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Hydrogen peroxide induces nucleus pulposus cell apoptosis by ATF4/CHOP signaling pathway.

ABSTRACT: Oxidative stress induces excessive apoptosis resulting in the reduction of intervertebral disc cells, the consequent reduction of extracellular matrix (ECM) synthesis, and compositional changes, which is the pathological basis for intervertebral disc degeneration (IVDD). The present study explored the activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway in the H2O2-induced nucleus pulposus (NP) cell apoptosis. Human degenerated intervertebral discs were collected from Lumbar disc surgery. NP cells isolated from the tissues were cultured with H2O2 to induce apoptosis in vitro. Malondialdehyde (MDA) analysis was performed to determine the reactive oxygen species (ROS) of the tissue. Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were performed to analyze collagen II, ATF4, CHOP, and caspase-9 gene expression. Flow cytometry was used to determine the apoptotic ratio of NP cells. siRNA was also used to silence ATF4 and CHOP gene expression. NP tissues in higher degenerated degree underwent much more MDA, expressed less collagen II, more ATF4, CHOP, and caspase-9 compared with the mildly degenerated tissues. H2O2 induced NP cell apoptosis by upregulating expression of ATF4, CHOP and caspase-9. The silencing of ATF4 or CHOP alleviated NP cell apoptosis by suppressing caspase-9 expression. Inhibiting caspase-9 did not affect ATF4 and CHOP expression but protected NP cells from apoptosis. In this study, we found H2O2 could promote NP cell apoptosis by activating the ATF4/CHOP signaling pathway resulting in the upregulation of caspase-9. Interdict of ATF4, CHOP, or caspase-9 contributed to the reduction of apoptosis caused by H2O2.

SUBMITTER: Liu Y

PROVIDER: S-EPMC7444416 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Hydrogen peroxide induces nucleus pulposus cell apoptosis by ATF4/CHOP signaling pathway.

Liu Yi Y

Experimental and therapeutic medicine 20200727 4

Oxidative stress induces excessive apoptosis resulting in the reduction of intervertebral disc cells, the consequent reduction of extracellular matrix (ECM) synthesis, and compositional changes, which is the pathological basis for intervertebral disc degeneration (IVDD). The present study explored the activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway in the H<sub>2</sub>O<sub>2</sub>-induced nucleus pulposus (NP) cell apoptosis. Human degenerated interver ...[more]

PMID: 32855694

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Project description:BackgroundIntervertebral 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.MethodProtein 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.ResultsIn 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.ConclusionIn 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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Hydrogen peroxide induces nucleus pulposus cell apoptosis by ATF4/CHOP signaling pathway.

Publications

Hydrogen peroxide induces nucleus pulposus cell apoptosis by ATF4/CHOP signaling pathway.

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