Protective role of 17β-estradiol on tumor necrosis factor-α-induced apoptosis in human nucleus pulposus cells

The molecular mechanisms underlying protection and pathogenesis in spinal degenerative diseases remain unclear. Tumor necrosis factor-α (TNF-α) has been demonstrated to induce Apoptosis of inte rvertebral disc (IVD) cells during IVD degeneration, and 17β‑estradiol (17β‑E2) has a protective effect against IVD cell Apoptosis. However, the underlying molecular mechanism by which 17β‑E2 protects nucleus pulposus (NP) cells remains to be investigated. The aim of the present study was to evaluate whether 17β‑E2 modulates Apoptosis of human NP cells induced by TNF‑α. In addition, the concentration‑response effect of 17β‑E2 on human NP cells was investigated. Human NP cells were cultured in complete medium, which was replaced every three days until the culture was ~80% confluent. Cells were treated with 100 ng/ml TNF‑α for 48 h, with or without pretreatment with various concentrations of 17β‑E2, and ICI 182,780, for 30 min. Morphologic alterations characteristic of Apoptosis were observed by inverted phase‑contrast microscopy and Hoechst 33258 staining; the Apoptosis rate was analyzed by flow cytometry. A Cell Counting kit‑8 assay was used to assess cell proliferation. Furthermore, caspase‑3 activity was determined and proteins associated with Apoptosis were analyzed by western blotting. The level of Apoptosis and caspase‑3 activity in human NP cells increased, whereas proliferation and the expression of poly ADP‑ribose polymerase decreased following TNF‑α treatment. These effects of TNF‑α were abolished by pretreatment with 17β‑E2 in a concentration‑dependent manner. The results of the present study indicated that 17β‑E2 serves a critical role in the survival of degenerative human NP cells.