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ObjectivesCircular RNAs (circRNAs) have been proven to function as competing endogenous RNAs to interact with microRNAs (miRNAs) and influence the expression of miRNA target mRNAs. In this study, we investigated whether circRNAs could act as competing endogenous RNAs to regulate the pathological process of intervertebral disc degeneration (IVDD).MethodsThe role and mechanism of a circRNA, circVMA21, in IVDD were explored in nucleus pulposus (NP) cells and degenerative NP tissues from patients and rat models. The interaction between circVMA21 and miR-200c as well as the target mRNA, X linked inhibitor-of-apoptosis protein (XIAP), was examined.ResultsThe decreased expression of XIAP in the inflammatory cytokines-treated NP cells and the degenerative NP tissues was directly associated with excessive apoptosis and imbalance between anabolic and catabolic factors of extracellular matrix. miR-200c regulated NP cell viability and functions through inhibiting XIAP. circVMA21 acted as a sponge of miR-200c and functioned in NP cells through targeting miR-200c and XIAP. Intradiscal injection of circVMA21 alleviated IVDD in the rat model.ConclusionsCircVMA21 could alleviate inflammatory cytokines-induced NP cell apoptosis and imbalance between anabolism and catabolism of extracellular matrix through miR-200c-XIAP pathway. It provides a potentially effective therapeutic strategy for IVDD.

Although mesenchymal stem cells (MSCs) transplantation into the IVD (intervertebral disc) may be beneficial in inhibiting apoptosis of nucleus pulposus cells (NPCs) and alleviating IVD degeneration, the underlying mechanism of this therapeutic process has not been fully explained. The purpose of this study was to explore the protective effect of MSC‐derived exosomes (MSC‐exosomes) on NPC apoptosis and IVD degeneration and investigate the regulatory effect of miRNAs in MSC‐exosomes and associated mechanisms for NPC apoptosis. MSC‐exosomes were isolated from MSC medium, and its anti‐apoptotic effect was assessed in a cell and rat model. The down‐regulated miRNAs in apoptotic NPCs were identified, and their contents in MSC‐exosomes were detected. The target genes of eligible miRNAs and possible downstream pathway were investigated. Purified MSC‐exosomes were taken up by NPCs and suppressed NPC apoptosis. The levels of miR‐21 were down‐regulated in apoptotic NPCs while MSC‐exosomes were enriched in miR‐21. The exosomal miR‐21 could be transferred into NPCs and alleviated TNF‐α induced NPC apoptosis by targeting phosphatase and tensin homolog (PTEN) through phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Intradiscal injection of MSC‐exosomes alleviated the NPC apoptosis and IVD degeneration in the rat model. In conclusion, MSC‐derived exosomes prevent NPCs from apoptotic process and alleviate IVD degeneration, at least partly, via miR‐21 contained in exosomes. Exosomal miR‐21 restrains PTEN and thus activates PI3K/Akt pathway in apoptotic NPCs. Our work confers a promising therapeutic strategy for IVD degeneration.

Intervertebral disc (IVD) degeneration is largely a process of destruction and failure of the extracellular matrix (ECM), and symptomatic IVD degeneration is thought to be one of the leading causes of morbidity or life quality deterioration in the elderly. To date, however, the mechanism of IVD degeneration is still not fully understood. Cellular loss from cell death in the process of IVD degeneration has long been confirmed and considered to contribute to ECM degradation, but the causes and the manners of IVD cell death remain unclear. Programmed cell death (PCD) is executed by an active cellular process and is extensively involved in many physiological and pathological processes, including embryonic development and human degenerative diseases. Thus, the relationship between PCD and IVD degeneration has become a new research focus of interest in recent years. By reviewing the available literature concentrated on PCD in IVD and discussing the methodology of detecting PCD in IVD cells, its inducing factors, the relationship of cell death to ECM degradation, and the potential therapy for IVD degeneration by modulation of PCD, we conclude that IVD cells undergo PCD via different signal transduction pathways in response to different stimuli, that PCD may play a role in the process of IVD degeneration, and that modulation of PCD might be a potential therapeutic strategy for IVD degeneration.

Intervertebral disc cell apoptosis occurs through either death receptor or mitochondrial pathway, but whether disc cell apoptosis is also mediated by the endoplasmic reticulum (ER) pathway remains unclear. The objective of this study was to investigate whether ER and mitochondria are co-involved in disc cell apoptosis and intervertebral disc degeneration (IVDD) in rats. Forty-eight rats were used for in vivo experiments. IVDD was characterized by X-ray and histomorphology examination, disc cell apoptosis was detected by TUNEL staining, and the co-involvement of ER and mitochondria in apoptosis was determined by immunohistochemical staining for GRP78, GADD153, caspase-12, and cytochrome C. Additional eight rats were used for annular cell isolation and culture. After sodium nitroprusside treatment, annular cell apoptosis was observed morphologically and quantified by flow cytometry; the expression of biomarkers of ER stress and mitochondrial dysfunction were analyzed by reverse transcriptase PCR (RT-PCR), fluorescence double labeling, and Western blot; and mitochondrial membrane potential was detected by 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbo cyanine iodide (JC-1) staining. Finally, NS3694 and Z-ATAD-FMK were employed to inhibit the formation of apoptosome complex and the activation of caspase-12, respectively, and apoptotic incidence and caspase-9 activity were assayed. We found that IVDD, induced by unbalanced dynamic and static forces in the rats, was accompanied by increased disc cell apoptosis and enhanced expression of GRP78, GADD153, caspase-12, and cytochrome C. Annular cell apoptosis induced by sodium nitroprusside was confirmed by morphologic observation and flow cytometry. With increased apoptosis, the expression of GRP78, GADD153, and caspase-12 upregulated, mitochondrial membrane potential decreased, and accumulation of cytochrome C in the cytosol manifested. Furthermore, NS3694 and Z-ATAD-FMK dramatically suppress annular cell apoptosis and caspase-9 activity. In conclusion, disc cell apoptosis mediated simultaneously by ER and mitochondria plays a potent role in IVDD.

Various mechanical stresses in vivo induce disc cell apoptosis and intervertebral disc (IVD) degeneration, but the underlying molecular mechanism is not fully known. The aim of this study was to investigate the role of endoplasmic reticulum stress in cyclic stretch-induced apoptosis of rat annulus fibrosus (AF) cells. Flexercell Tension Plus system was used to apply cyclic stretch to rat annulus fibrosus cells at a frequency of 0.5 Hz with 20% elongation for 12, 24, 36, or 48 h. Apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining and caspase-8, 9 activity assays. The expression of the markers of endoplasmic reticulum stress including CHOP, GRP78, and caspase-12 were determined by RT-PCR and Western blot. Mitochondrial membrane potential change was observed by JC-1 staining in situ. In addition, the levels of the nitric oxide (NO) were determined with the Griess reaction and fluorescence staining. The results indicated that cyclic stretch at a frequency of 0.5 Hz with 20% elongationinduced apoptosis in rat AF cells. Prolonged exposure of the unphysiologically cyclic stretch to AF cells caused NO overproduction, up-regulation of endoplasmic reticulum stress markers including CHOP, GRP78, and caspase-12, depolarization of mitochondria and activation of caspase-9. However, cyclic stretch at this level had no effect on caspase-8 activity. In addition, specific inhibitor of caspase-12 (Z-ATAD-FMK) and caspase-9 (Z-LEHD-FMK) partly suppressed cyclic stretch-induced AF cell apoptosis and the anti-apoptotic effects of the caspase inhibitors were additive. Our data suggest that endoplasmic reticulum stress, likely mediated by NO, contributes to the AF cell apoptosis induced by cyclic stretch in addition to the mitochondrial pathway. These findings could be helpful to understand the mechanism of disc cell apoptosis, the root cause of IVD degeneration.

Our results suggested that disc cells can express leptin and its functional receptor, that leptin can stimulate proliferation of disc cells in vitro, and therefore that leptin may play a role in the process of intervertebral disc degeneration by contributing to the formation of cell cluster and proliferating fibrocartilaginous tissue.

The expression of CHOP (C/EBP homologous protein), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress induced by cyclic stretch and leads to rat AF cells apoptosis. However, whether the suppression of CHOP can inhibit apoptosis and attenuates disc degeneration by cyclic stretch remains unclear. The aim of this study was to evaluate the suppressive effects of lentiviral CHOP shRNA on apoptosis induced by cyclic stretch in rat annulus fibrosus (AF) cells in vitro and disc degeneration of rat lumber spine in vivo. Lentiviral CHOP shRNA was constructed and introduced into AF cells. After stretched by the Flexcell Tension Plus system with 20% elongation for 36 h, silencing of the CHOP gene was identified by RT-PCR and Western blot. Inhibition of apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining. The effect of CHOP shRNA on disc degeneration was determined in vivo by using a rat model. At 7 weeks after intradiscal injection of the control or CHOP shRNA in the L4/L5 and L5/L6 discs, disc degeneration was assessed by X-ray examination, magnetic resonance imaging (MRI) assessment, and HE and TUNEL staining. A significant decrease in CHOP mRNA and protein expression was detected in AF cells with CHOP shRNA transfection after 36 h stretch. There was a significant decrease in apoptotic incidence in cells treated with CHOP shRNA, which was parallel to the expression of CHOP. Injection of CHOP shRNA in vivo resulted in the improvement in MRI and histologic score, and decrease in the apoptosis in the disc. No significant change in disc height was observed. In conclusion, a novel lentiviral vector expressing CHOP shRNA efficiently inhibits apoptosis in rat AF cells by silencing CHOP expression. In a rat model, intradiscal injection of CHOP shRNA induces the suppression of disc degeneration. The therapeutic effects of lentiviral CHOP shRNA should be further explored.