Inhibition of p53/p21 by TWIST alleviates TNF-α induced nucleus pulposus cell senescence in vitro.

Abstract

This study aims to reveal the TWIST protein expression in the degenerated nucleus pulposus (NP), its effect on the TNF-α treated NP cells, and to explore its specific mechanism of anti-senescence. NP tissues from spine fracture patients without intervertebral disc degeneration (IDD) and the IDD patients were collected to detect the TWIST1/2 protein expression by Western blot (WB). NP cells isolated from the healthy tissue was treated with TNF-α to induce senescence, and the TWIST1/2 protein expression was also analyzed. We transfected NP cells with the plasmid coding TWIST to upregulate its expression, which was also cultured in the TNF-α condition. Besides, the TNF-α pretreated NP cells were further stimulated with the recombinant human TWIST1/2 protein. The collagen II and senescent marker β-galactosidase (β-gal) were determined by immunofluorescence (IF); the MMP-13, TIMP-3, IL-10, IL-1β mRNA expression level was detected by quantitative Real Time PCR; the cell proliferation was analyzed by CCK8 assay; the cell cycle was measured by flow cytometry. TWIST1/2 protein was decreased both in the degenerated NP tissue, and TNF-α treated NP cells. The overexpression of TWIST1/2 could prevent the p53, p21, β-gal, MMP-13, and IL-1β expression, moreover, it protected the collagen II, TIMP-3, and IL-10 expression in the TNF-α treated NP cells. Additionally, TWIST overexpression also promoted cell proliferation by ensuring the process of the cell cycle. Furthermore, the supplement of TWIST protein was functional to reverse these senescent phenotypes caused by TNF-α partly. TWIST alleviates the TNF-αinduced NP cells senescence via the inhibition of the p53/p21 pathway.