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

Tsujimoto, Ryu; Yurube, Takashi; Takeoka, Yoshiki; Kanda, Yutaro; Miyazaki, Kunihiko; Ohnishi, Hiroki; Kakiuchi, Yuji; Miyazaki, Shingo; Zhang, Zhongying; Takada, Toru; Kuroda, R.; Kakutani, Kenichiro

doi:10.1016/j.joca.2021.12.004

Cited by 16 publications

(18 citation statements)

References 52 publications

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“…6 A). Atg5 silencing solely doesn’t lead to apoptosis of nucleus pulposus cells [ 26 , 31 ]. After 24 h of indicated treatment, cleaved caspase-9, B-cell lymphoma 2 (Bcl-2), BCL2 Associated X (Bax) were analyzed by western blot.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, cell membrane integrity was also disrupted if autophagy was blocked, in consistent with mitochondrial apoptosis in our study. Not only apoptosis, ATG5 and autophagy block also play an critical role in senescence [ 31 ]. Together, autophagy and ATG5 are crucial to apoptosis and other functional characteristics of NPCs and further contribute to the development of IDD.…”

Section: Discussionmentioning

confidence: 99%

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Celecoxib activates autophagy by inhibiting the mTOR signaling pathway and prevents apoptosis in nucleus pulposus cells

Chen

Yasen

Wang

et al. 2022

BMC Pharmacol Toxicol

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Background Intervertebral disc degeneration results from a variety of etiologies, including inflammation and aging. Degenerated intervertebral discs feature down-regulated extracellular matrix synthesis, resulting in losing their ability to retain water and absorb compression. Celecoxib is a well-known selective cyclooxygenase-2 inhibitor for treating arthritis and relieving pain. Nevertheless, the mechanism of Celecoxib for treating inflammation-related intervertebral disc degeneration has not yet been clarified. Method Protein synthesis was analyzed by western blot. Fluorescent probes DCFH-DA and MitoSox Red detected reactive oxygen species and were measured by flow cytometry. The activity of the kinase pathway was evaluated by protein phosphorylation. Autophagy was monitored by mRFP-GFP-LC3 transfection and LC3 analysis. Mitochondrial apoptotic proteins were analyzed by western blot and cell membrane integrity was measured by flow cytometry. The autophagic gene was silenced by siRNA. Results In this study, interleukin-1β stimulation reduced the synthesis of aggrecan, type I and II collagen and caused excessive production of reactive oxygen species. We looked for a therapeutic window of Celecoxib for nucleus pulposus cells to regain extracellular matrix synthesis and reduce oxidative stress. To look into nucleus pulposus cells in response to stimuli, enhancement of autophagy was achieved by Celecoxib, confirmed by mRFP-GFP-LC3 transfection and LC3 analysis. The mammalian target of rapamycin and a panel of downstream proteins responded to Celecoxib and propelled autophagy machinery to stabilize homeostasis. Ultimately, inhibition of autophagy by silencing autophagy protein 5 disrupted the protective effects of Celecoxib, culminating in apoptosis. Conclusion In summary, we have demonstrated a new use for the old drug Celecoxib that treats intervertebral disc degeneration by enhancing autophagy in nucleus pulposus cells and opening a door for treating other degenerative diseases.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Celecoxib activates autophagy by inhibiting the mTOR signaling pathway and prevents apoptosis in nucleus pulposus cells

Chen

Yasen

Wang

et al. 2022

BMC Pharmacol Toxicol

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“…Beclin 1, the mammalian orthologue of yeast Atg6, has a key function in autophagy, a pathway of programmed cell survival, which is increased during periods of cell stress and extinguished during the cell cycle (17). In some studies, it has mentioned compression induces the generation of morphological and biochemical markers of autophagy in rat NP cells, and triggers autophagosome formation by LC3B expression (13,18). From our study, we identified higher and more laterally expressed Beclin-1 protein in EF.…”

Section: Discussionmentioning

confidence: 99%

“…Hence, the IVD cells would demonstrate different forms of cells death, besides apoptosis, autophagy in response to compression (13).So, we hypothesized that EF and SI may exert autophagy and apoptosis mediated IVDD through NP cells injury in rat tail discs (Fig- 1). Rings were connected longitudinally with three threaded rods.…”

Section: Of 27mentioning

confidence: 99%

“…Stress can influence frequently both types of PCD. Under stress, autophagy provides energy by degrading with accumulating the miss-folded protein and damaged organelles (1,13).Many studies have been conducted regarding compression mediated apoptosis; conversely, a few studies have been conducted regarding role of autophagy in IVD cells. But, it has not been well established, when IVD discs expose to compression.…”

Section: Introductionmentioning

confidence: 99%

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Comparison Two Methods between Injection Saline and External Fixator in Rat Tail Disc Degeneration

Khaleque¹,

Choi²,

Kim³

et al. 2023

Preprint

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Purpose: Intervertebral disc is a leading avascular organ in the body. Hence, it may utilize autophagy and apoptosis to alive in unfavorable condition like stress and mechanical force. But excessive force and stress is a key cause to intervertebral disc degeneration (IVDD). Purpose of this study is to identify, external fixator (EF) and saline injection (SI) may induce autopahgy and apoptosis mediated nucleus pulposus (NP) cells death in rat tail’s disc and which method is better for studying autophagy and apoptosis. Methods: Sixteen 9-week-old male Sprague–Dawley rat tails were treated with 0.9% saline and EF (two-cross kirschner wires) for 6 weeks and 12 weeks as well. Treated discs were dissected for checking out participation of autopahgy and apoptosis to intervertebral disc degradation (IVDD). For identifying them, we performed H&E staining, Masson’s trichrome staining, and Immunohistochemistry (IHC) for LC3, Beclin-1and P62 as well as MMP-2, MMP-3 and TIMP-1.Furthermore, we performed quantitative polymerase chain reaction (qPCR)to check out autophagy related gene expression (beclin-1, LC3 and P62) and apoptosis related gene expression (MMP-2, MMP-3 and TIMP-1) respectively. Results: More insidious nucleus pulposus (NP) cell degeneration was indentified in (EF) group than in (Ctrl) control group. And rate of degradation was elevated with increasing duration of compression, but SI group cannot distinguish the margin of AF and NP cells. Highest along with lateral expression was found for LC3, Beclin-1, and P62 of both groups. Up-regulated and central expression was observed for MMP-2, MMP-3 and TIMP-1 of both groups, although SI group cannot recognize the margin between NP and AF cells. Utmost autophagy related gene expression was identified in EF group than in SI group. Besides, maximum autophagy accumulating materials was found in EF group than in SI group. And it was amplified along with increasing the duration of compression. Highest, apoptotic gene expression was observed in SI group, whereas, EF group showed lowest expression. Conclusion: External fixator (EF) method was better to study autophagy and apoptosis because it enhanced IVDD after compression which is actively linked with autophagy and apoptosis. Degeneration process was conversely proportional along with duration of compression. On contrary, saline injection (SI) could not distinguish AF and NP cell border.

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Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix

Kritschil,

Li,

Wang

et al. 2023

JOR Spine

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BackgroundIntervertebral 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.MethodsIn 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.ResultsHere, 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.ConclusionThe 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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Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5

Cited by 16 publications

References 52 publications

Celecoxib activates autophagy by inhibiting the mTOR signaling pathway and prevents apoptosis in nucleus pulposus cells

Celecoxib activates autophagy by inhibiting the mTOR signaling pathway and prevents apoptosis in nucleus pulposus cells

Comparison Two Methods between Injection Saline and External Fixator in Rat Tail Disc Degeneration

Impact of autophagy inhibition on intervertebral disc cells and extracellular matrix

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