Journal: Bioactive Materials

Article Title: Exosomes derived from MSCs exposed to hypoxic and inflammatory environments slow intervertebral disc degeneration by alleviating the senescence of nucleus pulposus cells through epigenetic modifications

doi: 10.1016/j.bioactmat.2025.02.046

Figure Lengend Snippet: Hi-Exos reduced the senescence of NPCs by transferring the miR-221-3p to decrease DDIT4 expression. a, Bioinformatic analysis to determine the targeted genes of miR-221-3p; b, Protein level of DDIT4 in non-IDD tissues and IDD tissues; c, Protein level of DDIT4 in normal NPCs and senescent NPCs treated with TNF-α; d,e, Western blots to detect the AGO2 (d) and qRT-PCRs to detect the miR-221-3p and DDIT4 expression level in RIP analysis (e); f, Dual-luciferase reporter assay to determine the binding sites between miR-221-3p and DDIT4; g, Protein level of DDIT4 after the transfection of siRNAs; h-l, Western blots to detect the protein level of p21 (h), EdU assay to detect the cell proliferation (i,j), and SA-β-gal staining to detect the SA-β-gal activity (k,l) after the knockdown of DDIT4 in senescent NPCs induced by TNF-α; m, Western blots to detect the protein level of DDIT4 after the overexpression of DDIT4; n-s, Western blots to detect the protein level of p21 (n), EdU assay to detect the cell proliferation (o,p), and SA-β-gal staining to detect the SA-β-gal activity (q,r) in rescue experiments between Hi-Exos and DDIT4 overexpression. ns, not significant (P < 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Article Snippet: The plasmids used for overexpressing DDIT4 were constructed by GenePharma (Shanghai, China).

Techniques: Transferring, Expressing, Western Blot, Luciferase, Reporter Assay, Binding Assay, Transfection, EdU Assay, Staining, Activity Assay, Knockdown, Over Expression

Journal: Bioactive Materials

Article Title: Exosomes derived from MSCs exposed to hypoxic and inflammatory environments slow intervertebral disc degeneration by alleviating the senescence of nucleus pulposus cells through epigenetic modifications

doi: 10.1016/j.bioactmat.2025.02.046

Figure Lengend Snippet: Hi-Exos reduced the senescence of NPCs through the miR-221-3p/DDIT4/NF-κB signalling pathway. a, GSEA enrichment of NF-κB signalling pathway; b-c, Western blots to detect the protein level of p-p65 and p65; d, Immunofluorescence to detect the nuclear translocation of p65; e-f, Western blots to detect the protein level of p-p65 and p65 after the knockdown of DDIT4; g, Immunofluorescence to detect the nuclear translocation of p65 after the knockdown of DDIT4; h-i, Western blots to detect the protein level of p-p65 and p65 in rescue experiments between Hi-Exos and DDIT4 overexpression; j, Immunofluorescence to detect the nuclear translocation of p65 in rescue experiments between Hi-Exos and DDIT4 overexpression. ns, not significant (P < 0.05); ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Article Snippet: The plasmids used for overexpressing DDIT4 were constructed by GenePharma (Shanghai, China).

Techniques: Western Blot, Immunofluorescence, Translocation Assay, Knockdown, Over Expression

Journal: Bioactive Materials

Article Title: Exosomes derived from MSCs exposed to hypoxic and inflammatory environments slow intervertebral disc degeneration by alleviating the senescence of nucleus pulposus cells through epigenetic modifications

doi: 10.1016/j.bioactmat.2025.02.046

Figure Lengend Snippet: Schematic diagram of Hi-Exos in relieving the senescence of NPCs and IDD. Hi-Exos was superior to Exos in relieving the senescence of NPCs and IDD for the increased epigenetic factor miR-221-3p level. In details, Hi-Exos transferred the miR-221-3p to the senescent NPCs to decrease the DDIT4 protein level in senescent NPCs, which further inhibited the activation of NF-κB signalling pathway to relieve the senescence of NPCs and repair the IDD.

Article Snippet: The plasmids used for overexpressing DDIT4 were constructed by GenePharma (Shanghai, China).

Techniques: Activation Assay