Ferroptosis is a necrotic form of regulated cell death that was associated with lipid peroxidation and free iron‐mediated Fenton reactions. It has been reported that iron deficiency had been implicated in the pathogenesis of intervertebral disc degeneration (IVDD) by activating apoptosis. However, the role of ferroptosis in the process of IVDD has not been illuminated. Here, we demonstrate the involvement of ferroptosis in IVDD pathogenesis. Our in vitro models show the changes in protein levels of ferroptosis marker and enhanced lipid peroxidation level during oxidative stress. Safranin O staining, hematoxylin‐eosin staining, and immunohistochemical were used to assess the IVDD after 8 weeks of surgical procedure in vivo. Treatment with ferrostatin‐1, deferoxamine, and RSL3 demonstrate the role of ferroptosis in tert‐butyl hydroperoxide (TBHP)‐treated annulus fibrosus cells (AFCs) and nucleus pulposus cells (NPCs). Ferritinophagy, nuclear receptor coactivator 4 (NCOA4)‐mediated ferritin selective autophagy, is originated during the process of ferroptosis in response to TBHP treatment. Knockdown and overexpression NCOA4 further prove TBHP may induce ferroptosis of AFCs and NPCs in an autophagy‐dependent way. These findings support a role for oxidative stress‐induced ferroptosis in the pathogenesis of IVDD.

The main pathological mechanism of intervertebral disc degeneration (IVDD) is the programmed apoptosis of nucleus pulposus (NP) cells. Oxidative stress is a significant cause of IVDD. Whether mitophagy is induced by strong oxidative stress in IVDD remains to be determined. This study aimed to investigate the relationship between oxidative stress and mitophagy and to better understand the mechanism of IVDD in vivo and in vitro. To this end, we obtained primary NP cells from the human NP and subsequently exposed them to TBHP. We observed that oxidative stress induced mitophagy to cause apoptosis in NP cells, and we suppressed mitophagy and found that NP cells were protected against apoptosis. Interestingly, TBHP resulted in mitophagy through the inhibition of the HIF-1α/NDUFA4L2 pathway. Therefore, the upregulation of mitochondrial NDUFA4L2 restricted mitophagy induced by oxidative stress. Furthermore, the expression levels of HIF-1α and NDUFA4L2 were decreased in human IVDD. In conclusion, these results demonstrated that the upregulation of NDUFA4L2 ameliorated the apoptosis of NP cells by repressing excessive mitophagy, which ultimately alleviated IVDD. These findings show for the first time that NDUFA4L2 and mitophagy may be potential therapeutic targets for IVDD.

Long-term and high dose glucocorticoid treatment can cause decreased viability and function of osteoblasts, which leads to osteoporosis and osteonecrosis. In this study, we investigated the role and mechanism of action of HIF-1α in glucocorticoid-induced osteogenic inhibition in MC3T3-E1 cells. Our results showed that HIF-1α protein expression was reduced when MC3T3-E1 cells were exposed to dexamethasone (Dex) at varying concentrations ranging from 10⁻⁹ to 10⁻⁶ M. PDK1 expression was also decreased in MC3T3-E1 cells after dexamethasone treatment. MC3T3-E1 cells when treated with the glucocorticoid receptor antagonist RU486 along with dexamethasone showed enhanced HIF-1α expression. In addition, upregulated expression of HIF-1α was capable of promoting the osteogenic ability of MC3T3-E1 cells and PDK1 expression. However, the HIF-1α antagonist 2-methoxyestradiol (2-ME) had a reverse effect in MC3T3-E1 cells exposed to dexamethasone. Furthermore, the PDK1 antagonist dichloroacetate could repress the osteogenic ability of MC3T3-E1 cells, although HIF-1α was upregulated when transduced with adenovirus-HIF-1α construct. The PDK1 agonist PS48 was able to promote the osteogenic ability of MC3T3-E1 cells treated with dexamethasone. Importantly, the protein levels of p-AKT and p-mTOR were increased in MC3T3-E1 cells treated with dexamethasone after PS48 treatment. in vivo, the PDK1 agonist PS48 could maintain the bone mass of mice treated with dexamethasone. This study provides a new understanding of the mechanism of glucocorticoid-induced osteoporosis.

Apoptosis of annulus fibrosus (AF) is observed widely in intervertebral disc degeneration (IVDD) which causes weaken of tension in the annulus of intervertebral disc. Previous studies reported that apoptosis of AF is induced mainly by oxidative stress. SIRT2 is a major regulator of mitochondria to mediate ROS production. However, the mechanism of SIRT2 in IVDD remains unclear. Here, the expression of SIRT2 was detected in AF cells exposed to tert-Butyl hydroperoxide (TBHP) by western blotting. Autophagic flux and apoptosis were assessed by western blotting, flow cytometry and immunofluorescence respectively. Safranin O staining, HE, and immunohistochemical were used to assess the IVDD after 3, 6 and 9 months of surgical procedure in vivo. The expression of SIRT2 was decreased in AF cells treated with TBHP. Repression of mitophagy alleviated the apoptosis of AF cells caused by TBHP. Overexpression of PGC-1α prevented AF cells from apoptosis and mitophagy after applying Lenti-PGC-1α to transfect AF cells. These protections of PGC-1α were reduced by FCCP. Furthermore, the expression of PGC-1α was reduced and the level of mitophagy was increased in IVDD models. In conclusion, this study indicates that the regulation of PGC-1α expression provide a new theoretical basis for the mechanism of IVDD.

N6-methyladenosine (m⁶A) is required for differentiation of human bone marrow mesenchymal stem cells (hBMSCs). However, its intrinsic mechanisms are largely unknown. To identify the possible role of m⁶A binding protein YTHDF1 in hBMSCs osteogenesis in vivo, we constructed Ythdf1 KO mice and showed that depletion of Ythdf1 would result in decreased bone mass in vivo. Both deletion of Ythdf1 in mouse BMSCs and shRNA-mediated knockdown of YTHDF1 in hBMSCs prevented osteogenic differentiation of cells in vitro. Using methylated RNA immunoprecipitation (Me-RIP) sequencing and RIP-sequencing, we found that ZNF839 (a zinc finger protein) served as a target of YTHDF1. We also verified its mouse homolog, Zfp839, was translationally regulated by Ythdf1 in an m⁶A-dependent manner. Zfp839 potentiated BMSC osteogenesis by interacting with and further enhancing the transcription activity of Runx2. These findings should improve our understanding of the mechanism of BMSC osteogenesis regulation and provide new ideas for the prevention and treatment of osteoporosis.