TGF-ß1 reduces the oxidative stress-induced autophagy and apoptosis in rat annulus fibrosus cells through the ERK signaling pathway.

ABSTRACT

BACKGROUND: The aim of this study is to explore the effects of TGF-ß1 on autophagy and apoptosis induced by exogenous hydrogen peroxide (H2O2) in annulus fibrosus (AF) cells and possible signal pathways involved in this process. METHODS: AF cells were isolated from rat lumbar discs and subjected to different concentrations of exogenous H2O2 (50, 100, 200 µmol/L) for different time periods (0.5, 1, 2, and 4 h). Cell viability was determined by CCK-8 assay, and the levels of autophagy and apoptosis were evaluated by Western blotting and caspase 3, 8, 9 activity assay. By administration with different concentrations of TGF-ß1 (5, 10, 20 ng/mL), the effects of TGF-ß1 on autophagy and apoptosis induced by H2O2 were observed, and the possible signaling pathways were also investigated by using various apoptosis inhibitors or an autophagy inhibitor Bafilomycin A (Baf A) in AF cells. RESULTS: H2O2 significantly impaired cell viability in a dose- and time-dependent manner. H2O2 also induced a sudden and the highest level of autophagy at 1 h, and gradually increased apoptosis through ERK pathway. The mitochondrial pathway was involved in H2O2-induced apoptosis in AF cells. TGF-ß1 reduced the expression of p-ERK and downregulated the expressions of Beclin-1, LC3 II/I, and mitochondrial-related apoptotic proteins (Bax/Bcl-2, caspase-9). Meanwhile, TGF-ß1 downregulated the level of intracellular H2O2 through upregulating the expression level of glutathione peroxidase-1 (GPx-1). CONCLUSIONS: TGF-ß1 reduced autophagy and apoptosis induced by exogenous H2O2 through downregulating the expression of ERK in AF cells. TGF-ß1 could downregulate the level of ERK and intracellular H2O2 by upregulating GPx-1.