Abstract: BACKGROUND: Salvianolic acid B can inhibit cell damage induced by H2O2, effectively remove excess reactive oxygen species, and exert antioxidant properties. It has been used in the treatment of many diseases. However, there are relatively few studies on the role and mechanism of salvianolic acid B in intervertebral disc degeneration.
OBJECTIVE: To observe the effect and mechanism of salvianolic acid B on oxidative stress-induced intervertebral disc degeneration by using gelatin methacryloyl hydrogel as a carrier through the in vitro cell experiment and the in vivo animal experiment.
METHODS: The gelatin methacryloyl hydrogel (drug-loaded hydrogel) loaded with salvianolic acid B was prepared. (1) In vitro cell experiment: The lumbar nucleus pulposus cells of adult SD rats were isolated and extracted, and passage 3 nucleus pulposus cells were selected and divided into groups: Group A was added complete medium. In group B, a complete medium containing H2O2 was added. Group C was inoculated on methylacrylylated gelatin hydrogel and added with a complete medium containing H2O2. Group D was inoculated on methyl acrylyl gelatin hydrogel loaded with salvianolic acid B and added into a complete medium containing H2O2. The E group was inoculated on the methylacrylyl gelatin hydrogel loaded with salvianolic acid B, and the complete medium containing H2O2 and the complete medium containing TLR4 signaling pathway inhibitor were added. Cell proliferation, oxidative stress, inflammatory response, gene expression of cell matrix-associated proteins and the protein expression of TLR4/nuclear factor-kB signaling pathway were detected. (2) Animal in vivo experiment: Sixty adult SD rats were randomly divided into normal group, acupuncture group, acupuncture + salvianolic acid group, acupuncture + hydrogel group and acupuncture + loading potion gel group, with 12 rats in each group. The last four groups were treated with acupuncture to establish models of intervertebral disc degeneration and then injected with normal saline, salvianolic acid B solution, non-drug loaded gel and drug-loaded gel in turn. Imaging examination and pathological observation were performed 4 weeks after surgery.
RESULTS AND CONCLUSION: (1) In vitro cell experiment: Compared with group A, the cell proliferation was decreased; the oxidative stress reaction and inflammation reaction were enhanced; the expression of extracellular matrix degrading enzymes (matrix metalloproteinase 3, matrix metalloproteinase 13, ADAMTS4, ADAMTS5) was increased in group B (P < 0.05), and the synthesis of extracellular matrix (type II collagen, proteoglycan) was decreased (P < 0.05). The protein expression of the TLR4/nuclear factor-kB signaling pathway was increased (P < 0.05). Compared with group B, the cell proliferation of groups D and E was increased, the oxidative stress response and inflammatory response were weakened, and the expression of extracellular matrix degrading enzymes (matrix metalloproteinase 3, matrix metalloproteinase 13, ADAMTS4, ADAMTS5) was decreased (P < 0.05), and the synthesis of extracellular matrix was increased (P < 0.05). The protein expression of TLR4/nuclear factor-kB signaling pathway was decreased (P < 0.05), and the effect was more significant in group E. (2) Animal in vivo experiment: 4 weeks after surgery, intervertebral disc height index, index of MRI and pathological and histological grading of the intervertebral disc had improved significantly in the acupuncture+drug-loaded hydrogel group, and simply injecting hydrogel or salvianolic acid B solution can to a certain extent improve the intervertebral disc degeneration, but they are not as good as the injection of the drug-loaded hydrogel. (3) It is concluded that gelatin methacryloyl hydrogel loaded with salvianolic acid B can inhibit oxidative stress and inflammation in the degenerated intervertebral disc tissue, inhibit the degradation of extracellular matrix, and alleviate the process of intervertebral disc degeneration, which may be accomplished by inhibiting the TLR4/nuclear factor-kB signaling pathway.

Key words: intervertebral disc degeneration, nucleus pulposus cell, salvianolic acid B, hydrogel, oxidative stress, gelatin methacryloyl