Autophagy protects osteoblasts from advanced glycation end products-induced apoptosis through intracellular reactive oxygen species

1. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 1

   The viability of osteoblastic MC3T3-E1 cells treated with increasing concentrations of AGEs was tested via an MTT assay. Cell viability decreased with increasing concentrations of AGEs. The viability of cells treated with 200 mg/L AGEs decreased to 50% of that of the control group. Cell viability in osteoblastic MC3T3-E1 cells treated with > 300 mg/L AGEs was nearly eliminated. Values represent mean ±s.e.d. of at least three independent experiments. *P<0.05 vs control cells. ***P<0.001 vs control cells.
2. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 2

   The autophagy level was detected by assessing LC3I and LC3II and p62/SQSTMI expression using western blotting. (A and B) Autophagy increased in osteoblastic MC3T3-E1 cells treated with AGEs (200 mg/L). The total LC3 level and the LC3II/LC3I ratio statistically significantly increased after 8 and 12 h of AGEs treatment, as compared with control cells. However, after >12 h of AGEs treatment, the autophagy level was increased as compared with control cells, but was not increased to the same level as seen after 12 h of AGEs treatment. p62/SQSTMI was decreased in osteoblastic MC3T3-E1 cells treated with AGEs for > 4 h. Autolysosomes were detected in cultured osteoblastic MC3T3-E1 cells by intracellular LC3 redistribution, and were calculated as the average number of LC3-labeled punctae per cell. The average number of LC3-labeled punctae per cell in AGEs-treated cells was substantially increased as compared with control cells (C). The p62 level, as detected by western blotting, was decreased statistically significantly after treatment with AGEs for 8 h. (C) An immunofluorescence assay showed that LC3 punctae were markedly increased in osteoblastic MC3T3-E1 cells treated with AGEs for 12 h. (D) Quantitation of LC3 punctae per cell. Values represent mean ±s.e.m. of at least three independent experiments. ***P<0.001 vs control cells.
3. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 3

   AGEs-induced apoptosis in osteoblastic MC3T3-E1 cells in a time-dependent manner. Moreover, AGEs-induced apoptosis was affected by cotreatment with the autophagy inducer RA or the autophagy inhibitor 3-MA. (A) Apoptosis in osteoblastic MC3T3-E1 cells treated with AGEs (200 mg/L) for 0, 4, 8, 12, 16, or 24 h was assessed using annexin V and propidium iodide staining. Apoptotic cells are located in the upper-right quadrant. (B) The percent of cells in apoptosis in the different treatment groups is shown. (C) Karyopyknosis was visible in osteoblastic MC3T3-E1 cells after treatment with AGEs for 24 h. (D and E) Total cell death was reduced from 13.56 to 7.74% in cells treated with AGEs and the autophagy inducer RA (2 μM), as compared with treatment with AGEs alone. Total cell death increased from 13.56 to 29.1% in cells treated with AGEs and the autophagy inhibitor 3-MA (2 mmol/L), as compared with treatment with AGEs alone. No change in the percent of apoptotic cells was observed when cells were treated with either RA or 3-MA alone. Data are representative of three independent experiments. **P<0.01 vs control cells, ***P<0.001 vs control cells. ^##P<0.01 vs AGEs-treated cells.
4. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 4

   Intracellular oxidative stress assay in AGEs-treated osteoblastic MC3T3-E1 cells. (A) AGEs increased intracellular ROS generation in osteoblastic MC3T3-E1 cells in a time-dependent manner. The ROS assay used 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA), a reliable fluorogenic marker for ROS. The peaks of groups treated with AGEs for different time periods shifted to the right, indicating that DCF-positive cells (intracellular ROS-positive cells) increased. Data are representative of three independent experiments. (B and C) A decrease in ΔΨm, which represents mitochondrial damage, was determined by staining for JC-1, a well-known marker of ΔΨm. Cells with normal ΔΨm are depicted as those with high red and high green fluorescence in the upper right quadrant of the scatterplots. In contrast, cells that had decreased ΔΨm are depicted as those with high green and low red fluorescence in the lower right quadrant in response to the treatment with AGEs. Data are representative of three independent experiments. (D, E and F) Intracellular ROS levels and ΔΨm were affected by the autophagy inhibitor 3-MA (2 mmol/L), and the autophagy inducer RA (2 μM). When AGEs-treated cells were cotreated with RA (2 μM), the number of DCF-positive cells decreased, which indicates that intracellular ROS were reduced. ΔΨm was stable, which indicates that mitochondrial damage was attenuated. In contrast, when AGEs-treated cells were cotreated with 3-MA, intracellular ROS levels were increased, and ΔΨm balance was damaged. Data are representative of three independent experiments. #P<0.05 vs AGEs-treated cells. ^###P<0.001 vs AGEs-treated cells
5. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 5

   Western blotting and immunofluorescence assays analyzing the effect of ROS inhibitor on autophagy level in AGEs-treated osteoblastic MC3T3-E1 cells. In cells cotreated with NAC (10 mM), an inhibitor of intracellular ROS, LC3 expression was reduced as compared with that of cells treated with AGEs alone. Treatment with NAC alone had no effect on the expression of LC3I and LC3II. An immunofluorescence assay also showed that LC3 punctae were markedly decreased in osteoblastic MC3T3-E1 treated with NAC and AGEs for 12 h. Data are representative of three independent experiments. **P<0.01 vs control cells.***P<0.001 vs control cells. ^##P<0.01 vs AGEs-treated cells. ^###P<0.001 vs AGEs-treated cells.

• © 2016 Society for Endocrinology