The role of autophagy in ossification of the human ligamentum flavum / 中国组织工程研究

ABSTRACT

BACKGROUND: Pathological mechanism of ossification of the ligamentum flavum is unclear. There is no effective drug or non-surgical treatment in clinical practice. Current studies have found that osteopontin and autophagy play an important role in the process of osteogenesis, but their role in ossification of the ligamentum flavum has not been elucidated. OBJECTIVE: To seek for the potential target of drug therapy by exploring the mechanism of ossification of the ligamentum flavum. METHODS: (1) Surgical specimens of the ligamentum flavum were taken from patients with ossification of the ligamentum flavum, thoracic vertebrae or simple lumbar disc herniation undergoing posterior total laminectomy and decompression. These specimens were divided into two groups An ossification group and a non-ossification group. Eight specimens from each group were collected. Osteopontin, osteocalcin and autophagy indexes Beclin-1, LC3 and P62 were stained by immunohistochemistry. (2) The ligamentum flavum cells were isolated and cultured by adherence method. The third generation cells were treated with osteopontin at different concentrations for different time to construct an in vitro model of ligamentum flavum ossification. (3) Autophagy inhibitor 3-methyladenine with different concentrations was used to intervene with non-ossified ligamentum flavum cells, followed by induction with 100 μg/L osteopontin. Western blot assay was used to detect the expression of alkaline phosphatase, osteocalcin. (4) Non-ossified ligamentum flavum cells were induced with 100 μg/L osteopontin, and the induction was terminated at 0, 15, 30, 60, and 120 minutes, respectively. The phosphorylation of ERK1/2, JNK and P38, which are important molecules in the MAPK signaling pathway, was detected by western blot. (5) Finally, after inhibition by ERK1/2 phosphorylation blocker U0126, the expression of alkaline phosphatase and osteocalcin was detected by western blot after induction with 100 μg/L steopontin. RESULTS AND CONCLUSION: (1) Immunohistochemical staining of osteopontin and osteocalcin in ossified and non-ossified ligamentum flavum was positive. In the ossified ligamentum flavum, Beclin-1 was positive, but LC3 and P62 were not. Beclin-1, LC3 and P62 were all positive in the non-ossified ligamentum flavum. (2) The expression of alkaline phosphatase and osteocalcin in the ossified ligamentum flavum cells was higher than that in the non-ossified ligamentum flavum cells. Osteopontin could induce ossification of the ligamentum flavum in a concentration- and time-dependent manner. (3) The degree of ossification was negatively correlated with the degree of autophagy, that is, the more obvious autophagy was, the weaker ossification was. (4) Osteopontin could phosphorylate the MAPK signaling pathway in a time-dependent manner. After inhibiting the phosphorylation of MAPK, osteopontin could still induce the ossification of ligamentum flavum cells. To conclude, in the process of ligamentum flavum ossification, the upstream and downstream relationships of ERK1/2, osteopontin, alkaline phosphatase and osteocalcin molecules in signaling pathway are ERK1/2→osteopontin→osteocalcin/alkaline phosphatase.