Stimulation by exosomes from hypoxia-preconditioned hair follicle mesenchymal stem cells facilitates mitophagy by inhibiting the PI3K/AKT/mTOR signaling pathway to alleviate ulcerative colitis

Abstract

Background: Ulcerative colitis (UC) is an intestinal inflammatory disease that is strongly associated with mitochondrial damage and dysfunction as well as mitophagy and lacks of satisfactory treatments. Hair follicle mesenchymal stem cell (HF-MSC)-derived exosomes owe benefit effectiveness on inflammatory therapies. Hypoxia-preconditioned HF-MSCs exhibit enhanced proliferation and migration abilities, and their exosomes exert stronger effects than normal exosomes. However, the therapeutic function of Hy-Exos in UC is unknown. Methods: The inflammation model was established with LPS-treated MODE-K cells, and the mouse UC model was established by dextran sulfate sodium (DSS) administration. The therapeutic effects of HF-MSC-derived exosomes (Exos) and hypoxia-preconditioned HF-MSC-derived exosomes (Hy-Exos) were compared in vitro and in vivo. Immunofluorescence staining and western blotting were used to explore the effects of Hy-Exos on mitochondrial function, mitochondrial fission and fusion and mitophagy. MiRNA sequencing analysis was applied to investigate the differences in components between Exos and Hy-Exos. Results: Hy-Exos had a better therapeutic effect on LPS-treated MODE-K cells and DSS-induced UC mice. Hy-Exos promoted colonic tight junction proteins expression, suppressed the oxidative stress response, and reduced UC-related inflammatory injury. Hy-Exos may exert these effects via miR-214-3p-mediated inhibition of the PI3K/AKT/mTOR signaling pathway, maintenance of mitochondrial dynamic stability, alleviation of mitochondrial dysfunction and enhancement of mitophagy. Conclusion: This study revealed a vital role for Hy-Exos in suppressing inflammatory progression in UC and suggested that miR-214-3p is a potential critical target for Hy-Exos in alleviating UC.

Keywords: Exosome; Hair follicle mesenchymal stem cell; Mitophagy; PI3K/AKT/mTOR signaling pathway; Ulcerative colitis.

Figure 1

Characterization of HF-MSCs, Exos and Hy-Exos. Primary (A) and P3 (B) HF-MSCs. Scale bar = 100 μm. C. Adipogenic differentiation of HF-MSCs. D. Osteogenic differentiation of HF-MSCs. E. Flow cytometry was used to detect HF-MSC-specific antigenic markers. F-G. Immunofluorescence staining were used to measure the expression of CD34 and CK15 in HF-MSCs. Scale bar = 100 μm. H. SOX9 and CK19 expression was measured by western blotting. I. Morphology of Exos by TEM. Scale bar = 100 μm. J. Nanoparticle tracking analysis of Exos. K. Morphology of Hy-Exos by TEM. Scale bar = 100 μm. L. Nanoparticle tracking analysis of Hy-Exos. M. CD63, TSG101, HSP70 and Calnexin expression were measured by western blotting. N. PKH26-labelled Exos and Hy-Exos were internalized by PKH67-labelled MODE-K cells. Scale bar = 50 μm.

Figure 2

Hy-Exos inhibit the PI3K/AKT/mTOR signaling pathway and promote LPS-induced MODE-K cells recovery. A-D. IL-1β, TNF-α, IL-4 and IL-10 expression in MODE-K cells was measured by PCR. E-F. MODE-K cells proliferation in each group was determined by EdU staining. Scale bar = 100 μm. G. MODE-K cells viability in each group was detected by CCK-8 assays. H. Volcano plot of DEGs in GSE75214. I. Heat map of DEGs in GSE75214. J-L. GO enrichment analysis of DEGs. M. KEGG enrichment analysis of DEGs. N-Q. Western blotting was used to measure p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR expression in MODE-K cells. Data are shown as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3

Hy-Exos maintain mitochondrial dynamic stabilization, alleviate mitochondrial dysfunction and enhance autophagy in MODE-K cells. A-F. Western blotting was used to measure Drp1, Fis1, Mfn1, Mfn2 and OPA1 expression in MODE-K cells. G, I. Mitochondrial membrane potential in MODE-K cells as measured by a JC-1 staining kit, and the ratio of aggregates (red) to monomers (green) was calculated. Scale bar = 50 μm. H, J. Mitochondrial ROS was detected in MODE-K cells. Scale bar = 50 μm. K. Determination of ATP contents in MODE-K cells. L-M. MODE-K cells were infected with mRFP-EGFP-LC3 adenovirus, and the number of yellow spots (autophagosomes) was observed under a microscope. Scale bar = 10 μm. Data are shown as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4

Hy-Exos enhance mitophagy in MODE-K cells. A-F. Beclin1, p62, LC3II/I, HSP60 and TOMM20 expression in MODE-K cells was measured by western blotting. G. Mitochondrial probe and lysosome probe staining in MODE-K cells. Scale bar = 20 μm. H-I. Analysis of the colocalization of the mitochondrial probe and lysosomal probe. J. Immunofluorescence staining for LC3 and COX IV in MODE-K cells. Scale bar = 50 μm. K-L. The colocalization of LC3 and COX IV analysis. Data are shown as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

DEMs identification and the role of miR-214-3p in Hy-Exos. A. Volcano plot of DEMs. B. Heat map of DEMs. C. PCR was used to detect the expression of miR-92b-3p, miR-484, miR-214-3p, miR-30a-5p and miR-205-5p in the Exos and Hy-Exos groups. D-G. Western blotting was used to measure p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR expression in MODE-K cells. H-M. Western blotting was used to measure Drp1, Fis1, Mfn1, Mfn2 and OPA1 expression in MODE-K cells. N-S. Western blotting was used to measure Beclin1, p62, LC3II/I, HSP60 and TOMM20 expression in MODE-K cells. Data are shown as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 6

Hy-Exos promote the amelioration of DSS-induced UC. A-B. Mice in the control group and UC group were given DiR-stained Exos and Hy-Exos via the tail vein. The mice were subjected to ex vitro organ imaging and quantitative analysis of fluorescence intensity. C-D. Comparison of the colon lengths of the mice in each group. E. Comparison of body weight changes in mice in each group. F. DAI scores were determined in mice by monitoring changes in body weight, faecal traits, and the degree of haematochezia. G-J. HE staining results of mice in each group. Scale bar = 500 μm (left). Scale bar = 100 μm (right). K-M. The area and height of the colonic villi of mice in each group were measured and analysed. N. MPO expression in colonic tissue homogenates from mice in each group was determined. Data are shown as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001, ^△△△△p < 0.0001).

Figure 7

Hy-Exos inhibit the PI3K/AKT/mTOR signaling pathway, maintain mitochondrial dynamic stabilization and alleviate mitochondrial dysfunction in UC mice. A-D. RT-PCR was performed to measure IL-1β, TNF-α, IL-4 and IL-10 expression in the colon tissues of mice. E-H. Western blotting was used to measure p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR expression in colon tissues. I-J. PKH26-stained Exos and Hy-Exos colocalized with Claudin1 and Occludin. Scale bar = 150 μm. K-L. Claudin1 and Occludin expression in colon tissues was determined by western blotting. M-R. Western blotting was used to measure the expression of Drp1, Fis1, Mfn1, Mfn2, and OPA1 in colon tissues. S-U. Determination of the CAT, GSH, and MDA contents in colon tissues. Data are shown as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 8

Hy-Exos enhance mitophagy in UC mice. A. TEM was performed to observe the ultrastructure of the colonic tissues of mice, and the formation of mitochondria (red arrows) and autophagosomes (yellow arrows) was observed. Scale bar = 1 μm. B-D. PKH26-stained Exos and Hy-Exos colocalized with Beclin1, p62 and LC3II/I. Scale bar = 150 μm. E-J. Western blotting was used to measure Beclin1, p62, LC3II/I, HSP60 and TOMM20 expression in colon tissues. K. Immunofluorescence staining for LC3 and COX IV in colon tissues. Scale bar = 150 μm. Data are shown as the mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).