Exosomes derived from MSC pre-treated with oridonin alleviates myocardial IR injury by suppressing apoptosis via regulating autophagy activation

Abstract

This study aimed to investigate the molecular mechanisms underlying the role of bone marrow mesenchymal stem cells (BMMSCs)-derived exosomes in ischaemia/reperfusion (IR)-induced damage, and the role of oridonin in the treatment of IR. Exosomes were isolated from BMMSCs. Western blot analysis was done to examine the expression of proteins including CD63, CD8, apoptotic-linked gene product 2 interacting protein X (AliX), Beclin-1, ATG13, B-cell lymphoma-2 (Bcl-2), apoptotic peptidase activating factor 1 (Apaf1) and Bcl2-associated X (Bax) in different treatment groups. Accordingly, the expression of CD63, CD81 and AliX was higher in BMMSCs-EXOs and IR + BMMSCs-EXOs + ORI groups compared with that in the BMMSCs group. And BMMSCs-derived exosomes inhibited the progression of IR-induced myocardial damage, while this protective effect was boosted by the pre-treatment with oridonin. Moreover, Beclin-1, ATG13 and Bcl-2 were significantly down-regulated while Apaf1 and Bax were significantly up-regulated in IR rats. And the presence of BMMSCs-derived exosomes partly alleviated IR-induced dysregulation of these proteins, while the oridonin pre-treatment boosted the effect of these BMMSCs-derived exosomes. The inhibited proliferation and promoted apoptosis of H9c2 cells induced by hypoxia/reperfusion (HR) were mitigated by the administration of BMMSCs-derived exosomes. Meanwhile, HR also induced down-regulation of Beclin-1, ATG13 and Bcl-2 expression and up-regulation of Apaf1 and Bax, which were mitigated by the administration of BMMSCs-derived exosomes. And oridonin pre-treatment boosted the effect of BMMSCs-derived exosomes. In conclusion, our results validated that BMMSCs-derived exosomes suppressed the IR-induced damages by participating in the autophagy process, while the pre-treatment with oridonin could boost the protective effect of BMMSCs-derived exosomes.

Keywords: apoptosis; autophagy; exosomes; mesenchymal stem cells.; myocardial ischaemia/reperfusion; oridonin.

FIGURE 1

Exosomes isolated from BMMSCs were visualized by a transmission electron microscope, and the expression of CD63, CD81 and AliX was compared among the BMMSCs, BMMSCs‐EXOs and BMMSCs‐EXOs + ORI groups (n = 3; *P value < .05 vs BMMSCs group). A, The general shape of exosomes isolated from BMMSCs‐EXOs group and BMMSCs‐EXOs + ORI group; B, Western blot of CD63, CD81 and AliX expression in BMMSCs, BMMSCs‐EXOs and BMMSCs‐EXOs + ORI groups; C, Relative density of CD63 in BMMSCs, BMMSCs‐EXOs and BMMSCs‐EXOs + ORI groups; D, Relative density of CD81 in BMMSCs, BMMSCs‐EXOs and BMMSCs‐EXOs + ORI groups; E, Relative density of AliX in BMMSCs, BMMSCs‐EXOs and BMMSCs‐EXOs + ORI groups

FIGURE 2

H&E staining indicated that BMMSCs‐derived exosomes inhibited the progression of IR‐induced myocardial damage in IR + BMMSCs‐EXOs and IR + BMMSCs‐EXOs + ORI groups, and the pre‐treatment with oridonin enhanced the effect of BMMSCs‐derived exosomes. A, H&E staining results of longitudinal section of myocardial tissues collected from the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; B, Quantitative H&E staining results of longitudinal myocardial tissues in different rat groups; C, H&E staining results of cross section of myocardial tissues collected from the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; D, Quantitative H&E staining results of cross‐sectional myocardial tissues in different rat groups

FIGURE 3

TUNEL staining and Masson's trichrome staining indicated that IR‐induced apoptosis and fibrosis, as well as damaged cardiac functions, were restored by BMMSCs‐derived exosomes in IR + BMMSCs‐EXOs and IR + BMMSCs‐EXOs + ORI groups, and the pre‐treatment with oridonin enhanced the effect of BMMSCs‐derived exosomes (n = 3; *P value < .05 vs SHAM group; **P value .05 vs IR group; ^# P value < .05 vs IR + BMMSCs‐EXOs group). A, TUNEL staining of IR‐induced apoptosis of myocardial tissues collected from the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group (Brown indicates TUNEL positive cells, and blue indicates TUNEL negative cells); B, Masson's trichrome staining of IR‐induced fibrosis of myocardial tissues collected from the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group (Blue indicate fibrosis); C, Heart rate of the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; D, LVSP of the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; E, LVFS of the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; F, LVEF of the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; G, LVWT of the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group

FIGURE 4

The dysregulation of Beclin‐1, ATG13, Apaf1, Bcl‐2, Bax and Ki67 expression was reversed by BMMSCs‑derived exosomes in vivo (n = 3; *P value < .05 vs SHAM group; **P value .05 vs IR group; ^# P value < .05 vs IR + BMMSCs‐EXOs group). A, Western blot of Beclin‐1, ATG13 and Apaf1 expression in the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; B, Protein expression of Beclin‐1 in the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; C, Protein expression of ATG13 in the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; D, Protein expression of Apaf1 in the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; E, Protein expression of Bcl‐2 in the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; F, Protein expression of Bax in the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group; G, IHC assay of Ki67 in the sham group, IR group, IR + BMMSCs‐EXOs group and IR + BMMSCs‐EXOs + ORI group

FIGURE 5

Administration of BMMSCs‐derived exosomes affected the proliferation and apoptosis of H9c2 cells (*P value < .05 vs control group; **P value .05 vs HR group; ^# P value < .05 vs HR + BMMSCs‐EXOs group). A, Ratio of EdU positive cells in the control group, HR group, HR + BMMSCs‐EXOs group and HR + BMMSCs‐EXOs + ORI group; B, Apoptosis rate of H9c2 cells in the control group, HR group, HR + BMMSCs‐EXOs group and HR + BMMSCs‐EXOs + ORI group

FIGURE 6

The dysregulation of Beclin‐1, ATG13, Apaf1, Bcl‐2 and Bax expression was reversed by BMMSCs‑derived exosomes in vitro (*P value < .05 vs control group; **P value .05 vs HR group; ^# P value < .05 vs HR + BMMSCs‐EXOs group). A, Western blot of Beclin‐1, ATG13 and Apaf1 expression in the control group, HR group, HR + BMMSCs‐EXOs group and HR + BMMSCs‐EXOs + ORI group; B, Protein expression of Beclin‐1 in the control group, HR group, HR + BMMSCs‐EXOs group and HR + BMMSCs‐EXOs + ORI group; C, Protein expression of ATG13 in the control group, HR group, HR + BMMSCs‐EXOs group and HR + BMMSCs‐EXOs + ORI group; D, Protein expression of Apaf1 in the control group, HR group, HR + BMMSCs‐EXOs group and HR + BMMSCs‐EXOs + ORI group; E, Protein expression of Bcl‐2 in the control group, HR group, HR + BMMSCs‐EXOs group and HR + BMMSCs‐EXOs + ORI group; F, Protein expression of Bax in the control group, HR group, HR + BMMSCs‐EXOs group and HR + BMMSCs‐EXOs + ORI group