Mechanism research of senescence of NP cells mediated by A20 in TNF-ɑ inflammation microenvironment

English Keywords:Nucleus pulposus cells  Zinc finger protein A20  TNF-α: Inflammatory response  Cell senescence

English Abstract:

  【Abstract】 Objectives: To investigate the mechanism of zinc finger protein A20(also known as tumor necrosis factor-inducible protein 3, TNFAIP3) involved in the senescence of nucleus pulposus cells(NPCs) in the tumor necrosis factor α(TNF-α) inflammatory microenvironment. Methods: Isolated NPCs of sprague-dawley rat (SD) were cultured with or without TNF-α in vitro. The experiment was divided into three groups: normal control group(0ng/ml), TNF-α intervention group(10ng/ml, 50ng/ml, 100ng/ml), and natural aging group(p20 generation). CCK-8 cell proliferation assay, β-galactosidase staining, Western blot(WB), QT-PCR and immunofluorescence(IF) were used to assess senescence changes in NP cells from genes, proteins and cell function levels. The expressions of p53, A20, NF-ｋB(p65) and NF-ｋB (p-p65/phospho S536) were observed by Western Blotting, QT-PCR and IF. Results: Compared with the normal control group, the proliferation ability of NP cells significantly decreased after TNF-α intervention for 48h and 72h. The positive rate of β-galactose staining in the TNF-α intervention group and the aging group was significantly higher than that of the control group. The results of QT-PCR demonstrated that compared with the control group, the expression of p53 in the TNF-α intervention group increased(P<0.05), while the expression of A20 decreased with the increase of TNF-α concentration. The expression of A20 in the aging group decreased compared with that in the control group, while p53 expansion increased. WB showed that TNF-α could induce up-regulated expressions of p53, A20 and p-p65, while the expression of A20 decreased with the increase of TNF-αconcentration. Meanwhile, the expression of A20 in the aging group decreased compared with that in the control group, while the expression of p-p65 and p53 increased(P<0.05). IF showed that compared with the control group, the expressions of A20 and p-p65 increased with TNF-α treatment, but the expression of A20 decreased with the increase of TNF-α. The A20 of aging group also decreased significantly and the expression of p-p65 increased. Conclusions: There is a dose relationship between senescence of NP cells and TNF-α intervention. TNF-α can stimulate the expression of A20 in NP cells and activate NF-?资B signaling pathway. In addition, the expression of A20 is affected by the degree of cellular senescence, with aging of NP cells, the mechanism of action of A20 is decreasing.