The Role of Smad2 in Transforming Growth Factor-β1 Induced Hypertrophy of Ligamentum Flavum.

BACKGROUND Hypertrophy of ligamentum flavum (LF) contributes to the development of spinal stenosis. Smad proteins can mediate the fibrogenesis activity through transforming growth factor-β1 (TGF-β1) pathway, but which Smad protein plays a more important role in the hypertrophy process of LF remained unclear. METHODS The LF samples were obtained from 50 patients. After culturing the ligamentum flavum cells (LFC), small interfering ribonucleic acid (siRNA) that target human phosphorylated－Smad2, 3, or 4(p- Smad2,3,4) genes was transfected into LFCs. After that, proteins from cells were extracted and the protein levels of Smad2, 3, and 4 were detected by western blot. The messenger ribonucleic acid (mRNA) level of TGF-β1 was measured by real-time polymerase chain reaction (PCR) Furthermore, enzyme linked immunosorbent assay (ELISA) assay was performed to test the impact of Smad2 on downstream of TGF-β1 signaling pathway. RESULTS Degeneration of the ligamentum flavum was characterized by an increase in disorganized elastic fibers and fibrotic transformation by extracellular collagen deposition. The gene expression analysis of fibrotic genes in LFC showed that knockdown of p-Smad2 by siRNA significantly reduced the protein expression level of TGF-β1 compared to other groups. The ELISA assay suggested that protein expression level of Smad2 can influence the downstream events of TGF-β1 signaling pathway in the LFC. CONCLUSION Our findings suggest that Smad2 plays a potential role in the pathological development of hypertrophy of LF. We also found that Smad2 knockdown by Smad-siRNA can influence TGF-β1 signaling pathway through decreasing expression of TGF-β1, TNF-α and NF- κb.