Effect of andrographolide on TBHP-induced apoptosis of intervertebral disc nucleus pulposus cells and its mechanism

Abstract:

Objective To investigate the effect and mechanism of andrographolide（ANDRO） on intervertebral disc nucleus pulposus cell（NPC） apoptosis induced by tert-butyl hydroperoxide（TBHP）.Methods The cell model of NPCs induced by TBHP was established. NPCs were divided into five groups： control group，model group（100 μmol/L TBHP），low-dose ANDRO group（100 μmol/L TBHP+9 μmol/L ANDRO），middle-dose ANDRO group（100 μmol/L TBHP+18 μmol/L ANDRO），and high-dose ANDRO group（100 μmol/L TBHP+36 μmol/L ANDRO）. Cell apoptosis and reactive oxygen species（ROS） levels were determined by flow cytometry. The levels of oxidative stress related indicators，such as superoxide dismutase（SOD），glutathione peroxidase（GSH-PX），and catalase（CAT），were determined by an enzyme-linked immunosorbent assay kit. Mitochondrial membrane potential was measured by a JC-1 probe. Western blot was used to determine the expression levels of dynamin-related protein 1（Drp1），phosphorylated Drp1（p-Drp1），and mitofusin 2（Mfn2）.Results Compared with the control group，the model group had significantly increased apoptosis rate of NPCs，ROS level，and proportion of cells with decreased mitochondrial membrane potential（all P=0.000），significantly decreased SOD，GSH-PX，and CAT levels（all P=0.000），a significantly up-regulated p-Drp1/Drp1 level（P=0.000），and significantly down-regulated protein expression of Mfn2（P=0.000）. Compared with the model group，the high-dose ANDRO group had significantly decreased apoptosis rate of NPCs［（41.37±0.84）% vs.（26.36±1.33）%］，ROS level［（42.62±0.71）% vs.（22.50±0.37）%］，and proportion of cells with decreased mitochondrial membrane potential［（43.85±0.71）% vs.（24.96±1.04）%］（all P=0.000），significantly increased SOD［（288.16±14.59） ng/mL vs.（658.87±13.22） ng/mL］，GSH-PX［（6.66±0.37） ng/mL vs. （15.66±0.56） ng/mL］，and CAT［（40.89±1.04） pg/mL vs. （87.91±1.56） pg/mL］ levels（all P=0.000），a significantly down-regulated p-Drp1/Drp1 level（0.96±0.05 vs. 0.63±0.01）（P=0.001，0.000），and significantly up-regulated protein expression of Mfn2（0.39±0.01 vs. 0.64±0.02）（all P=0.000）.Conclusion ANDRO can inhibit TBHP-induced NPC apoptosis，and the mechanism may be related to the regulation of mitochondrial function and the relief of oxidative stress.