Figure 5.

ADR protects NPC from static mechanical pressure-induced damage. (A) Effect of ADR on NPC viability. NPCs were cultured in medium with ADR concentrations ranging from 0 µM to 30 µM for 24 h, and cell viability was measured using the CCK-8 kit; the results are expressed as the mean ± standard deviation, n=6 for each group. A significant decrease in cell viability was observed at ADR concentrations > 10 µM (P < 0.05). NPCs were pretreated with or without ADR or NAC for 8 h and then incubated in a 1.0 MPa environment for 24 h. (B) Cell viability was assayed using the CCK-8 kit. The results are expressed as the mean ± standard deviation, n=6 for each group. The cell viability was significantly inhibited by static mechanical pressure (P < 0.01), but significantly improved after pretreatment with ADR or NAC (P < 0.01). (C) ROS content was measured using ROS assay kit. The results are expressed as the mean ± standard deviation, n=6 for each group. The ROS content was significantly increased by static mechanical pressure (P < 0.01), but significantly decreased after pretreatment with ADR or NAC (P < 0.01). (D and E) Detection of apoptosis using the Annexin V-FITC/PI Apoptosis Detection Kit. The results are expressed as the mean ± standard deviation, n=3 for each group.The apoptosis rate was significantly increased by static mechanical pressure (P < 0.01), but significantly decreased after pretreatment with ADR or NAC (P < 0.05). (F) Apoptosis was detected again using TUNEL fluorescent staining. Sporadic nuclear fixation was observed in the control group, which showed red fluorescent highlights along with TUNEL assay solution. The number of red fluorescent bright spots increased significantly increased in the pressure group, but significantly decreased after pretreatment with ADR or NAC. *P < 0.05, **P < 0.01.

Abbreviations: ADR, andrographolide; NPC, nucleus pulposus cell; ROS, reactive oxygen species; NAC, N-acetylcysteine.