title: Increased macroautophagy in the pathological process of intervertebral disc degeneration in rats.
abstract: OBJECTIVE
  Macroautophagy increases with age in rat intervertebral discs; however, the effect of macroautophagy on the process of intervertebral disc degeneration (IVDD) is still unclear. The aim of this study was to examine the presence of autophagosome, as well as the levels of Beclin-1 and LC3 proteins, in vivo. Additionally, in vitro evidence of macroautophagy and GRP78 and GADD153 protein levels were investigated to explore the mechanism of macroautophagy in the process of IVDD.
  
  
  METHODS
  Male Sprague-Dawley rats, aged 2 months, were randomly divided into six groups (three control and three model groups, n = 8 per group). At the 6-, 12-, and 18-week time points, autophagosomes in nucleus pulposus cells were detected with transmission electron microscope (TEM). Expression of Beclin-1 and LC3 protein levels within intervertebral disc was detected using Western blotting analysis. Then, the rat annulus fibrosus cells were isolated and cultured with Earle's balanced salt solution. At 1, 2, and 3 hr of culture, autophagosomes were detected using monodansylcadaverine assay, and LC3, Beclin-1, GRP78, and GADD153 protein levels were detected using Western blotting analysis.
  
  
  RESULTS
  Transmission electron microscopy revealed autophagosomes within nucleus pulposus cells in both the control and model groups. At 6-, 12-, and 18-week posttreatments, the levels of Beclin-1 and the LC3-II/LC3-I protein ratio in the model groups were higher than those in the control groups (p < 0.05). Compared with the control rats, amino acid starvation increased the number of monodansylcadaverine-positive cells and the LC3-II/LC3-I protein ratio in the model rats. Moreover, the in vitro levels of Beclin-1, GRP78, and GADD153 proteins were increased with the prolongation of amino acid starvation (p < 0.05).
  
  
  CONCLUSIONS
  Macroautophagy was present and was associated with increased pathological process of IVDD in rats. Macroautophagy of intervertebral disc cells is possibly secondary to endoplasmic reticulum stress.
authors: Department of Spine Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, P.R. China.
authors: Wei
authors: W
authors: Ye
authors: Weirong
authors: W
authors: Zhu
authors: Kang
authors: K
authors: Xu
authors: Anjing
authors: A
authors: Liang
authors: Yan
authors: Y
authors: Peng
authors: Dongsheng
authors: D
authors: Huang
authors: Chunhai
authors: C
authors: Li
mesh_terms: D000596:Amino Acids / Q000172:deficiency; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy; D000071186:Beclin-1; D015153:Blotting, Western; D002103:Cadaverine / Q000031:analogs & derivatives / Q000032:analysis; D002478:Cells, Cultured; D004195:Disease Models, Animal; D006360:Heat-Shock Proteins / Q000378:metabolism; D007403:Intervertebral Disc / Q000000981:diagnostic imaging / Q000378:metabolism / Q000473:pathology*; D055959:Intervertebral Disc Degeneration / Q000000981:diagnostic imaging / Q000378:metabolism / Q000473:pathology*; D008159:Lumbar Vertebrae / Q000000981:diagnostic imaging / Q000473:pathology; D008297:Male; D046529:Microscopy, Electron, Transmission; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism / Q000648:ultrastructure*; D011859:Radiography; D051381:Rats; D017207:Rats, Sprague-Dawley; D051742:Transcription Factor CHOP / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP
chemical_list: D000596:Amino Acids; D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; D002103:Cadaverine; D006360:Heat-Shock Proteins; D008869:Microtubule-Associated Proteins; D051742:Transcription Factor CHOP; C512829:Becn1 protein, rat; C497251:Ddit3 protein, rat; C051684:GRP78 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; C526109:Anxa3 protein, rat; C008542:monodansylcadaverine