Shuo Zhang,1,2,* Miaoheng Yan,3,* Xiao Lv,2,* Peng Wang,2 Weijian Liu,2 Binwu Hu,2 Songfeng Chen,3 Zengwu Shao2 

1School of Medicine, Nankai University, Tianjin, 300071, People’s Republic of China; 2Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, People’s Republic of China; 3Department of Orthopaedics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Songfeng Chen, Department of Orthopaedics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, People’s Republic of China, Email fccchensf@zzu.edu.cn Zengwu Shao, Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, People’s Republic of China, Email szwpro@163.com

Purpose: To investigate the role of macrophage-derived small extracellular vesicles (MΦ-sEVs) in nucleus pulposus (NP) cell (NPC) senescence and screen the pro-senescent micro-RNA (miRNA) in MΦ-sEVs and potential mRNA targets.
Methods: Bone marrow-derived macrophage (BMDM)-derived sEVs were isolated by differential centrifugation, and the phenotypes of MΦ-sEVs were identified. NPCs were treated with MΦ-sEVs, and cellular senescence levels were examined by senescence‐associated β‐galactosidase (SAβGal) staining and Western blotting (WB). Activation of the senescence-associated secretory phenotype (SASP) was tested using qRT-PCR and cytometric bead arrays (CBA). LPS+IFNγ-MΦ-sEVs or IL-4-MΦ-sEVs were injected into the rat coccygeal NP tissues to determine the in vivo effects of MΦ-sEVs on intervertebral disc degeneration (IVDD) and NPC senescence. The miRNA levels in MΦ-sEVs were evaluated using PANDORA sequencing. NPCs were transfected with miRNA mimics or inhibitors to screen the miRNAs with pro-senescence effects.
Results: MΦ-sEVs displayed the cup-shaped morphology, with diameters mainly ranging from 40 to 200 nm. Both LPS+IFNγ-MΦ-sEVs and IL-4-MΦ-sEVs impaired NPC viability and accelerated NPC senescence. The expression levels of SASP and senescence-related proteins, including p16, p21, and p53, were elevated by MΦ-sEVs treatment. Animal experiments indicated that LPS+IFNγ-MΦ-sEVs or IL-4-MΦ-sEVs exacerbated IVDD with increased p16-positive cell ratio and activated SASP. PANDORA sequencing of MΦ-sEVs revealed high levels of let-7i-5p, which exerted pro-senescence effects by downregulating LIN28A expression. Inhibiting or silencing LIN28A by C1632 or specific siRNAs also triggered NPC senescence.
Conclusion: Both LPS+IFNγ-MΦ-sEVs and IL-4-MΦ-sEVs induced NPC senescence by delivering miRNA let-7i-5p to inhibit LIN28A.

Keywords: macrophage-derived small extracellular vesicle, intervertebral disc degeneration, nucleus pulposus cell, senescence, let-7i-5p