Abstract: BACKGROUND: Stem cell transplantation is a new way to prevent and cure intervertebral disc degeneration. However, whether the transplanted stem cells can survive, proliferate, differentiate, and restore the function of nucleus pulposus cells after transplantation, is the key and difficult point to overcome. 
OBJECTIVE: To explore the effects of Bushenhuoxue decoction on survival, proliferation, and nucleus pulposus-like differentiation of adipose-derived stem cells.
METHODS: A Transwell chamber was used to construct a co-culture model of human adipose-derived stem cells and human degenerative nucleus pulposus cells. The experiment was divided into control group, model group, drug-containing serum group, and drug-free serum group. Except for the control group, the co-culture system of other groups was treated with 50 μmol/L tert-butyl hydrogen peroxide for 24 hours. The drug-containing serum group and drug-free serum group were treated with DMEM low-glucose complete culture medium containing drug-containing serum of Bushenhuoxue decoction or drug-free serum with 20% volume fraction for 48 hours. The sublayer adipose-derived stem cells were taken. Toluidine blue staining was used to detect proteoglycan synthesis levels. Real-time PCR method was used to detect mRNA expression of type II collagen, proteoglycan and SRY-box transcription factor 9. The protein expression of SOX9 was detected by western blot assay. Lactate dehydrogenase assay was used to detect cytotoxicity. Flow cytometry was used to detect reactive oxygen species, and β-galactosidase staining was used to detect cell senescence. 
RESULTS AND CONCLUSION: (1) Compared with the control group, the proportion of necrotic cells in the model group increased; toluidine blue staining became lighter, and the expression levels of type II collagen, proteoglycan, SOX9 mRNA and SOX9 protein decreased (P < 0.05). Compared with the model group, the drug-containing serum of Bushenhuoxue decoction could significantly reduce cell injury and promote the expression of type II collagen, proteoglycan, SOX9 mRNA, and SOX9 protein (P < 0.05), but the improvement in the drug-free serum group was not significant (P > 0.05). (2) Compared with the control group, the contents of cytotoxicity, reactive oxygen species, and cell senescence in the model group were significantly increased. Compared with the model group, the microenvironment of the coculture system was significantly improved by drug-containing serum of Bushenhuoxue decoction (P < 0.05), while drug-free serum had no significant effect on the microenvironment of the co-culture system (P > 0.05). (3) The results show that Bushenhuoxue decoction can promote the survival, proliferation, and nucleus pulposus-like differentiation of adipose-derived stem cells.

Key words: adipose-derived stem cell, nucleus pulposus cell, co-culture, intervertebral disc degeneration, Bushenhuoxue decoction, oxidative damage