Confocal microscopy analysis of PKH26-labeled BMSC-EVs localization in colon after injection.

<p>The localization was measured 12h after intravenous injection. (A-I) Representative micrograph of inflammatory colon with the treatment of PKH26-labeled BMSC-EVs (red) at the dose of 50, 100 and 200μg. In merge images, nuclei were stained with DAPI (blue). Original magnification×200.</p

Extracellular Vesicles Derived from Bone Marrow Mesenchymal Stem Cells Protect against Experimental Colitis via Attenuating Colon Inflammation, Oxidative Stress and Apoptosis

<div><p>The administration of bone mesenchymal stem cells (BMSCs) could reverse experimental colitis, and the predominant mechanism in tissue repair seems to be related to their paracrine activity. BMSCs derived extracellular vesicles (BMSC-EVs), including mcirovesicles and exosomes, containing diverse proteins, mRNAs and micro-RNAs, mediating various biological functions, might be a main paracrine mechanism for stem cell to injured cell communication. We aimed to investigate the potential alleviating effects of BMSC-EVs in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model. Intravenous injection of BMSC-EVs attenuated the severity of colitis as evidenced by decrease of disease activity index (DAI) and histological colonic damage. In inflammation response, the BMSC-EVs treatment significantly reduced both the mRNA and protein levels of nuclear factor kappaBp65 (NF-κBp65), tumor necrosis factor-alpha (TNF-α), induciblenitric oxidesynthase (iNOS) and cyclooxygenase-2 (COX-2) in injured colon. Additionally, the BMSC-EVs injection resulted in a markedly decrease in interleukin-1β (IL-1β) and an increase in interleukin-10 (IL-10) expression. Therapeutic effect of BMSC-EVs associated with suppression of oxidative perturbations was manifested by a decrease in the activity of myeloperoxidase (MPO) and Malondialdehyde (MDA), as well as an increase in superoxide dismutase (SOD) and glutathione (GSH). BMSC-EVs also suppressed the apoptosis via reducing the cleavage of caspase-3, caspase-8 and caspase-9 in colitis rats. Data obtained indicated that the beneficial effects of BMSC-EVs were due to the down regulation of pro-inflammatory cytokines levels, inhibition of NF-κBp65 signal transduction pathways, modulation of anti-oxidant/ oxidant balance, and moderation of the occurrence of apoptosis.</p></div

The morphology and identification of BMSCs.

<p>(A) Representative micrographs of photics microscopy obtained on BMSCs (original magnification ×200) at passage 4. (B and C) Characterization of BMSCs at passage 4 were evaluated by flow cytometric analysis using antibodies against CD90, CD29, CD11b and CD45. 89.8% (P1) of the BMSCs expressed surface markers phenotype of CD29 and CD90, while 97.5%(P2) did not have the lineage marker expression of CD11b or CD45.</p

Effects of BMSC-EVs therapies on mRNA expression of inflammatory mediators in TNBS-induced colitis.

<p>The mRNA expression of NF-κBp65(A), TNF-α(B), iNOS(C) and COX-2(D) were detected by standard RT-PCR methods. β-actin was used as a control. Values mean ± SD, n = 10 for each group, differences are evaluated using the one-way ANOVA on ranks test. ^##P<0.01, vs control group. ^△p>0.05,*P<0.05, **P<0.01 vs TNBS group.</p

Effects of BMSC-EVs therapies on expression of IL-1βand IL-10 in TNBS-induced colitis.

<p>ELISA analysis was performed to detect IL-1β(A) and IL-10(B) expression. Values mean ± SD, n = 10 for each group, differences are evaluated using the one-way ANOVA on ranks test. ^##P<0.01, vs control group. ^△p>0.05, *P<0.05, **P<0.01 vs TNBS group.</p

The characterization of BMSC-EVs.

<p>(A) Transmission electron microscopy analysis of purified EVs showed a spheroid shape. The black line refers to200 nm. (B and C) Flow cytometry was used to characterize the proper size of BMSC-EVs. Analyzed BMSC-EVs (black) were compared with Sulfate-modified polystyrene latex beads with a mean size of 1 μm (fluorescent red, λ_ex ~575 nm; λ_em ~610 nm). P1 represents the gate of the fluorescent beads. (D) FACS analysis of BMSC-EVs surface protein expression. The results indicated that BMSC-EVs were positive for CD90(76.8%), CD29(86.4%) and negative for CD11b(10.3%) and CD45(9.54%).</p

Effects of BMSC-EVs therapies on protein expression of inflammatory mediators in TNBS-induced colitis.

<p>(A) Immunohistochemical detection of NF-κBp65, TNF-α, iNOS and COX-2 protein expression. (B) Western blot detection of NF-κBp65, TNF-α, iNOS and COX-2 protein expression. (C) Grey value histogram of western blot detection. β-actin was used as a control. Values mean ± SD, n = 10 for each group, differences are evaluated using the one-way ANOVA on ranks test. ^##P<0.01, vs control group. ^△p>0.05,*P<0.05, **P<0.01 vs TNBS group.</p

Scoring of disease activity index (DAI).

<p>Scoring of disease activity index (DAI).</p

The effects of BMSC-EVs on oxidative stress and antioxidant defenses in TNBS-induced colitis.

<p>ELISA analysis was performed to detect MPO(A), MAD(B), GSH(C) and SOD(D) expression. Values mean ± SD, n = 10 for each group. ^##P<0.01, vs control group. ^△p>0.05,*P<0.05, **P<0.01 vs TNBS group.</p

Diagram depicting the alleviating actions of BMSC-EVs in TNBS-induced colitis.

<p>Diagram depicting the alleviating actions of BMSC-EVs in TNBS-induced colitis.</p