Abstract: BACKGROUND: Huangqi Guizhi Wuwu Decoction has a certain protective effect on intervertebral disc degenerative diseases. Mitophagy plays an important role in regulating mitochondrial damage and oxidative stress. However, the effect of Huangqi Guizhi Wuwu Decoction on mitophagy in chondrocytes of intervertebral disc endplate is still unclear. 
OBJECTIVE: To investigate the effect of Huangqi Guizhi Wuwu Decoction on hydrogen peroxide-induced injury of intervertebral disc endplate chondrocytes and its possible molecular mechanism. 
METHODS: Chondrocytes were obtained from the intervertebral disc endplate of rats and divided into five groups. Cells in low-, medium-, and high-dose groups were treated with 6.25, 12.5, and 25 mg/mL Huangqi Guizhi Wuwu Decoction for 24 hours, respectively. Cells in normal and models groups were cultured with no treatment. Except for the normal group, the cells in the model group and Huangqi Guizhi Wuwu Decoction groups were treated with 400 μmol/L H2O2 for 2 hours. Cell proliferation was then detected by cell counting kit-8 assay. Lactate dehydrogenase, adenosine triphosphate, superoxide dismutase, catalase and malondialdehyde levels were detected by colorimetric assay. The changes in mitochondrial membrane potential were detected by immunofluorescence. Reactive oxygen species and apoptosis were detected by flow cytometry. Mitophagy was detected by immunofluorescence double staining. Cells were transfected with siRNA, and the protein expressions of Parkinson-related gene (Parkin), LC3 and p62 were detected by western blot. Cells were transfected with Parkin adenovirus overexpression vector, and the survival rate of cells was detected. 
RESULTS AND CONCLUSION: Compared with the model group, Huangqi Guizhi Wuwu Decoction treatment significantly improved the survival rate of chondrocytes, increased the levels of adenosine triphosphate, superoxide dismutase, catalase and mitochondrial membrane potential in chondrocytes, and effectively attenuated the apoptotic rate of chondrocytes (P < 0.05, P < 0.01). After treatment with Huangqi Guizhi Wuwu Decoction, the activity of lactate dehydrogenase and the levels of reactive oxygen species and malondialdehyde in chondrocytes were significantly decreased compared with those in the model group (P < 0.01). Parkin siRNA significantly decreased mitophagy induced by hydrogen peroxide (P < 0.01), increased cell survival rate and adenosine triphosphate level (P < 0.01), and decreased reactive oxygen species level, malondialdehyde level and apoptosis rate (P < 0.01). Compared with the model group, the treatment with Huangqi Guizhi Wuwu Decoction could attenuate the increase of Parkin and LC3 II protein levels caused by hydrogen peroxide (P < 0.05, P < 0.01), while the protein level of p62 in the Huangqi Guizhi Wuwu Decoction treatment groups was significantly increased compared with the model group (P < 0.01). On the contrary, overexpression of Parkin attenuated the protective effect of Huangqi Guizhi Wuwu Decoction on the survival rate of cells treated with hydrogen peroxide. To conclude, Huangqi Guizhi Wuwu Decoction can effectively inhibit hydrogen peroxide-induced mitochondrial damage and oxidative stress in intervertebral disc endplate chondrocytes, and its protective mechanism may be related to attenuating Parkin-regulated mitophagy.

Key words: Huangqi Guizhi Wuwu Decoction, intervertebral disc, mitochondrial damage, oxidative stress, Parkin