title: High glucose induces autophagy of MC3T3-E1 cells via ROS-AKT-mTOR axis.
abstract: In the present study, we investigate the function of ROS-AKT-mTOR axis on the apoptosis, proliferation and autophagy of MC3T3-E1 cells, and the proliferation of MC3T3-E1 cells after autophagy inhibition under high glucose conditions. MC3T3-E1 cells cultured in vitro were divided into the following groups: normal control group, N-acetylcysteine (NAC) group, 11.0 mM high glucose group, 11.0 mM high glucose + NAC group, 22.0 mM high glucose group, 22.0 mM high glucose + NAC group, CQ group, 22.0 mM high glucose + CQ group, 3-MA group and 3-MA + 22.0 mM high glucose group. ROS production was measured by DCFH-DA fluorescent probe. Cell proliferation was measured by MTT assay. Cells in different groups were stained with Annexin V-FITC/PI, and then apoptosis rate was detected by flow cytometry. Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest33258. Protein expression was measured using Western blotting and immunofluorescence. Cell apoptosis and proliferation in high glucose group were increased and decreased, respectively, in a dose-dependent manner. Autophagy was significantly induced in high glucose group, even though different concentration of glucose induced autophagy in different stages of autophagy. ROS production in MC3T3-E1 cells was remarkably increased in high glucose group, but not in a dose-dependent manner. NAC, as an antioxidant, reduced ROS production and ameliorated cell apoptosis, proliferation abnormity and autophagy caused by high glucose. Expression of p-AKT and p-mTOR proteins were dramatically decreased in high glucose group, and NAC reversed their expression. In addition, 3-MA, an inhibitor of autophagy, significantly decreased the proliferation of MC3T3-E1 cells. When cocultured with 22.0 mM glucose that induced autophagy, proliferation of MC3T3-E1 cells was not affected compared to 22.0 mM high glucose group. Our present findings reveal that high glucose affects apoptosis, proliferation and autophagy of MC3T3-E1 cells through ROS-AKT-mTOR axis. In addition, autophagy inhibition does not affect the proliferation of MC3T3-E1 cells under high glucose conditions.
authors.affiliation: Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Xiaoju
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China. Electronic address: fengzhengping112@163.com.
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Feng
authors.affiliation: Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Jiling
authors.initials: J
authors.lastname: Li
authors.affiliation: Central Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Lixue
authors.initials: L
authors.lastname: Chen
authors.affiliation: Central Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
authors.forename: Weixue
authors.initials: W
authors.lastname: Tang
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1 / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D053144:Cell Nucleus Shape / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000633:toxicity*; D051379:Mice; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013194:Staining and Labeling; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D000071186:Beclin-1; C115010:Becn1 protein, mouse; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D017382:Reactive Oxygen Species; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D005947:Glucose; D000111:Acetylcysteine
keywords: AKT-mTOR; Apoptosis; Autophagy; High glucose; Osteoblast; Proliferation; Reactive oxygen species