Proanthocyanidins inhibit high glucose-induced apoptosis in nucleus pulposus cells in a rabbit

doi:10.3969/j.issn.2095-4344.1747

Abstract

Abstract:

BACKGROUND: Proanthocyanidins are an internationally recognized active oxygen free radical scavenger in the human body due to their high antioxidant activity.
OBJECTIVE: To investigate whether proanthocyanidins has a significant inhibitory effect on high glucose-induced apoptosis in nucleus pulposus cells and to investigate its molecular mechanism.
METHODS: Healthy 2-month-old New Zealand white rabbits were provided by Shandong Kond Biotechnology Co., Ltd., and the study protocol was approved by Health Care Technology Association of Shandong Province, China. Nucleus pulposus cells were isolated, cultured and passaged to the 5 generation in vitro. Passage 3 nucleus pulposus cells at a concentration of 1×10⁸/L were inoculated into six 4-well culture plates and cultured in proanthocyanidins at different concentrations (0, 10, 20, 30, 40, 50, 60 µmol/L). Cell proliferation was detected using cell counting kit-8, to determine the proper concentration of proanthocyanidins. Cells were divided into four groups and cultured normally, in the medium containing 100 mmol/L glucose, in the medium containing 40 µmol/L proanthocyanidins, and in the medium containing 40 µmol/L proanthocyanidins and 100 mmol/L glucose (combination group, in which 100 mmol/L glucose was added after 6-hour pretreatment with 40 µmol/L proanthocyanidins), respectively. The morphology of nucleus pulposus cells was observed by inverted microscope, and the cell growth curve was drawn. The apoptotic rate, intracellular reactive oxygen species and expression of heme oxygenase in the cells were detected.
RESULTS AND CONCLUSION: Nucleus pulposus cells from rats showed polygonal and short spindle shapes and cellular pseudopod appeared gradually and then became slim with increasing passage numbers. The cells grew in an “S”-shaped curve. The cells grew slowly on day 1 after passage, and grew rapidly from day 2 to day 5. As the number of passages increased, the cell viability gradually decreased. It was found that the proper concentration of proanthocyanidins was 40 μmol/L. Flow cytometry results showed that the apoptosis rate of nucleus pulposus cells in the high glucose group was significantly increased. Pretreatment with proanthocyanidins significantly decreased the apoptosis rate as compared to the high glucose group (P < 0.05). Results of western blot assay showed that proanthocyanidins significantly increased the expression of heme oxygenase-1, and moreover, the expression of heme oxygenase 1 was negatively correlated with the apoptotic rate, active oxygen free radical accumulation and cell proliferation activity. All the findings indicate that proanthocyanidins could inhibit the apoptosis of nucleus pulposus cells induced by high glucose, and decrease intracellular reactive oxygen species accumulation and proliferation activity by up-regulating the expression of heme oxygenase 1.

Key words: tissue construction, chondrocytes, nucleus pulposus cells, apoptosis, cell proliferation, reactive oxide species, intervertebral disc, proanthocyanidin, heme oxygenase-1, glucose

CLC Number:

R453
R318

Xu Qing, Fang Haolin, Liu Yang, Zhang Cunxin, Tian Baofang. Proanthocyanidins inhibit high glucose-induced apoptosis in nucleus pulposus cells in a rabbit[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3426-3431.

Figures/Tables 3

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