A20 ameliorates disc degeneration by suppressing mTOR/BNIP3 axis-mediated mitophagy.

BACKGROUND: The pathological mechanism of intervertebral disc degeneration (IDD) is an unanswered question that we are committed to exploring. A20 is an anti-inflammatory protein of nucleus pulposus (NP) cells and plays a protective role in intervertebral disc degeneration. OBJECTIVE: This study aims to investigate the molecular mechanism by which A20 attenuates disc degeneration. METHODS: The proteins of interest were measured by immunoblotting, immunofluorescence, ELISA assay, and immunohistochemical technique to conduct related experiments. Immunofluorescence assays and mitochondrial membrane potential (JC-1) were used to assess mitophagy and mitochondrial fitness, respectively. RESULTS: Here, we demonstrated that A20 promoted mitophagy, attenuated pyroptosis, and inhibited the degradation of the extracellular matrix, consequently significantly ameliorating disc degeneration. Mechanistically, A20 reduces pyroptosis and further suppresses cellular mTOR activity. On the one hand, A20-induced mTOR inhibition triggers BNIP3-mediated mitophagy to ensure mitochondrial fitness under LPS stimulation, as a result of mitigating mitochondrial dysfunction induced by LPS. On the other hand, A20-induced mTOR inhibition reduces the loss of mitochondrial membrane potential and the generation of Mitochondrial ROS. CONCLUSION: The study revealed that A20 promotes BNIP3-mediated mitophagy by suppressing mTOR pathway activation against LPS-induced pyroptosis.

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p.

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. STATEMENT OF SIGNIFICANCE: Intervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

The mitochondrial antioxidant SS-31 attenuated lipopolysaccharide-induced apoptosis and pyroptosis of nucleus pulposus cells via scavenging mitochondrial ROS and maintaining the stability of mitochondrial dynamics.

Evidence has shown that effects from inflammation and mitochondrial dysfunction lead to pyroptosis and apoptosis of nucleus pulposus (NP) cells. Damaged mitochondria release dangerous molecules such as reactive oxygen species (ROS), activating the NLRP3 inflammasome. SS-31 is a mitochondria-targeting peptide that has been used in the treatment of many diseases by scavenging ROS and ameliorating mitochondrial function. This study found that SS-31 ameliorated lipopolysaccharide (LPS)-induced loss of cell viability, ROS production, and apoptosis in NP cells. Moreover, mitochondrial dynamics and ATP synthesis were restored on pretreatment with SS-31 compared with the LPS group. For the molecular mechanism research, SS-31 stabilized mitochondrial morphology and inhibited the activation of the NF-κB pathway and the activation of the NLRP3 inflammasome. To evaluate whether the inhibition of NLRP3 inflammasome activation by SS-31 is dependent on the clearance of mitochondrial ROS, we comparatively analyzed the activation of NLRP3 inflammasome in NP cells pretreated with SS-31 and the ROS scavenger N-acetyl-L-cysteine (NAC). The results indicate that SS-31 could inhibit NLRP3 inflammasome activation by limiting the production of mitochondrial ROS. To sum up, our results revealed that SS-31 inhibits LPS-induced apoptosis, pyroptosis, and inflammation in NP cells via scavenging ROS and maintaining the stability of mitochondrial dynamics, which could be considered a promising therapeutic intervention for disk degeneration.