Fig. 5.

Mechanical forces regulate the elimination of apoEVs through the skin. (A) Schematic diagram of mechanical forces applied to mice. Treadmill running exercise as a force augmentation model; tail suspension as a weightless model. (B) Immunofluorescent images show that the number of GFP-ApoEV migrating to the stratum corneum was increased in running group and reduced in tail-suspension group at 3 days post-injection. White dotted line represents the dividing layer between stratum corneum and the other layers of epidermis. The quantity graph shows the relative intensity of apoEVs in the skin. n = 5. **P < 0.01, ***P < 0.001, one-way ANOVA test. Data shown as mean ± SD. (C) Western blotting confirms the level of GFP in the skin tissue is increased in running group and reduced in tail suspension group. (D, E) PKH67-labeled apoEVs (4 × 10⁶) were injected into C57BL/6 mice via the tail vein. After treadmill running exercise for 7 days, blood samples were collected. The flow cytometric calculation showed that the number of PKH67⁺/Annexin V⁺/CD62P⁻ apoEVs accumulated in the blood was significantly reduced compared to the control group (D). After tail suspension for 3 days, flow cytometry analysis showed that the number of PKH67⁺/Annexin V⁺/CD62P⁻ apoEVs in the blood was significantly increased compared to the control group (E). n = 3. *P < 0.05, Student's t-test. Data shown as mean ± SD. (F, G) DIR-labeled apoEVs (4 × 10⁶) were injected into immunocompromised mice via the tail vein. Ex vivo fluorescent images showed that the elimination of apoEVs through the skin was increased after treadmill exercise while the migration of apoEVs through the skin was decreased after tail suspension compared to the control group, as indicated by quantitative graph. n = 3. *P < 0.05, Student's t-test. Data shown as mean ± SD. (H) ELISA analysis showed the level of DKK1 in the circulation of C57BL/6 mice was reduced after treadmill running, but enhanced after tail suspension. n = 3. *P < 0.05, **P < 0.01, Student's t-test. Data shown as mean ± SD. (I) Immunofluorescent images show the expression level of active-β-catenin (green) in skin was enhanced after treadmill running, but reduced after tail suspension. The DKK1 expression level in the skin showed opposite trends of active-β-catenin. SC, stratum corneum; BM, basement membrane; D, dermis. Scale bar (B), 50 μm; Scale bar (I), 40 μm.