Rabbit Nucleus Pulposus Cells

Lupeol Against High-Glucose-Induced Apoptosis Via Enhancing the Anti-Oxidative Stress in Rabbit Nucleus Pulposus Cells

IDD-induced secondary spinal instability and spinal stenosis are the main causes of low back pain. The reduced number and function of nucleus pulposus cells (NPCs) are the major reasons for IDD. Lupeol, a triterpenoid substance in fruits, vegetables, and Chinese herbal medicines, has been reported to have anti-oxidative, anti-inflammatory, antibacterial, and other benefits in several animal experiments.

Guo's team hypothesized that lupeol can reduce ROS production and inhibit apoptosis of NPCs in a high-glucose environment by antagonizing oxidative stress. The CCK-8 assay showed that cell viability was reduced by concentrations of lupeol more than 60 μg/mL (P < 0.01, Fig. 1a) and had no cytotoxicity by concentrations less than 60 μg/mL. Thus, 60 μg/mL of lupeol was selected for the following experiments. The assay showed no significant difference in cell proliferation activity between the LUP and CON groups, but the HG group had significantly lower activity (P < 0.01). The HG + LUP group had increased activity compared to the HG group (P < 0.05) (Fig. 1b). ROS levels were not significantly different between the LUP and CON groups, but were significantly higher in the HG group (P < 0.01) and lower in the HG + LUP group compared to the HG group (P < 0.05) (Fig. 1c). The study concluded that hyperglycaemia inhibits NPC growth and leads to IDD. Therefore, it was concluded that hyperglycaemia could significantly inhibit the growth of NPCs and lead to IDD.

Fig. 1. Lupeol (LUP) protected NPCs from high-glucose-induced cytotoxicity (Guo MB, Wang DC, et al., 2018).

Effects of Hypoxia and ASIC3 on Nucleus Pulposus Cells

Intervertebral disc degeneration, a leading cause of osphyalgia, occurs in an ischemic, hypoxic, acidic environment. This microenvironment, especially acidity, inhibits cell activity and is linked to ASIC3 activation. ASIC3 forms Ca²⁺-permeable channels at low pH, activating downstream pathways like MAPK and influencing cell behavior. Wang's team explored the effects of hypoxia and acid-sensing ion channel 3 (ASIC3) on nucleus pulposus cells from cell behavior to molecular mechanism.

The mRNA expression of ASIC3 in cells treated with different ASIC3 shRNAs is shown in Figure 2. Three interference sequences for ASIC3 were designed online, with shRNA-ASIC3 2 significantly reducing ASIC3 mRNA expression (P < 0.05), making it suitable for further experiments. Figure 2B demonstrates that the empty vector and shRNA-NC resulted in similar ASIC3 levels as control, whereas shRNA-ASIC3 reduced and ASIC3 expression vector increased ASIC3 expression significantly (P < 0.05), confirming the effectiveness of these interventions. Figure 3 illustrates cell cycle, viability, and apoptosis in various groups. As seen in Figure 3A, 2% O₂ treatment increased G1 phase and decreased S phase cell numbers compared to control, with ASIC3+2% O₂ showing the most G1 and least S phase cells (P < 0.05). shRNA-ASIC3 relieved G1 phase blockage caused by ASIC3 expression and hypoxia. Figure 3B shows that 48-hour 2% O₂ treatment significantly inhibited cell viability (P < 0.05), and Figure 3C reveals increased apoptosis with 2% O₂ treatment compared to control (P < 0.05). ASIC3 expression and hypoxia elevated apoptosis, while shRNA-ASIC3 reduced it.

Fig. 2. The expression of ASIC3 in primary rabbit nucleus pulposus cells treated with different ASIC3 shRNA (A) and different ASIC3 expression vectors and interference sequences (B) (Wang D, Zhu H, et al., 2019).

Fig. 3. The cycle (A), viability (B) and apoptosis (C) of primary rabbit nucleus pulposus cells in various groups which were evaluated by flow cytometry and CCK-8 assay (Wang D, Zhu H, et al., 2019).