Intervertebral disc degeneration is closely associated with the functional changes in nucleus pulposus stem cells (NPSCs), yet traditional planar culture fails to simulate their genuine 3D microenvironment. This study aims to investigate the endoplasmic reticulum stress (ERS) response of NPSCs under 3D spheroid culture conditions, as well as its effects on cell proliferation and cell cycle changes.

NPSCs were isolated from 36 male SD rats (aged 8 weeks, weighing 350±50 g), and then a 3D spheroid culture model was constructed. The ERS, stress signals, cell cycle, and proliferation status of NPSCs under different culture conditions (3D spheroid culture vs planar culture) were compared. The morphological changes in endoplasmic reticulum were observed by electron microscopy. Cell viability and apoptosis rates were assessed using live/dead cell staining and Annexin V-FITC/PI flow cytometry. The expression of stress markers such as GRP78, ATF6, and PERK was detected by qPCR and Western blotting. Cell proliferation was analyzed by EdU and Ki67 flow cytometry. Calcium ion fluorescent probe plasmids were transfected to observe the changes in intracellular calcium dynamics. Finally, the endoplasmic reticulum function inhibitor 4-PBA was used to verify the role of ERS in cell cycle regulation.

① Light microscopy showed that the cells formed compact spheroids in the 3D culture dishes. Live/dead cell staining demonstrated favorable cytocompatibility of 3D spheroid culture, with no statistical difference compared with the planar culture group (P>0.05). The apoptosis rate remained at an extremely low level of 1.6%, and no significant difference was observed between groups (P>0.05). ② Compared with planar culture, NPSCs under 3D spheroid culture exhibited obvious expansion of the endoplasmic reticulum (observed by electron microscopy) and upregulation of GRP78, ATF6, and PERK (P<0.001), suggesting ERS activation. ③ Cell cycle analysis revealed a significant decrease in proliferation, upregulation of P21 expression (P<0.001) and downregulation of PCNA, CDK4 and CDK5 expression (P<0.001) in the spheroid culture group. EdU and flow cytometry demonstrated that spheroid cells showed gradually declined proliferative capacity over time, indicating cell cycle arrest. ④ Confocal microscopy showed that the intracellular calcium concentration was increased under 3D spheroid culture (P<0.01), suggesting that ERS may regulate the cell cycle through calcium signaling. ⑤ 4-PBA treatment partially restored cell proliferation and decreased the expression of P21 and P27 (P<0.001), further demonstrating the role of ERS in cell cycle inhibition.

3D structural microenvironment can induce the imbalance of endoplasmic reticulum homeostasis in NPSCs. ERS induced by 3D spheroid culture drives NPSCs into a reversible low-proliferation, adaptive quiescent state.

To explore the role and possible mechanism of lysyl oxidase（LOX）in age-related degeneration of intervertebral disc.

Six SD rats, aged of 2, 8 and 20 months respectively, were subjected, and then underwent intervertebral disc MRI and X-ray to evaluate the effect of age on intervertebral disc degeneration. Then, the total proteins of intervertebral disc from different age groups were isolated and detected for the expression of LOX protein by Western blotting. The hypoxia apoptosis model of nucleus pulposus cells was established by CoCl₂ treatment. Through LOX protein inhibitor BAPN and plasmid pcDNA3.1（+）-LOX, the effect of LOX protein on apoptosis of nucleus pulposus cells under different hypoxia conditions was studied. Fifteen 6-week-old SD rats were randomly divided into blank control group, vehicle group and BAPN group, with 5 animals in each group. PBS buffer of 20 μL or BAPN of 100 μg/mL was injected into the rats from the vehicle or BAPN group via caudal vertebrae. X-ray film examination was taken at 2 and 4 weeks after operation to evaluate intervertebral disc degeneration.

With the increase of age, X-ray films showed that the intervertebral disc height of 20-month-old rats was decreased significantly compared with 2-month-old rats（P<0.01）, and MRI displayed reduced T2 signals, indicating appearance of intervertebral disc degeneration. The protein level of LOX was decreased significantly in intervertebral disc of 20-month-old rats（P<0.01）. Treatment of 300 mmol/L CoCl₂ induced obvious apoptosis of nucleus pulposus cells as severe hypoxic condition（P<0.05）and subsequently down-regulated the expression of LOX protein（P<0.05）. In vivo experiment showed that after LOX inhibitor BAPN treatment for 4 weeks, the height of intervertebral disc was significantly declined in the BAPN group when compared with the PBS group（P<0.01）, suggesting more serious degeneration of intervertebral disc.

LOX protein can regulate the apoptosis of nucleus pulposus cells induced by hypoxia and thus affect intervertebral disc degeneration.