Study on the mechanism of asporin in degeneration of intervertebral disc / 中华骨科杂志

ABSTRACT

Objective To explore the molecular regulation mechanism of asporin in the matrix synthesis and secretion of the intervertebral disc,and to clarify its role in degenerative lesions of intervertebral disc.Methods There were 8 cases of intervertebral disc tissue in patients with severe intervertebral disc herniation (including typical clinical symptoms,signs and Pfirrmann's grade Ⅲ).There were 6 male and 2 female with an average age of (20.25 ± 3.37) years old (ranged from 11 to 28 years).After primary culture and redifferentiation in alginate beads,cells were reseeded and treated with different concentrations of TGF-β1 for 6,12,18 and 24 h.Total RNA extracted from the cells and was subjected to real-time PCR analysis to examine the expression of asporin.After silencing the expression of endogenous asporin by siRNA,the cells stimulated 24 h with TGF-β1.Total RNA extracted from the cells and was subjected to real-time PCR analysis to examine the expression of asporin,collagen Ⅱ and proteoglycans.After treatment of specific p38 inhibitor or ERK inhibitor for 12 h,cells were stimulated with TGF-β1 for 24h.Protein extracted from the cells by protein extraction kit to examine the level of asporin.Results In the primary intervertebral disc cell experiment,TGF-β1 stimulation induced asporin transcription significantly in a dose and time dependent manner.After 24 h stimulation,a significant difference between different concentration groups (5,10 and 15 ng/ml) was observed,2.754±0.24,3.651 ±0.319 and 4.583±0.38,respectively (F=24.782,P=0.001).Knockdown of endogenous asporin led to the upregulated expression of aggrecan and collagen]Ⅱ (aggrecant=7.387,P=0.002,collagen Ⅱt=4.443,P=0.0113).Specific p38 inhibitor was used to block p38 phosphorylation,and TGF-β1 on asporin induction was significantly inhibited.Conclusion Our results have verified a functional feedback loop between TGF-β1 and asporin in human intervertebral annulus cells indicating that TGF-β1 can increase asporin expression,whereas asporin inhibits TGF-β 1 signaling pathway by negative feedback,thereby inhibiting TGF-β1 mediated synthesis of extracellular matrix,and TGF-31 can increase asporin expression by p38 in human intervertebral disc cells.