Immortalized Human Nucleus Pulposus Cells-SV40

Downregulation of hsa_circ_0059955 Induced Apoptosis of NP Cells

Low back pain, often caused by intervertebral disc degeneration (IVDD), presents significant health and economic challenges globally. IVDD features decreased extracellular matrix and increased cell death. Recent studies highlight circRNAs' involvement in various diseases, including IVDD. However, the role of specific circRNAs like hsa_circ_0059955 in IVDD remains underexplored.

Kong's team identified hsa_circ_0059955 in immortalized human nucleus pulposus cells (NP) cells through a FISH assay and found its main location to be the cytoplasm (Fig. 1A). They used two shRNAs designated as hsa_circ_0059955 shRNA1 and hsa_circ_0059955 shRNA2 to reduce hsa_circ_0059955 expression in NP cells and found shRNA1 to be more effective (Fig. 1B), thus chosen for further experiments. The CCK-8 and Ki67 assays demonstrated that hsa_circ_0059955 reduction led to substantial inhibition of NP cell proliferation (Fig. 1C-E). JC-1 staining demonstrated that the downregulation led to a reduction in mitochondrial membrane potential (MMP) (Fig. 2A) demonstrated that NP cell apoptosis rose due to higher Bax levels and increased cleaved caspase 3 and caspase 9 activity while Bcl-2 expression decreased (Fig 2B-H). Research shows that knocking down hsa_circ_0059955 expression triggers apoptosis in NP cells.

Fig. 1. Downregulation of hsa_circ_0059955 inhibited proliferation of NP cells (Kong DL, Gu R, et al., 2020).

Fig. 2. Downregulation of hsa_circ_0059955 induced apoptosis of NP cells (Kong DL, Gu R, et al., 2020).

Evo Inhibits LPS-induced NPCs Apoptosis

IDD, linked to chronic back pain, lacks effective treatments. Nucleus pulposus cells regulate ECM components and are influenced by apoptosis and inflammation. Evo, with known protective effects, could impact these processes via SIRT1 and PI3K/Akt pathways.

The effect of Evo on immortalized human nucleus pulposus cells (NPCs) viability was detected. The results showed that Evo at 5, 10 and 20 μM had no significant effect on NPCs viability, but Evo at 40 μM caused damage on NPCs (Fig. 3A). Therefore, the maximum Evo concentration was 20 μM in the following experiments. Subsequently, CCK-8 assay (Fig. 3B) and TUNEL staining (Fig. 3C and D) showed significant apoptosis of NPCs induced by LPS. It should be noted that Evo enhanced NPCs viability in a concentration-dependent manner. In addition, caspase-3 activity and the expression levels of apoptosis-related proteins in NPCs were also assessed (Fig. 3E and F). Following LPS induction, caspase-3 activity was increased, Bax protein was significantly upregulated and Bcl-2 protein was downregulated, indicating that LPS induced apparent apoptosis. Similarly, this change also could be reversed by Evo.

Fig. 3. Evo inhibits LPS-induced NPCs apoptosis (Kuai J and Zhang N, 2022).