Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix.
Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix.
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作者：
Kritschil, Rebecca;Li, Vivian;Wang, Dong;Dong, Qing;Silwal, Prashanta;Finkel, Toren;Lee, Joon;Sowa, Gwendolyn;Vo, Nam
椎间盘退行性变(IDD)是导致下腰痛(LBP)的主要因素。自噬被低氧和营养饥饿强烈激活，是一个重要的细胞内质量控制过程，它去除受损的蛋白质和细胞器，使其循环用于细胞生物合成和能量生产。虽然自噬被公认为许多年龄相关疾病的主要驱动力，但自噬调节失调或缺乏仍被证实是导致IDD的原因。在体外，用巴菲洛霉素A1抑制自噬的大鼠髓核(NP)细胞被检测糖胺多聚糖(GAG)含量、蛋白多糖合成和细胞活力。在体内，成功地产生了一种转基因小鼠(Col2a1-Cre；ATG7fl/fl)，主要在NP组织中抑制自噬。对12 月龄的Col2a1-Cre；ATG7f1/f1小鼠椎间盘(IVD)进行了细胞凋亡和细胞衰老的生物标志物、凝集素含量和组织学变化的评估。在这里，我们证明了巴菲霉素抑制自噬在大鼠NP细胞中产生了IDD特征，包括增加了细胞凋亡和细胞衰老(P21CIP1)，以及减少了盘基质基因Col2a1和Acan的表达。H&E组织学染色显示，在6月龄和12 月龄时，Col2a1-Cre；ATG7f1/fl小鼠的NP组织与对照组相比有显著但轻微的变性变化。有趣的是，12个月大的Col2a1-Cre；ATG7fl/fl小鼠并没有表现出NP蛋白多糖丢失的增加。此外，与对照组相比，12月龄Col2a1-Cre；ATG7f1/f1小鼠的细胞凋亡标志(裂解caspase-3，TUNEL)和细胞衰老标志(P53，p16INK4a，IL-1β，肿瘤坏死因子-α)没有受到影响。然而，12月龄Col2a1-Cre；ATG7f1/fl小鼠的NP组织中p21CIP1和Mmp13基因的表达与对照组相比上调，提示p21CIP1介导的细胞衰老可能与NP靶向ATG7基因敲除导致的组织学变化有关。在NP组织中，缺乏明显的IDD特征来干扰ATG7介导的巨大自噬，这暗示了其他补偿机制，突显了需要更多的研究来阐明自噬在调节年龄依赖的IDD中的复杂生物学。这是首次使用一种新的NP靶向自噬消融转基因啮齿动物模型来研究自噬是否对体内IVD健康是必不可少的。我们发现只有温和的退变特征的Col2a1-Cre；Atg7fl/fl小鼠直到12 月龄的小鼠的椎间盘组织，这表明与IVD领域的普遍想法相反，NP自噬抑制不会在12 月龄的小鼠中加速缺碘。
Intervertebral disc degeneration (IDD) is a leading contributor to low back pain (LBP). Autophagy, strongly activated by hypoxia and nutrient starvation, is a vital intracellular quality control process that removes damaged proteins and organelles to recycle them for cellular biosynthesis and energy production. While well‐established as a major driver of many age‐related diseases, autophagy dysregulation or deficiency has yet been confirmed to cause IDD. In vitro, rat nucleus pulposus (NP) cells treated with bafilomycin A1 to inhibit autophagy were assessed for glycosaminoglycan (GAG) content, proteoglycan synthesis, and cell viability. In vivo, a transgenic strain (Col2a1‐Cre; Atg7 fl/fl ) mice were successfully generated to inhibit autophagy primarily in NP tissues. Col2a1‐Cre; Atg7 fl/fl mouse intervertebral discs (IVDs) were evaluated for biomarkers for apoptosis and cellular senescence, aggrecan content, and histological changes up to 12 months of age. Here, we demonstrated inhibition of autophagy by bafilomycin produced IDD features in the rat NP cells, including increased apoptosis and cellular senescence (p21 CIP1 ) and decreased expression of disc matrix genes Col2a1 and Acan. H&E histologic staining showed significant but modest degenerative changes in NP tissue of Col2a1‐Cre; Atg7 fl/fl mice compared to controls at 6 and 12 months of age. Intriguingly, 12‐month‐old Col2a1‐Cre; Atg7 fl/fl mice did not display increased loss of NP proteoglycan. Moreover, markers of apoptosis (cleaved caspase‐3, TUNEL), and cellular senescence (p53, p16 INK4a , IL‐1β, TNF‐α) were not affected in 12‐month‐old Col2a1‐Cre; Atg7 fl/fl mice compared to controls. However, p21 CIP1 and Mmp13 gene expression were upregulated in NP tissue of 12‐month‐old Col2a1‐Cre; Atg7 fl/fl mice compared to controls, suggesting p21 CIP1 ‐mediated cellular senescence resulted from NP‐targeted Atg7 knockout might contribute to the observed histological changes. The absence of overt IDD features from disrupting Atg7‐mediated macroautophagy in NP tissue implicates other compensatory mechanisms, highlighting additional research needed to elucidate the complex biology of autophagy in regulating age‐dependent IDD. This is the first study to investigate whether autophagy is essential for IVD health in vivo using a novel transgenic rodent model of NP‐targeted autophagy ablation. We found only modest degenerative features disc tissue of Col2a1‐Cre; Atg7fl/fl mice up to 12 months of age suggesting that contrary to the prevailing thinking in the IVD field, NP autophagy inhibition does not accelerate IDD in mice up to 12 months of age.
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影响因子：
5
作者：
Hashemzaei, Mahmoud;Heravi, Reza Entezari;Karimi, Gholamreza
通讯作者：
Karimi, Gholamreza
影响因子：
3.7
作者：
Melgoza IP;Chenna SS;Tessier S;Zhang Y;Tang SY;Ohnishi T;Novais EJ;Kerr GJ;Mohanty S;Tam V;Chan WCW;Zhou CM;Zhang Y;Leung VY;Brice AK;Séguin CA;Chan D;Vo N;Risbud MV;Dahia CL
通讯作者：
Dahia CL
影响因子：
16
作者：
Kroemer G;Mariño G;Levine B
通讯作者：
Levine B
影响因子：
5.6
作者：
Kritschil R;Scott M;Sowa G;Vo N
通讯作者：
Vo N
影响因子：
4.3
作者：
Congdon EE
通讯作者：
Congdon EE