抑制自噬对椎间盘细胞和细胞外基质的影响

IF 3.4 3区 医学 Q1 ORTHOPEDICS JOR Spine Pub Date : 2023-09-13 DOI:10.1002/jsp2.1286
Rebecca Kritschil, Vivian Li, Dong Wang, Qing Dong, Prashanta Silwal, Toren Finkel, Joon Lee, Gwendolyn Sowa, Nam Vo
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引用次数: 0

摘要

背景 椎间盘退变(IDD)是导致腰背痛(LBP)的主要原因。自噬是一种重要的细胞内质量控制过程,可清除受损蛋白质和细胞器,使其循环用于细胞生物合成和能量生产。自噬是许多与年龄有关的疾病的主要驱动因素,但自噬失调或缺乏尚未被证实会导致 IDD。 方法 在体外,用巴佛洛霉素 A1 处理大鼠髓核(NP)细胞以抑制自噬,评估糖胺聚糖(GAG)含量、蛋白多糖合成和细胞活力。在体内,成功培育出了转基因品系(Col2a1-Cre; Atg7fl/fl)小鼠,主要在NP组织中抑制自噬。我们对 Col2a1-Cre; Atg7fl/fl 小鼠椎间盘(IVD)进行了评估,检测其细胞凋亡和细胞衰老的生物标记物、骨胶原含量以及 12 个月大的组织学变化。 结果 在这里,我们证实了巴佛洛霉素抑制自噬作用会在大鼠 NP 细胞中产生 IDD 特征,包括细胞凋亡和细胞衰老(p21CIP1)增加以及椎间盘基质基因 Col2a1 和 Acan 表达减少。H&E组织学染色显示,与对照组相比,6个月和12个月大的Col2a1-Cre; Atg7fl/fl小鼠的NP组织发生了显著但温和的退行性变化。耐人寻味的是,12 个月大的 Col2a1-Cre; Atg7fl/fl 小鼠并没有表现出 NP 蛋白多糖丢失增加。此外,与对照组相比,12月龄的Col2a1-Cre; Atg7fl/fl小鼠的细胞凋亡标志物(裂解的caspase-3、TUNEL)和细胞衰老标志物(p53、p16INK4a、IL-1β、TNF-α)均未受到影响。然而,与对照组相比,12月龄 Col2a1-Cre; Atg7fl/fl 小鼠的 NP 组织中 p21CIP1 和 Mmp13 基因表达上调,这表明 NP 靶向 Atg7 基因敲除导致的 p21CIP1 介导的细胞衰老可能是观察到的组织学变化的原因之一。 结论 在NP组织中破坏Atg7介导的大自噬作用并没有导致明显的IDD特征,这意味着还存在其他代偿机制,突出表明还需要进行更多的研究来阐明自噬在调节年龄依赖性IDD方面的复杂生物学作用。
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Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix

Background

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.

Methods

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; Atg7fl/fl) mice were successfully generated to inhibit autophagy primarily in NP tissues. Col2a1-Cre; Atg7fl/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.

Results

Here, we demonstrated inhibition of autophagy by bafilomycin produced IDD features in the rat NP cells, including increased apoptosis and cellular senescence (p21CIP1) 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; Atg7fl/fl mice compared to controls at 6 and 12 months of age. Intriguingly, 12-month-old Col2a1-Cre; Atg7fl/fl mice did not display increased loss of NP proteoglycan. Moreover, markers of apoptosis (cleaved caspase-3, TUNEL), and cellular senescence (p53, p16INK4a, IL-1β, TNF-α) were not affected in 12-month-old Col2a1-Cre; Atg7fl/fl mice compared to controls. However, p21CIP1and Mmp13 gene expression were upregulated in NP tissue of 12-month-old Col2a1-Cre; Atg7fl/fl mice compared to controls, suggesting p21CIP1-mediated cellular senescence resulted from NP-targeted Atg7 knockout might contribute to the observed histological changes.

Conclusion

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.

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来源期刊
JOR Spine
JOR Spine ORTHOPEDICS-
CiteScore
6.40
自引率
18.90%
发文量
42
审稿时长
10 weeks
期刊最新文献
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