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. 2019 May 10;39(5):BSR20190126.
doi: 10.1042/BSR20190126. Print 2019 May 31.

Resveratrol attenuates inflammation environment-induced nucleus pulposus cell senescence in vitro

Xiaoming Li  1 , Feixiang Lin  1 , Yaohong Wu  1 , Ning Liu  1 , Jun Wang  1 , Rongchun Chen  1 , Zhijun Lu  2
Affiliations

Resveratrol attenuates inflammation environment-induced nucleus pulposus cell senescence in vitro

Xiaoming Li et al. Biosci Rep. .

Retraction in

Abstract

Intervertebral disc degeneration is a disease identified as an inflammation response-participated pathological process. As a classical cellular feature, disc cell senescence is reported to be closely related with disc cell senescence. Resveratrol has a protective role against inflammation in some cells. However, its biological effects on disc cells remain largely unclear. The present study was aimed to study the effects of resveratrol on disc nucleus pulposus (NP) cell senescence in an inflammation environment. Isolated NP cells were cultured in cultured medium with (control group) or without (inflammation group) inflammatory cytokine TNF-α and IL-1β for 14 days. Resveratrol was added along with the NP cells treated with inflammatory cytokines to investigate its effects. NP cell senescence was analyzed by senescence-associated β-Galactosidase (SA-β-Gal) staining, cell proliferation, G0/1 cell cycle arrest, telomerase activity, gene/protein expression of senescence markers (p16 and p53) and NP matrix biosynthesis. In addition, the intracellular reactive oxygen species (ROS) was also analyzed. Compared with the control group, inflammation group significantly increased SA-β-Gal activity and ROS content, decreased cell proliferation and telomerase activity, promoted G0/1 cell cycle arrest, up-regulated gene/protein expression of senescence markers (p16 and p53) and matrix catabolism enzymes (MMP-3, MMP-13 and ADAMTS-4), and down-regulated gene/protein expression of NP matrix macromolecules (aggrecan and collagen II). However, resveratrol partly reversed the effects of inflammatory cytokine on these cell senescence-associated parameters. Together, resveratrol was effective to suppress cell senescence in an inflammatory environment. The present study shows new knowledge on how to retard inflammation response-initiated disc degeneration.

Keywords: cell senescence; intervertebral disc degeneration; nucleus pulposus; resveratrol.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. Measurement of intracellular ROS content
The intracellular ROS content in NP cells was measured by the fluorescent probe DCFH-DA. The numeric Data are expressed as mean ± S.D. (n=3). *: Indicates a statistical difference (P<0.05) between two groups.
Figure 2
Figure 2. Cell proliferation analysis
NP cell proliferation potency was evaluated by the CCK-8 assay. The numeric Data are expressed as mean ± S.D. (n=3). *: Indicates a statistical difference (P<0.05) between two groups.
Figure 3
Figure 3. Analysis of SA-β-Gal activity
SA-β-Gal activity of NP cells was measured using a senescence β-Galactosidase staining kit. The numeric Data are expressed as mean ± S.D. (n=3). *: Indicates a statistical difference (P<0.05) between two groups.
Figure 4
Figure 4. Cell cycle analysis
NP cell cycle was evaluated by flow cytometry. Cell fraction in the G0/G1 phase, G2/M phase and S phase was calculated.
Figure 5
Figure 5. Telomerase activity analysis
Telomerase activity of NP cells was evaluated using a chemical kit. The numeric Data are expressed as mean ± S.D. (n=3). *: Indicates a statistical difference (P<0.05) between two groups.
Figure 6
Figure 6. Expression of senescence markers
(A) Gene expression of p16 and p53 in NP cells was analyzed by real-time PCR analysis. (B) Protein expression of p16 and p53 in NP cells was analyzed by Western blot assay. The numeric Data are expressed as mean ± S.D. (n=3). *: Indicates a statistical difference (P<0.05) between two groups.
Figure 7
Figure 7. Expression of matrix catabolism enzymes
Gene expression of MMP-3, MMP-13 and ADAMTS-4 in NP cells was analyzed by real-time PCR analysis. The numeric Data are expressed as mean ± S.D. (n=3). *: Indicates a statistical difference (P<0.05) between two groups.
Figure 8
Figure 8. Expression of matrix macromolecules
(A) Gene expression of aggrecan and collagen II in NP cells was analyzed by real-time PCR analysis. (B) Protein deposition of aggrecan and collagen II in NP cells was analyzed by Western blot assay. The numeric Data are expressed as mean ± S.D. (n=3). *: Indicates a statistical difference (P<0.05) between two groups.
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