Parathyroid hormone 1­34 inhibits senescence in rat nucleus pulposus cells by activating autophagy via the m­TOR pathway.

Osteoporosis is closely associated with intervertebral disc degeneration. While parathyroid hormone (PTH) 1­34, which is an established drug used to treatosteoporosis, is thought to inhibit the disc degeneration associated with osteoporosis, the precise mechanism involved remains unclear. In the present study, primary Sprague­Dawley rat nucleus pulposus cells (NPCs) were cultured, phenotyped and then treated with dexamethasone (DXM) for 48 h. Cell area analysis and ß­galactosidase staining were used to investigate the effect of DXM on the senescence of NPCs. In addition, the protein levels of LC3­II, Beclin­1, P62, p­mTOR and p­p70S6k were determined by western blotting and analyzing the regulatory effect of PTH upon autophagy and the mTOR signaling pathway in cells treated with DXM. Following autophagic inhibition induced by ATG5 siRNA transfection, the regulatory effect of PTH on senescence in NPCs were investigated in addition to the potential role of autophagy. As the concentration of DXM increased, the size of the NPCs was significantly enlarged and the proportion of cells with positive ß­galactosidase staining increased significantly (P<0.05). In terms of protein expression, PTH treatment led to an increase in LC3­II and Beclin­1 proteins, a reduction in P62 protein, and inhibited p­mTOR and p­p70S6k protein expression in DXM­treated NPCs (P<0.05). PTH attenuated the effect of DXM according to the cell size and percentage of ß­galactosidase­positive cells. However, the inhibition of autophagy via ATG5 siRNA transfection reversed the protective effect of PTH on cell senescence (P<0.05). Collectively, the present findings suggest that PTH may inhibit the senescence of NPCs induced by DXM by activating autophagy via the mTOR pathway.

Pterostilbene inhibits reactive oxygen species production and apoptosis in primary spinal cord neurons by activating autophagy via the mechanistic target of rapamycin signaling pathway.

Autophagy is an important self-adaptive mechanism that is involved in inhibiting reactive oxygen species (ROS) in spinal cord neurons. Pterostilbene, a natural plant extract, has been demonstrated to possess antioxidant effects; however, it has not yet been investigated whether pterostilbene could activate autophagy and protect spinal cord neurons from oxidative stress. In the present study, primary spinal cord neurons of Sprague Dawley rats were cultured. Cell counting kit­8 analysis was used to detect cytotoxicity of pterostilbene. Cells were treated with various doses of pterostilbene for 24 and 48 h, respectively, and H2O2 was used to induce ROS production. Western blot analysis was performed to assess the protein expression of microtubule­associated protein 1 light chain 3 (LC3)­II, Beclin­1, p62, p­p70S6K and p­mechanistic target of rapamycin (mTOR). Furthermore, the green fluorescent protein (GFP)­LC3 assay was used to detect the level of autophagy level and activation mechanism. 2',7'­Dichlorofluorescin diacetate and MitoSOX Red staining were used to detect ROS production, and Terminal deoxynucleotidyl­transferase­mediated dUTP nick end labelling assay was used to analyze apoptosis percentage. ATG5 small interfering (si)RNA transfection was used to analyze the involvement of autophagy. A dose­dependent increase in the expression of LC3­II and Beclin­1, as well as the p62 decline, were observed in the pterostilbene­treated neurons; however, p­p70S6K and p­mTOR expression was inhibited by pterostilbene. Pterostilbene increased the expression of LC3­II in H2O2­treated cells, and GFP­LC3 analysis demonstrated an increased number of autophagosomes. Furthermore, pterostilbene significantly inhibited the ROS production and apoptosis induced by H2O2; however, ATG5 siRNA transfection significantly reversed the protection of pterostilbene. These results indicate that pterostilbene may inhibit the ROS production and apoptosis in spinal cord neurons by activating autophagy via the mTOR signaling pathway.