptosis and proliferation of rabbit nucleus pulposus cell in vitro

Ke NIE; Shuhua YANG; Liming XIONG; Bing GUO; Jianguo ZHOU

ABSTRACT

BACKGROUND:

It has reported that nicotinamide is capable of protecting intervertebral disc (IVD) against interleukin-1β (IL-1β) or tumor necrosis factor-alpha (TNF-α) induced degeneration. However, the protective mechanism of nicotinamide on IVD cells apoptosis and proliferation remains unclear.

OBJECTIVE:

To investigate regulatory effects of nicotinamide on rabbit nucleus pulposus cell apoptosis and proliferation in vitro.DESIGN, TIME AND

SETTING:

Randomized control grouping design, which was carried out in the Laboratory of Orthopaedics and Stem Cell Center, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from April to October 2007.MATERIALS Ten Japanese white rabbits (aged 2-3 months weighing 1.5-2.0 kg) were used in this study. Furthermore, nucleus pulposus cells obtained from L1-6 lumbar spine were harvested and cultured for further experiments.

METHODS:

The NP cells were divided into 6 groups, including control group (without any drug as control), nicotinamide group (0.5 g/L nicotinamide), IL-1β group (10 μg/L IL-1β), IL-1β + caspase group (10 μg/L IL-1β and non-specific caspase inhibitor Z-VAD-FMK), IL-1β + small-dose nicotinamide group (10 μg/L IL-1β and 0.05 g/L nicotinamide), and IL-1β + large-dose nicotinamide group (10 μg/L IL-1β and 0.5 g/L nicotinamide). After 3 days of culture, the cells were examined with Annexin V-PI staining, caspase-3, 8 and 9 activity staining and MTT assay.MAIN OUTCOME

MEASURES:

The apoptotic rates, the positive rates of caspase-3, 8 and 9 activity staining and the absorbance of MTT assay of each group.

RESULTS:

① As compared to IL-1β group, the apoptotic rates were decreased in the IL-1β + caspase group and IL-1β + large-dose nicotinamide group (P < 0.01). ②As compared to IL-1β group, the positive rates of caspase-3, 8 and 9 activity staining were decreased in the IL-1β + caspase group, IL-1β + large-dose and small-dose nicotinamide groups (P < 0.01 or P < 0.01). ③As compared to IL-1β group, the absorbance was increased in the IL-1β + caspase group and IL-1β + large-dose nicotinamide group (P < 0.01).

CONCLUSION:

Nicotinamide is capable of promoting cell proliferation and inhibiting IL-1β induced apoptosis of nucleus pulposus cells in vitro. The inhibition of apoptosis mainly acts via inhibition of the mitochondrial pathway.