The Role Of Autophagy In Oxidative Damage To Osteoblasts And Its Contribution To Osteoporosis

Part â…  Increased activity of osteocyte autophagy in ovariectomized rats and itscorrelation with oxidative stress status and bone lossIntroduction:The objectives of the present study were to investigate the autophagy activity of osteocytes in ovariectomized rats and to examine whether autophagy of osteocytes is associated with bone loss and oxidative stress status.Methods:36 female Sprague-Dawley rats were randomly divided into 3 groups: sham-operated (Sham), ovariectomized (OVX), and OVX with 17-(3-estradiol. Six weeks later, bone mineral density (BMD) and bone micro-architecture in the proximal tibia were assessed by micro-CT and DEXA. The autophagy activity of osteocytes in the proximal tibia was evaluated by Transmission Electron Microscopy (TEM), Immunofluorescent histochemistry (IHC), qRT-PCR and Western blot. Serum 17-(3-estradiol (E2) level was measured by enzyme linked immunosorbent assay (ELISA). Total Antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, and catalase (CAT) activity in the proximal tibia were also determined by ELISA. In addition, we analyzed the correlation between oxidative stress status and autophagy.Result:Ovariectomy increased the mRNA and protein expressions of BECN-1 and microtubule associated protein 1 light chain 3 (Mapl-LC3)) while decreased the expressions p62/SQSTM1 in the proximal tibia. Ovariectomy also declined the activities of T-AOC, CAT, and SOD. Treatment with E2 prevented the reduction in bone mass as well as restored the autophagy level. Furthermore, Mapl-LC3B expression was inversely correlated with T-AOC, CAT, and SOD activities. Mapl-LC3B expression negatively correlated with BV/TV, Tb.N, and BMD in proximal tibias.Conclusion:Ovariectomy induced oxidative stress and increased the autophagy activity of osteocytes. The autophagy activity was inversely correlated with the oxidative stress status of osteocytes, and with the microarchitectural deterioration of bone.Part â…¡ Oxidative damage to osteoblasts can be alleviated by early autophagy through the endoplasmic reticulum stress pathwayIntroduction:Oxidative stress can damage various cellular components of osteoblasts, and is regarded as a pivotal pathogenic factor for bone loss. However, the role of autophagy in the osteoblasts under oxidative stress remains to be clarified. The current study was designed to investigate the role of autophagy in oxidative damage to osteoblast and the interrelationship between autophagy and apoptosis.Methods:Osteoblastic Mc3T3-E1 cells were exposed to hydrogen peroxide in different concentrations (0.1,1,10 mM). At indicated time point (0,2,6,8,12hrs), Ultrastructural analysis, flow cytometry and lysosomal activity assessment were performed after the in vitro treatment to determine the presence and levels of autophagy. Reactive oxygen species level was evaluated by flow cytometry after DCFH-DA staining. Next, the expressions of autophagy-related proteins (BECN-1 and Mapl-LC3) were assessed using western blot. Rapamycin (autophagy inducer), 3-methyladenine (the early steps of autophagy inhibitor) and bafilomycin A1 (the last steps of autophagy inhibitor) were used to observe the interactions between autophagy and apoptosis. Meanwhile, we evaluated the expression of the endoplasmic reticulum-stress markers, such as p-PERK, PERK, PDI, GRP-78. Subsequently, PERK, GRP-78 and IRE1 genes were knocked down using Talen technique and then the autophagy activity were assessed by western blot and fuorescence microscopy to determine the interplay between autophagy and endoplasmic reticulum-stress. Additionally, we investigated the co-localization of mitochondria and autophagosomes in Mc3T3-E1 cells under oxidative stress in order to explore the link between mitochondria damage and autophagy.Results:In this study, we verified that hydrogen peroxide induced autophagy and apoptosis in a dose-time dependent manner in the osteoblastic Mc3T3-E1 cells. Both 3-methyladenine and bafilomycin A1 enhanced the cell apoptosis and reactive oxygen species level in the osteoblasts insulted by hydrogen peroxide. However, promotion of autophagy with either pharmacologic inducer (rapamycin) or Beclin-1 over-expressing technique rescued the cell apoptosis and reduced the reactive oxygen species level in the cells. The mitochondrial membrane potential decreased significantly while the mitochondria-mediated apoptosis markers increased significantly in the cells under oxidative stress. Furthermore, the molecular levels of the endoplasmic reticula stress signaling pathway changed in hydrogen peroxide-treated Mc3T3-El cells, and blocking this stress signaling pathway by Talen technique against candidates of glucose-regulated protein 78 and protein kinase-like endoplasmic reticulum kinase decreased autophagy while increased apoptosis in the cells. The result of fuorescence microscopy showed that 23% co-localization of GFP-LC3 and RFP-mito after 2 hrs H2O2 treatment.Conclusions:Oxidative damage to osteoblasts could be alleviated by early autophagy through the endoplasmic reticulum stress pathway. Autophagy may represent a protective mechanism against ROS-mediated apoptosis and modulation of osteoblast autophagy might have a potentially therapeutic value for osteoporosis.Part â…¢ Effects of BECN-1 on the proliferation, differentiation and apoptosis of preosteoblastic Mc3T3-ElcellsIntroduction:Autophagy could protect against oxidative stress damage to osteoblasts, but the mechanism is still unknown. BECN-1 is a well-known key regulator of autophagy and previous studies demonstrated that BECN-1 was involved in many pathological processes including aging, osteoarthritis and degenerative diseases. However, the function of BECN-1 in osteoporosis remains unclear. Thus, the objective of the present study was to investigate whether BECN-1 acted directly on the proliferation, differentiation and apoptosis of osteoblasts.Methods:We overexpressed and knocked down BECN-1 in Mc3T3-E1 cells by using the lentiviral overexpression system and Talen technique respectively. Cell viability was estimated by measuring the cell count kit-8 (CCK-8). The effects of overexpression and knockdown of BECN-1 on osteoblastic cell cycle distributions were analyzed by using flow cytometry assay. During osteogenic induction, the effects of BECN-1 on osteoblast differentiation and mineralization were determined by qRT-PCR, western blot assay, and Alizarin Red staining. Finally, using flow cytometry assay by staining with Annexin V-PE/7AAD staining, we addressed the role of BECN-1 in the osteoblast apoptosis under oxidative stress.Results:CCK-8 assay demonstrated that the proliferation of Mc3T3-E1 cells was suppressed by knockdown of BECN-1, but not changed by BECN-1 overexpression. Overexpression of BECN-1 did not alter cell cycle while knockdown of BECN-1 caused an arrest of S phase. Under oxidatve stress condition, overexpression of BECN-1 reduced the cell apoptosis incidence while knockdown of BECN-1 rendered them more sensitive to hydrogen peroxide. Mineralized nodule after osteogenic induction for 4 weeks were independently suppressed by knockdown of BECN-1, but not changed after overexpression of BECN-1. After osteogenic induction for 48 hrs, the mRNA and protein expressions of Runx2, OCN, COL-1 were suppressed by knockdown of BECN-1, whereas the expressions were not affected by overexpression of BECN-1.Conclusions:These studies suggested that BECN-1 knockdown suppressed not only proliferation, but also osteogenic differentiation of osteoblats. BECN-1 knockdown rendered Mc3T3-E1 cells more sensitive to hydrogen peroxide whereas BECN-1 overexpression partly rescued the cell apoptosis.