Mouse Nucleus Pulposus Cells

High Cholesterol can Directly Promote Metabolic Disorders in NPCs

Intervertebral disc degeneration (IVDD) is a major cause of low back pain (LBP). Dyslipidaemia can induce chronic inflammation, promote macrophage polarization, and may affect intervertebral disc (IVD) homeostasis. However, the relationship between dyslipidaemia and IVDD is unclear.

In this retrospective study, the baseline characteristics of 196 subjects were included and high TC, TG and LDL-C were identified as independent risk factors for IVDD. TC was selected as the indicator for further experiments. Mouse nucleus pulposus cells (NPCs) were cultured with water-soluble cholesterol. CCK-8 assays were used to evaluate the toxicity to NPCs and there was no significant difference under the concentration of cholesterol 5000 µg/mL at 24 h (Fig. 1A) and 2500 µg/mL at 48 h (Fig. 1B). The Chinese guidelines for lipid management (2023) was searched and hypercholesterolemia was diagnosed with TC > 6.2 mmol/L (~2400 µg/mL). Therefore, 2500 µg/mL of cholesterol was used for further experiments. Compared mRNA levels showed that cholesterol-treated NPCs had lower levels of benign matrix markers (COL2A1 and ACAN) and higher levels of MMP13 (~3 folds) and senescence markers (P21 and P16) (Fig. 1C). Immunofluorescence staining confirmed these results (Fig. 1D, E). γ-H2AX staining, a marker of cellular senescence, demonstrated significant senescence of NPCs under high-cholesterol condition, which is consistent with qPCR and Western blot analysis (Fig. 1D, E). EdU assays showed that NPCs had an inhibited proliferation ability in high-cholesterol condition (Fig. 1D, E). β-Galactosidase staining of NPCs showed a ~4-fold increase of senescent cells under cholesterol treatment (Fig. 1F). Western blotting also confirmed the above results (Fig. 1G). The results indicate that high cholesterol can directly accelerate metabolic disorders in NPCs, promote NPC aging and inhibit NPC proliferation, and finally accelerate IVDD.