Objective

In the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5).

Design

In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1β) stimulation. In vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence.

Results

In disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval: −51.7, −37.1], 51.5% [−80.5, −22.5], 62.3% [−96.6, −28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [−0.8, 21.4]) but in serum-deprived medium (66.4% [−29.8, −8.6]) further with IL-1β (44.5% [−36.9, −23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [−52.8, −5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([−43.9, −0.8]), histological damage ([−5.5, −0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56 d.

Conclusions

This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.

中文翻译：

---

自噬参与维持大鼠椎间盘稳态：Atg5 的体外和体内 RNA 干扰研究

客观的

在最大的无血管低营养椎间盘中，常驻细胞将利用自噬，这是一种通过自我消化和回收废物的应激反应生存机制。我们的目标是通过自噬相关基因 5 (Atg5) 的 RNA 干扰来阐明自噬在椎间盘稳态中的作用。

设计

在体外，将靶向自噬必需 Atg5 的小干扰 RNA (siRNA) 转染到大鼠椎间盘细胞中。在血清饥饿和/或促炎性白细胞介素 1 β (IL-1β) 刺激下评估细胞活力和自噬水平，包括 Atg5 表达、细胞凋亡和衰老。在体内，在将 Alexa Fluor® 555 标记的 Atg5-siRNA 注射到大鼠尾椎间盘后监测自噬通量的时程。此外，通过放射照相、组织形态学和免疫荧光观察到 24 小时临时静态压缩诱导的 Atg5 siRNA 注射椎间盘破坏。

结果

在椎间盘细胞中，三种不同的 Atg5 siRNA 通过 Atg5 蛋白敲低持续抑制自噬（平均 44.4% [95% 置信区间：-51.7，-37.1]，51.5% [-80.5，-22.5]，62.3% [-96.6，-28.2 ]）。然后，Atg5 敲低通过细胞凋亡和衰老降低细胞活力，而不是在补充血清的培养基中（93.6% [-0.8, 21.4]），而是在血清剥夺培养基（66.4% [-29.8, -8.6]）中进一步加入 IL-1β（ 44.5% [-36.9, -23.5]）。在椎间盘组织中，即使在注射后 56 天，免疫荧光也检测到标记 siRNA 的椎间盘内信号。免疫印迹发现 56 天自噬抑制与延长的 Atg5 敲低 (33.2% [-52.8, -5.3])。压缩后，注射 Atg5 siRNA 的椎间盘出现放射学高度损失 ([-43.9, -0.8])、组织学损伤 ([-5.5, -0.2]) 和免疫荧光细胞凋亡 ([2.2, 22.2]) 和衰老 ([4.1, 19.

结论

这项功能丧失研究表明 Atg5 依赖性自噬介导的抗细胞凋亡和抗衰老。自噬可能是退行性椎间盘疾病的分子治疗靶点。