The mechanism of naringin on the apoptosis of nucleus pulposus cells of human degenerative intervertebral disc by Wnt/β-catenin signaling pathway

Abstract

Objective To study the effects of naringin on the apoptosis of nucleus pulposus cells of human degenerative intervertebral disc. Methods Nucleus pulposus cells were identified via the toluidine blue and safranin O. Next, P3 cells were cultured with naringin 0 μg/ml (control group), naringin group (10, 20, 40, 80, 100 μg/ml). After 48 hours, the best concentration of naringin was selected by cell counting kit-8 (CCK-8) and flow cytometry. β-catenin, Cyclin D1, c-Myc gene expression was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The expression of B cell lymphoma/leukemia-2 (bcl-2), bcl-2 associated X protein (bax), Caspase-3 protein, type Ⅱ collagen and proteoglycan was determined by enzyme linked immunosorbent assay (ELISA). Western blotting detected of type Ⅱ collagen and proteoglycan expression. Results The results of cells staining were positive. CCK-8 and flow cytometry were used to determine the optimum concentration of naringin (20 μg/ml) to inhibit the apoptosis of nucleus pulposus cells. In the human degeneration nucleus pulposus cells, the expression of β-catenin gene was overexpressed and the expression of Cyclin D1 and c-Myc gene was down regulated. 20g/ml naringin could down regulate the expression of β-catenin gene (P=0.000), and up regulate the expression of Cyclin D1 and c-Myc gene (P=0.000, 0.001). The naringin (20 μg/ml) could obviously regulate up the expression of bcl-2, type Ⅱ collagen and proteoglycan protein (P=0.001, 0.000, 0.023), and regulate down the expression of bax and Caspase-3 protein, (P=0.001, 0.001). Western blotting showed that 20 μg/ml naringin could promote the expression of type Ⅱ collagen and proteoglycan. Conclusion Naringin may inhibit the apoptosis of nucleus pulposus cells by regulating the Wnt/β-catenin pathway and the mitochondrial apoptosis pathway. Key words: Naringin; Human nucleus pulpous; Apoptosis; Intervertebral disc; Degeneration