TGF-β1 reduces the oxidative stress-induced autophagy and apoptosis in rat annulus fibrosus cells through the ERK signaling pathway.

Abstract

The aim of this study is to explore the effects of TGF-β1 on autophagy and apoptosis induced by exogenous hydrogen peroxide (H₂O₂) in annulus fibrosus (AF) cells and possible signal pathways involved in this process. 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-β1 (5, 10, 20 ng/mL), the effects of TGF-β1 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. 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-β1 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-β1 downregulated the level of intracellular H₂O₂ through upregulating the expression level of glutathione peroxidase-1 (GPx-1). TGF-β1 reduced autophagy and apoptosis induced by exogenous H₂O₂ through downregulating the expression of ERK in AF cells. TGF-β1 could downregulate the level of ERK and intracellular H₂O₂ by upregulating GPx-1.