Department of Spine Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, P.R. China.
Connect Tissue Res. 2013;54(1):22-8. doi: 10.3109/03008207.2012.715702. Epub 2012 Aug 23.
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.
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.
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).
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.
大自噬在大鼠椎间盘中随年龄增长而增加；然而，大自噬对椎间盘退变（IVDD）过程的影响尚不清楚。本研究旨在体内检测自噬体的存在以及 Beclin-1 和 LC3 蛋白的水平。此外，还研究了体外大自噬和 GRP78 和 GADD153 蛋白水平的证据，以探讨大自噬在 IVDD 过程中的机制。
雄性 Sprague-Dawley 大鼠，2 月龄，随机分为 6 组（每组 3 只对照和 3 只模型组，每组 8 只）。在 6、12 和 18 周时，用透射电镜（TEM）检测髓核细胞中的自噬体。Western 印迹分析检测椎间盘内 Beclin-1 和 LC3 蛋白水平的表达。然后，分离并培养大鼠纤维环细胞，用 Earle 的平衡盐溶液培养。在培养 1、2 和 3 小时时，用单丹磺酰尸胺检测自噬体，Western 印迹分析检测 LC3、Beclin-1、GRP78 和 GADD153 蛋白水平。
透射电镜显示对照组和模型组髓核细胞内均有自噬体。在治疗后 6、12 和 18 周时，模型组 Beclin-1 和 LC3-II/LC3-I 蛋白比值均高于对照组（p<0.05）。与对照组大鼠相比，氨基酸饥饿增加了模型大鼠单丹磺酰尸胺阳性细胞数和 LC3-II/LC3-I 蛋白比值。此外，随着氨基酸饥饿的延长，体外 Beclin-1、GRP78 和 GADD153 蛋白水平升高（p<0.05）。
大鼠椎间盘中存在大自噬，与 IVDD 的病理过程增加有关。椎间盘细胞的大自噬可能继发于内质网应激。
