Polyphyllin I suppressed the apoptosis of intervertebral disc nucleus pulposus cells induced by IL-1β by miR-503-5p/Bcl-2 axis

Abstract

Background: There are no studies that have shown the role and underlying mechanism of Polyphyllin I (PPI)-mediated anti-apoptosis activity in nucleus pulposus cells (NPCs). The research aimed to evaluate the effects of PPI in interleukin (IL)-1β-induced NPCs apoptosis in vitro.

Methods: Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability, and cell apoptosis was evaluated by double-stained flow cytometry (FITC Annexin V/PI). The expression of miR-503-5p was quantified by real-time quantitative PCR (qRT-PCR), and the expression of Bcl-2, Bax, and cleaved caspase-3 was quantified by Western blot. Dual-luciferase reporter gene assay was used to detect the targeting relationship between miR-503-5p and Bcl-2.

Results: PPI at 40 μg·mL^-1 markedly promoted the viability of NPCs (P < 0.01). Also, PPI inhibited apoptosis and reduction in proliferative activity induced by IL-1β in the NPCs (P < 0.001, 0.01). PPI treatment significantly inhibited the expression of apoptosis-related protein Bax, cleaved caspase-3 (P < 0.05, 0.01), and enhanced the level of anti-apoptotic protein Bcl-2 (P < 0.01). The proliferative activity of NPCs was significantly decreased and the apoptosis rate of NPCs was increased under IL-1β treatment (P < 0.01, 0.001). Moreover, miR-503-5p was highly expressed in IL-1β-induced NPCs (P < 0.001). Furthermore, the effect of PPI on NPCs viability and apoptosis in IL-1β treatment was dramatically reversed by the overexpression of miR-503-5p (P < 0.01, 0.01). The targeted binding of miR-503-5p to the 3'UTR of Bcl-2 mRNA was confirmed by dual-luciferase reporter gene assays (P < 0.05). In further experiments, compared with miR-503-5p mimics, the effects of PPI on IL-1β-induced NPCs viability and apoptosis were greatly reversed by the co-overexpression of miR-503-5p and Bcl-2 (P < 0.05, 0.05).

Conclusion: PPI suppressed the apoptosis of intervertebral disk (IVD) NPCs induced by IL-1β via miR-503-5p/Bcl-2 molecular axis.

Keywords: Bcl-2; IL-1β; NPCs apoptosis; Polyphyllin I; miRNA-503-5p.