Self-amplifying loop of NF-κB and periostin initiated by PIEZO1 accelerates mechano-induced senescence of nucleus pulposus cells and intervertebral disc degeneration.

Abnormal mechanical load is a main risk factor of intervertebral disc degeneration (IDD), and cellular senescence is a pathological change in IDD. In addition, extracellular matrix (ECM) stiffness promotes human nucleus pulposus cells (hNPCs) senescence. However, the molecular mechanism underlying mechano-induced cellular senescence and IDD progression is not yet fully elucidated. First, we demonstrated that mechano-stress promoted hNPCs senescence via NF-κB signaling. Subsequently, we identified periostin as the main mechano-responsive molecule in hNPCs through unbiased sequencing, which was transcriptionally upregulated by NF-κB p65; moreover, secreted periostin by senescent hNPCs further promoted senescence and upregulated the catabolic process in hNPCs through activating NF-κB, forming a positive loop. Both Postn (encoding periostin) knockdown via siRNA and periostin inactivation via neutralizing antibodies alleviated IDD and NPCs senescence. Furthermore, we found that mechano-stress initiated the positive feedback of NF-κB and periostin via PIEZO1. PIEZO1 activation by Yoda1 induced severe IDD in rat tails without compression, and Postn knockdown alleviated the Yoda1-induced IDD in vivo. Here, we reported for the first time that self-amplifying loop of NF-κB and periostin initiated via PIEZO1 under mechano-stress accelerated NPCs senescence, leading to IDD. Furthermore, periostin neutralizing antibodies, which may serve as potential therapeutic agents for IDD, interrupted this loop.

Rhodium-Catalyzed Intramolecular [5+2] Cycloaddition of Inverted 3-Acyloxy-1,4-enyne and Alkyne: Experimental and Theoretical Studies.

By switching the position of the alkene and alkyne, a new type of 3-acyloxy-1,4-enyne (ACE) five-carbon building block was developed for Rh-catalyzed intramolecular [5+2] cycloaddition. An electron-withdrawing acyl group on the alkyne termini of the ACE was essential for a regioselective 1,2-acyloxy migration. This new method provided bicyclic [5.3.0]decatrienes that are different from previous methods because of the positions of the alkenes and the acyloxy group. Multiple mechanistic pathways become possible for this new [5+2] cycloaddition and they are investigated by computational studies.

PtI2-catalyzed cyclization of 3-acyloxy-1,5-enynes with the elimination of HOAc and a benzyl shift: synthesis of unsymmetrical m-terphenyls.

A novel cyclization of 3-acyloxy-1,5-enynes is developed in the presence of PtI2 for the synthesis of substituted unsymmetrical m-terphenyls in good to excellent yields. Two unique steps are involved in this transformation, which includes the elimination of HOAc and benzyl group migration. DFT calculations indicated that the rate-determining step is the migration of the benzylic carbocation to form a zwitterionic intermediate followed by the elimination of HOAc. The subsequent cyclopropanation of the zwitterionic intermediate is the regioselectivity-determining step.

Mechanism and Reactivity of Rh-Catalyzed Intermolecular [5+1] Cycloaddition of 3-Acyloxy-1,4-Enyne (ACE) and CO: A Computational Study.

The first theoretical study on the mechanism of [RhCl(CO)2]2-catalyzed [5 + 1] cycloadditions of 3-acyloxy-1,4-enyne (ACE) and CO has been performed using density functional theory (DFT) calculations. The effect of ester on reactivity of this reaction has been investigated. The computational results have revealed that the preferred catalytic cycle involves the sequential steps of 1,2-acyloxy migration, CO insertion, reductive elimination to form ketene intermediate, 6π-electroncyclization, and aromatization to afford the resorcinol product. The 1,2-acyloxy migration is found to be the rate-determining step of the catalytic cycle. The electron-rich p-dimethylaminobenzoate substrate promotes 1,2-acyloxy migration and significantly increases the reactivity by stabilizing the positive charge building up in the oxocyclic transition state.

Spatiotemporal Characterization of Human Early Intervertebral Disc Formation at Single-Cell Resolution.

The intervertebral disc (IVD) acts as a fibrocartilaginous joint to anchor adjacent vertebrae. Although several studies have demonstrated the cellular heterogeneity of adult mature IVDs, a single-cell transcriptomic atlas mapping early IVD formation is still lacking. Here, the authors generate a spatiotemporal and single cell-based transcriptomic atlas of human IVD formation at the embryonic stage and a comparative mouse transcript landscape. They identify two novel human notochord (NC)/nucleus pulposus (NP) clusters, SRY-box transcription factor 10 (SOX10)+ and cathepsin K (CTSK)+ , that are distributed in the early and late stages of IVD formation and they are validated by lineage tracing experiments in mice. Matrisome NC/NP clusters, T-box transcription factor T (TBXT)+ and CTSK+ , are responsible for the extracellular matrix homeostasis. The IVD atlas suggests that a subcluster of the vertebral chondrocyte subcluster might give rise to an inner annulus fibrosus of chondrogenic origin, while the fibroblastic outer annulus fibrosus preferentially expresseds transgelin and fibromodulin . Through analyzing intercellular crosstalk, the authors further find that notochordal secreted phosphoprotein 1 (SPP1) is a novel cue in the IVD microenvironment, and it is associated with IVD development and degeneration. In conclusion, the single-cell transcriptomic atlas will be leveraged to develop preventative and regenerative strategies for IVD degeneration.

Dihydroartemisinin Attenuated Intervertebral Disc Degeneration via Inhibiting PI3K/AKT and NF-κB Signaling Pathways.

Intervertebral disc degeneration (IDD) is the leading cause of low back pain (LBP). However, effective therapeutic drugs for IDD remain to be further explored. Inflammatory cytokines play a pivotal role in the onset and progression of IDD. Dihydroartemisinin (DHA) has been well reported to have powerful anti-inflammatory effects, but whether DHA could ameliorate the development of IDD remained unclear. In this study, the effects of DHA on extracellular matrix (ECM) metabolism and cellular senescence were firstly investigated in nucleus pulposus cells (NPCs) under tumor necrosis factor alpha (TNFα)-induced inflammation. Meanwhile, AKT agonist sc-79 was used to determine whether DHA exerted its actions through regulating PI3K/AKT and NF-κB signaling pathways. Next, the therapeutic effects of DHA were tested in a puncture-induced rat IDD model. Finally, we detected the activation of PI3K/AKT and NF-κB signaling pathways in clinical degenerative nucleus pulposus specimens. We demonstrated that DHA ameliorated the imbalance between anabolism and catabolism of extracellular matrix and alleviated NPCs senescence induced by TNFα in vitro. Further, we illustrated that DHA mitigated the IDD progression in a puncture-induced rat model. Mechanistically, DHA inhibited the activation of PI3K/AKT and NF-κB signaling pathways induced by TNFα, which was undermined by AKT agonist sc-79. Molecular docking predicted that DHA bound to the PI3K directly. Intriguingly, we also verified the activation of PI3K/AKT and NF-κB signaling pathways in clinical degenerative nucleus pulposus specimens, suggesting that DHA may qualify itself as a promising drug for mitigating IDD.

Computational Prediction of One-Step Synthesis of Seven-membered Fused Rings by (5+2) Cycloaddition Utilising Cycloalkenes.

The (5+2) cycloaddition reaction utilising cycloalkenes is rare, although it is one of the most efficient methods of constructing seven-membered fused rings because of its high atom- and step-economy. In this study, we used quantum mechanical calculations to predict the plausibility of using the Rh-catalysed intermolecular (5+2) cycloaddition of 3-acyloxy-1,4-enynes and cycloalkenes to produce fused seven-membered carbocycles. The calculation results suggest a convenient, highly efficient and energetically practical approach. Strained cycloalkenes, such as cyclopropene, have been predicted to be active, and the desired bicyclic product should be favoured, accompanied by the formation of byproducts from rearrangement reactions. The energy barriers of the alkene insertion step were analysed by the distortion/interaction model to disclose the origins of the different reactivities of cycloalkenes with different ring sizes.