Extracellular Vesicles From Adipose Tissue-Derived Stem Cells Affect Notch-miR148a-3p Axis to Regulate Polarization of Macrophages and Alleviate Sepsis in Mice

Figures

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  Figure 1. Characterization of ADSCs. (A) Flow cytometry analysis showed that these cells were highly positive for CD29 and CD105 but negative for CD31 and CD34 compared with isotype control. (B) Representative photographs of lipid droplets stained with Oil red O and calcium deposits stained with Alizarin Red S. Scale bar = 100 μm. (C) The morphology of the ADSC-EVs was detected by TEM. Scale bar= 1 μm. (D) The diameter distribution of EVs (nm). Four fields were chosen randomly, eight vesicles in each field were randomly chosen to measure the size of the ADSC-EVs. (E) CD9, CD63, CD81 and Alix expression in EVs was confirmed by western blotting. n = 4.

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  Figure 2. ADSC-EVs treatment alleviated inflammation induced by LPS in macrophages. (A) Macrophages were treated with LPS (1 μg/ml) and ADSC-EVs in different concentration gradients, and then, the mRNA levels of IL-1β, TNF-α, IL-6, and IL-10 were assessed by RT-PCR. (B,C) Macrophages were treated with LPS (1 μg/ml) or LPS + 25 μg/ml ADSC-EVs, and then, the protein levels of p-IKKα/β, as well as p65 in the nucleus and cytosol, were revealed separately by western blotting. *p < 0.05, **p < 0.01 compared with LPS group; n = 6.

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  Figure 3. ADSC-EVs alleviated inflammation and organ injury induced by LPS. Mice were treated with LPS (10 mg/kg body weight) or LPS + EVs (200 μg) for 24 h. (A) Blood from the left ventricle of the mice was collected, and the serum IL-1β, IL-6, and TNF-α levels were measured using commercial ELISA kits. (B) Morphological changes of liver, lung, and kidney tissues from mice after intraperitoneal injection of 10 mg/kg body weight LPS or LPS + 200 μg EVS. Sections were examined by H&E staining and photographed using an FSX100 microscope (Olympus, Japan). *p < 0.05, **p < 0.01 compared with LPS-injected mice; n = 6. Scale bar = 50 μm.

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  Figure 4. ADSC-EVs treatment led to reduced Notch signaling and miR-148a-3p expression in macrophages. (A) Macrophages were treated with LPS (1 μg/ml) and ADSC-EVs at different concentrations, and then, the mRNA levels of Notch1 and Notch2 were assessed by RT-PCR. (B) The protein levels of NICD were revealed by western blotting. (C) Levels of miR-148a-3p in LPS-treated macrophages with ADSC-EVs administration in different concentration. **p < 0.01; n = 6.

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  Figure 5. RBP-J−/− alleviated LPS induced inflammatory cytokine expression and NF-κB activation. Macrophages from RBP-J−/− mice and WT mice were treated with LPS (1 μg/ml). (A) The mRNA levels of IL-1β, IL-6, and TNF-α were tested by RT-PCR. (B,C) The phosphorylation of IKKα/β and nucleus import of p65 were tested by western blotting. **p < 0.01; n = 6.

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  Figure 6. RBP-J−/− alleviated inflammation and organ injury induced by LPS in vivo. RBP-J−/− mice and WT mice were treated with LPS (10 mg/kg body weight) for 24 h. (A) Blood from the left ventricle of mice was collected, and serum IL-1β, IL-6, and TNF-α levels were measured using commercial ELISA kits. (B) Morphological changes of liver, lung, and kidney tissues from mice after intraperitoneal injection of 10 mg/kg body weight LPS. Sections were examined by H&E staining and photographed using an FSX100 microscope (Olympus, Japan). *p < 0.05, **p < 0.01 compared with LPS-injected mice; n = 6. Scale bar = 50 μm.

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  Figure 7. RT-PCR showed the level of miR-148a-3p in LPS-treated (1 μg/ml) macrophages from WT mice or RBP-J−/− mice. *p < 0.05, **p < 0.01 compared with LPS group; n = 6.

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  Figure 8. miR-148a-3p could promote inflammation and M1 polarization. (A,B) Macrophages were treated with miR-148a-3p mimic or NC, and then, PBS or LPS (1 μg/ml) was added. The mRNA levels of IL-1β, TNF-α, and IL-6 were determined by RT-PCR. The protein level of p-IKKα/β in cell and p65 in nucleus were detected by western blotting. (C,D) Macrophages were treated with miR-148a-3p inhibitor or NC, and then, and then, PBS or LPS (1 μg/ml) was added. The mRNA levels of IL-1β, TNF-α and IL-6 were determined by RT-PCR and the protein levels of p-IKKα/β and p65 in nucleus were revealed by western blotting. *p < 0.05, **p < 0.01; n = 6.

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  Figure 9. miR-148a-3p agomiR diminished the protective effect of ADSC-EVs in LPS induced sepsis. Mice were treated with LPS (10 mg/kg body weight) and then divided into four groups as NC group, miR-148a-3p agomiR group, NC +EVs group and miR-148a-3p agomiR + EVs group. (A) The morphological changes of liver, lung, and kidney tissues in NC group, NC + EVs group, miR-148a-3p agomiR group and miR-148a-3p agomiR + EVs group were shown by H&E staining. Sections were observed and photographed using an FSX100 microscope (Olympus, Japan). (B) The protein levels of IL-1β and TNF-α in these four groups of mice were determined by ELISA. *p < 0.05, **p < 0.01; n = 6. Scale bar = 50 μm.

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  Figure 10. ADSC-EVs treatment attenuated inflammation induced by lipopolysaccharide (LPS). In LPS-induced inflammation, Notch signaling was activated. ADSC-EVs suppressed LPS induced inflammation through Notch/miR-148a-3p signaling, which could also affect macrophage polarization.