Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5

• DOI: 10.1016/j.joca.2021.12.004
• Publication Date: 2021-12-24T08:27:14Z
• Authors: R. Tsujimoto; T. Yurube; Y. Takeoka; Y. Kanda; K. Miyazaki; H. Ohnishi; Y. Kakiuchi; S. Miyazaki; Z. Zhang; T. Takada; R. Kuroda; K. Kakutani
• Keywords: Viability assay

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 involvement of autophagy in disc homeostasis through RNA interference autophagy-related gene 5 (Atg5).In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat cells. Cell viability with levels including expression, apoptosis, senescence assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1β) stimulation. vivo, time-course autophagic flux monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection tail discs. Furthermore, 24-h temporary static compression-induced disruption siRNA-injected discs observed radiography, histomorphology, immunofluorescence.In cells, three different siRNAs consistently suppressed 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, reduced cell apoptosis not serum-supplemented medium (93.6% [-0.8, 21.4]) but serum-deprived (66.4% [-29.8, -8.6]) further IL-1β (44.5% [-36.9, -23.5]). tissues, immunofluorescence detected intradiscal signals for labeled siRNA even at 56-d post-injection. Immunoblotting found suppression prolonged (33.2% [-52.8, -5.3]). With compression, presented radiographic height loss ([-43.9, -0.8]), histological damage ([-5.5, -0.2]), immunofluorescent ([2.2, 22.2]) ([4.1, 19.9]) induction compared control 56 d.This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis anti-senescence. Autophagy could be molecular therapeutic target degenerative disease.