Abstract: BACKGROUND: Studies have shown that intradiscal injection of syringin solution can improve the structure and function of the intervertebral disc, prevent and slow down the process of intervertebral disc degeneration in rats. However, the biological half-life of syringin is short and it is difficult for it to continue to play a role in the intervertebral disc. Its bioavailability needs to be further improved.
OBJECTIVE: To observe the effect of syringin-chitosan hydrogel on intervertebral disc degeneration in rats and the mechanism of syringin in the treatment of intervertebral disc degeneration. 
METHODS: (1) Cell experiment: Passages 2-5 rat nucleus pulposus cells were divided into four groups for treatment. The normal control group did not undergo any treatment. The degeneration group was added with interleukin-1β (to establish the intervertebral disc degeneration cell model). The drug group was added with interleukin-1β and syringing. The inhibitor group was added with interleukin-1β, syringing, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. After 24 hours of treatment, apoptosis, extracellular matrix, oxidative stress, and apoptosis-related proteins and PI3K/protein kinase B (AKT) signaling pathway proteins were detected respectively. (2) Animal experiment: Syringin-chitosan hydrogels were prepared, and the micromorphology and slow-release properties of the hydrogels were tested. Thirty SD rats were randomly divided into model control group, model intervention group, hydrogel group, syringin solution group, and syringin hydrogel group, with 6 rats in each group. The intervertebral disc degeneration model was established by the acupuncture method. Immediately after model establishment, the rats in model intervention group, hydrogel group, syringin solution group, and syringin hydrogel group were injected with PBS, chitosan hydrogel, syringin solution, and syringin-chitosan hydrogel, respectively. The samples were taken 8 weeks after modeling for histological detection.
RESULTS AND CONCLUSION: (1) Cell experiment: Compared with the normal control group, apoptosis rate, reactive oxygen species level, and expression of BAX, cleaved caspase-9, cleaved caspase-3, and matrix metalloproteinase 13 protein were increased in the nucleus pulpocytes in the degeneration group (P < 0.05), and the expression levels of p-PI3K, p-AKT, BCL-2, and type II collagen were decreased (P < 0.05). Superoxide dismutase activity decreased (P < 0.05). Compared with the degeneration group, apoptotic rate, reactive oxygen species level, and expression of BAX, cleaved caspase-9, cleaved caspase-3, and matrix metalloproteinase 13 protein were decreased in the syringin solution and syringin solution groups (P < 0.05), and expression levels of p-PI3K, p-AKT, BCL-2, and type II collagen were increased (P < 0.05). Superoxide dismutase activity increased (P < 0.05). LY294002 could partially inhibit the effect of syringin. (2) Animal experiment: Syringin-chitosan hydrogel had a loose porous structure and good slow-release performance. Hematoxylin-eosin and safranin O-fast green staining showed that compared with the model control group and model intervention group, chitosan hydrogel, syringin solution and syringing-chitosan hydrogel could improve the intervertebral disc degeneration in different degrees, and the therapeutic effect of syringing-chitosan hydrogel was better than that of hydrogel and drug solution alone. (3) These findings indicate that syringin can regulate apoptosis of nucleus pulposus cells and extracellular matrix degradation induced by oxidative stress by activating PI3K/AKT signaling pathway, thus delaying disc degeneration. Compared with syringin injection alone, syringin loading in chitosan hydrogel can further delay the progression of intervertebral disc degeneration in rats. 

Key words: nucleus pulposus cell, intervertebral disc degeneration, syringin, chitosan, hydrogel, engineered intervertebral disc