[EFFECT OF VITAMIN C ON APOPTOSIS OF NUCLEUS PULPOSUS CELLS INDUCED BY TUMOR NECROSIS FACTOR a AND SERUM DEPRIVATION].

Objective: To explore the effect of Vitamin C (Vit C) on the apoptosis of human nucleus pulposus (NP) cells induced by tumor necrosis factor a (TNF-alpha) and serum deprivation.

Methods: The NP cells were isolated from patients undergoing spine corrective operation by collagenase trypsin. The experiment was divided into 3 groups: Vit C group (group A), TNF-alpha group (group B), and serum deprivation group (group C). Group A was reassigned to Al subgroup (basic medium), A2 subgroup (100 pg/mL Vit C), and A3 subgroup (200 pg/mL Vit C). Group B was reassigned to B0 subgroup (control group), Bi subgroup (100 ng/mL TNF-alpha), B2 subgroup (100 microg/mL Vit C+100 ng/mL TNF-alpha), and B3 subgroup (200 microg/mL Vit C+100 ng/mL TNF-alpha). Group C was reassigned to C0 subgroup (Control group), C1 subgroup (2% FBS), C2 subgroup (2% FBS+100 microg/mL Vit C), and C3 subgroup (2% FBS+200 microg/mL Vit C). After application of 100 pg/mL or 200 microg/mL Vit C for 24 hours, NP cells were stimulated by TNF-alpha and serum deprivation, then the apoptosis rate of NP cells was detected by a flow cytometry, and the gene expressions of the extracellular matrix of NP cells (collagen type I, collagen type II, aggrecan, and Sox9) and apoptosis related genes (p53, FAS, and Caspase 3) were detected by real-time fluoroscent quantitative PCR. Results Group A: Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 of NP cells in A2 and A3 subgroups when compared with Al subgroup (P<0.05), but there was no significant difference between A2 subgroup and A3 subgroup (P>0.05); Vit C could promote the expressions of the extracellular matrix (collagen type I, collagen type II, aggrecan, and Sox9) of NP cells in a concentration dependent manner (P<0.05). Group B: TNF-alpha significantly increased the apoptosis rate and the gene expressions of p53, FAS, and Caspase 3 in B1 subgroup when compared with B0 subgroup (P<0.05); however, Vit C significantly increased the apoptosis rate and the gene expressions in B2 subgroup, and significantly decreased them in B3 subgroup when compared with B1 subgroup (P<0.05). Group C: 2% FBS significantly increased the apoptosis rate of NP cells and significantly reduced the gene expressions of p53, FAS, and Caspase 3 in C1 subgroup when compared with C0 subgroup (P<0.05); Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 in C3 subgroup, but it could significantly increase them in C2 subgroup when compared with C1 subgroup (P<0.05).

Conclusion: Vit C can promote the synthesis and secretion of extracellular matrix of NP cells. 200 microg/mL Vit C may delay the apoptosis induced by TNF-alpha and serum deprivation, indicating the potential therapeutic effect of Vit C on intervertebral disc degeneration.