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Abstract
Objective: Excessive apoptosis plays an important role in the progression of intervertebral disc degeneration. However, the effect of autophagy, another type of programmed cell death, on the pathogenesis of disc degeneration is still unclear. Macroautophagy and chaperone-mediated autophagy (CMA) change and intervertebral disc degeneration aggravates with age. This study aims at examining the expression changes of light chain 3 (LC3), lysosome-associated membrane protein 2A (LAMP-2A), and Hsc70, the indicator substrates of macroautophagy and CMA, in rat nucleus pulposus (NP) to prove that macroautophagy and CMA are both related with age. Methods: Female Sprague–Dawley rats of 3, 12, and 24 months (n = 8 per age) were used in this study. Autophagic vacuoles in NP cells were detected by transmission electron microscopy. In NP, the expressions of LC3-II and LAMP-2A protein and mRNA were examined by immunohistochemistry and reverse transcription polymerase chain reaction, respectively. LC3-II, LC3-I, and LAMP-2A protein were also measured by western blot. The mRNA and protein level of myocyte enhancer factor-2D regulated by LAMP-2A and Hsc70 were detected by reverse transcriptase polymerase chain reaction and western blot, respectively. Results: Transmission electron microscopy showed more autophagic vacuoles in 12- and 24-month groups than in 3-month group. Expression of LC3-II and LC3-II/LC3-I in 24-month group was significantly higher than in 3-month group (p < 0.05). Meanwhile, LAMP-2A expression was significantly higher in 24-month group than in 3-month group (p < 0.05). However, lower expression of Hsc70 and myocyte enhancer factor-2D was found in the 24-month rats than in 3-month group (p < 0.05, p < 0.05, respectively). Conclusion: Macroautophagy and CMA were present and increased with age in rat NP.
Acknowledgments
This work was supported by grants from the New Teacher's Doctoral Fund of Ministry of Education of China (no. 20090171120077), the Natural Science Foundation of Guangdong Province, China (no. 10151008901000084), the Science and Technology Planning Project of Guangdong Province, China (nos. 2008B060600059, 2009B060700097), and Medical Scientific Research Foundation of Guangdong Province, China (no. A2009188).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.