Fig. 9
ADMSCyoung-EVs regulated the NAMPT/SIRT1/Nf-κb p65/NLRP3 pathway in macrophages and alleviated tendinopathy in vitro through direct and indirect pathways. a The relative SASP status, including IL-6, IL-8, TGF-β1, MCP-1, MMP3, TNF-α and CXCL1 in the control, IL-1β, IL-1β + ADMSCyoung-EV and IL-1β + ADMSCold-EV macrophage groups, n = 3. b The relative phagocytosis ability in the control, IL-1β, IL-1β + ADMSCyoung-EV and IL-1β + ADMSCold-EV macrophage groups. Scale bar = 50 μm and n = 4. c Western blotting and quantification of NAMPT, SIRT1, p65 Nf-κb K310Ac, NLRP3 and ASC in the control, IL-1β, IL-1β + ADMSCyoung-EV, IL-1β + ADMSCold-EV, IL-1β + ADMSCyoung-EVs + NAMPT inhibitor and IL-1β + ADMSCyoung-EVs + SIRT1 inhibitor macrophage groups, n = 3. d A schematic diagram of the indirect contact macrophage-tenocyte crosstalk. e JC-1 staining of tenocytes in the control, IL-1β-treated macrophage medium, IL-1β-treated macrophage medium + ADMSCyoung-EV and IL-1β-treated macrophage medium + ADMSCold-EV groups and the IL-1β-treated macrophage medium + ADMSCyoung-EVs + NAMPT inhibitor and IL-1β-treated macrophage medium + ADMSCyoung-EVs + SIRT1 inhibitor groups. Aggregates are shown in red, and monomers are shown in green. The relative JC-1 intensity was quantified, and n = 4. Scale bar = 50 μm. f Live/dead assay of tenocytes in the control, IL-1β-treated macrophage medium, IL-1β-treated macrophage medium + ADMSCyoung-EV, IL-1β-treated macrophage medium + ADMSCold-EV, IL-1β-treated macrophage medium + ADMSCyoung-EVs + NAMPT inhibitor and IL-1β-treated macrophage medium + ADMSCyoung-EVs + SIRT1 inhibitor groups. Live cells are indicated in green, and dead cells are indicated in red. The rate of cell death was quantified. Scale bar = 100 μm and n = 4. (*: control vs. IL-1β treatment; #: IL-1β treatment (or treated macrophage medium) vs. IL-1β (or treated macrophage medium) + ADMSCyoung-EV treatment; &: IL-1β (or treated macrophage medium) + ADMSCyoung-EV vs. IL-1β (or treated macrophage medium) + ADMSCold-EV treatment groups, $: IL-1β (or treated macrophage medium) + ADMSCyoung-EVs vs. IL-1β (or treated macrophage medium) + ADMSCyoung-EVs + NAMPT inhibitor; ^: IL-1β (or treated macrophage medium) + ADMSCyoung-EVs vs. IL-1β (or treated macrophage medium) + ADMSCyoung-EVs + SIRT1 inhibitor, P < 0.05; **: P < 0.01; ***: P < 0.001)