Figure 6.

Restraint stressed-circulating exosomes induced MSC autophagy. (A) MSCs were treated with plasma exosomes derived from restraint stressed or control mice for 6 or 12 h. The expression of autophagy-related proteins including LC3-I/II, BECN1, p-STAT3, and STAT3 were assessed by western blotting. (B) MSCs were treated with plasma exosomes derived from restraint stressed or control mice for 12 h in the absence or presence of bafilomycin A1 (BafA1). BafA1 (50 nM) was added 2 h before cell harvest. The expression of LC3-I/II was assessed by western blotting. The histogram shows quantification of LC3-II after normalization to β-actin. (C) MSCs were transfected with a lentiviral plasmid encoding mCherry-GFP-LC3B and the autophagy flux was analyzed after the control and stressed exosomes treatment for 12 h (scale bars: 10 μm). The graph on the right shows the quantification of LC3 dots (**P < 0.01 for the total LC3 dots; ##P < 0.01 for red dots). (D) Western blotting showing the levels of BECN1 and LC3-I/II in control and Becn1 siRNA-treated MSCs. Becn1 siRNA-treated MSCs were infused into colitis mice subjected to restraint stress 3 days after DSS induction. (E) Body weight loss and (F) disease activity index (DAI) were monitored daily during the entire experiment (n = 3 for DSS and DSS+MSC group; n = 4 for DSS+Stress+MSC and DSS+Stress+siBecn1-MSC group) (* P < 0.05, ** P < 0.01, DSS+Stress+siBecn1-MSC versus DSS+Stress+MSC). At 10 days after DSS induction, mice were euthanized; (G) colon length was recorded, and (H) colonic epithelial/crypt destruction and inflammatory cell infiltration were visualized using HE staining (yellow arrows: goblet cells; green arrows: destruction of epithelial layer; red arrows: inflammatory cell infiltration). (I) Colonic damage was analyzed using histological activity index (HAI). (J, K) Splenocytes were harvested and the ratios of CD4⁺ IL17A⁺ Th17 cells and CD4⁺ CD25⁺ FOXP3⁺ Tregs among CD4⁺ T cells in the spleen were determined using flow cytometry. All results were verified in three independent experiments. A one-way ANOVA with Bonferroni’s comparison test was used for statistical analysis. Error bars represent the mean ± s.d. **P < 0.01; *P < 0.05