Intramyocardial injection of Exosomes derived from FNDC5-MSCs significantly attenuated cardiomyocyte apoptosis in MI mice
Exosomes were isolated by density-gradient ultracentrifugation. As shown in Supplemental Fig. 1A, 1B, exosomes showed a cup-shaped morphology by TEM, and the size of exosomes was about 100 nm by NTA. Meanwhile, markers of exosomes (CD63, CD81 and ALIX) were highly expressed in FNDC5-MSCs‐Exo groups compared with MSCs-Exo group (Supplemental Fig. 1C). 50 µg MSCs-Exo or FNDC5-MSCs-Exo dissolved in 50 µL PBS was injected into the infarct border zone at four spots. (Fig. 1A).
TUNEL assay was used to detect the cardiomyocyte apoptosis after MI. As shown in Fig. 1B, TUNEL-positive (green) cardiomyocytes in MSCs-Exo and FNDC5-MSCs-Exo group were decreased compared with PBS group. Meanwhile, the quantitative analysis reveals that the percentages of TUNEL-positive cells exposed to MSCs-Exo, and FNDC5-MSCs-Exo group were 11.37 ± 0.59% and 6.97 ± 0.46% respectively, significantly decreased as opposed to the PBS group (13.5 ± 0.43%, p < 0.05). What is more, compared with MSCs-Exo group, the apoptosis rate of cells was remarkably decreased in FNDC5-MSCs-Exo group (P < 0.05). Western blot analysis further confirmed that the cardioprotective effect of MSCs-Exo and FNDC5-MSCs-Exo with reducing protein amounts of Bax and cleaved caspase-3 in ischemic heart tissue (Fig. 1C and 1D, P < 0.05). Taken together, FNDC5-MSCs-Exo could attenuate cardiomyocyte apoptosis after MI.
FNDC5-MSCs-Exo administration reduced post‐infarction inflammation and increased M2 macrophage polarization in vivo
MI injury induced post-infarction inflammation, and a great amount of inflammatory cells assembled and infiltrated into the infarction area. H&E staining showed that in both MSCs-Exo and FNDC5-MSCs-Exo group, FNDC5-MSCs-Exo obviously alleviated the infiltration of inflammatory cells to protect myocardium from further damage (Fig. 2A). Furthermore, the pro-inflammatory cytokines, TNF‐α and IL-6, were detected in serum at 3 and 7 days. Compared with MSCs-Exo group, the secretions of TNF‐α and IL-6 were significantly reduced in FNDC5-MSCs-Exo group (Fig. 2B-E, P < 0.05).
Macrophage polarization played an essential role in inflammation. Thus, we explored the subsets of macrophages accumulating in the ischemic heart. Western blot analysis verified that the protein expression of CD206 (M2 marker) in ischemic heart tissue was elevated under MSCs-Exo and FNDC5-MSCs-Exo group (Fig. 2F, 2G). Furthermore, FNDC5-MSCs-Exo presented better effects than MSCs-Exo on facilitating M2 macrophage polarization after MI (Fig. 2H and 2I, P < 0.05). Collectively, compared with MSCs-Exo, FNDC5-MSCs-Exo reduced post-infarction inflammation and promoted M2 macrophage polarization in mice.
FNDC5-MSCs-Exo ameliorates inflammation responses by increasing anti‐inflammatory cytokines, as well as decreasing pro‐inflammatory cytokines in Raw264.7 cells
To further investigate the effects of MSCs-Exo, FNDC5-MSCs-Exo on inflammation responses, exosomes were administrated to Raw264.7 cells under LPS treatment (100 ng/mL). To optimize the reasonable dose of exosomes, we detected the effects of different doses of exosomes (5, 10, 15 and 20 µg/mL) on inflammatory cytokines. ELISA analysis showed that the pro-inflammatory cytokines (IL‐6, TNF‐α and IL-1β) were decreased while the anti-inflammatory cytokine (IL-10) was increased in both Exo and L-Exo groups by a dose-dependent manner (Fig. 3A‐D, P < 0.05). Moreover, the pro‐inflammatory cytokines were dramatically decreased by exosomes at 20 µg/mL (P < 0.05). Thus, the dose of exosomes was performed to further study.
CCK-8 assay was performed to confirm that MSCs-Exo and FNDC5-MSCs-Exo reduced pro-inflammatory cytokines without affecting cell viability. Data showed no significant influence on cell viability ((Supplemental Fig. 2).
In summary, our data indicated that FNDC5-MSCs-Exo had superior therapeutic effects on anti-inflammation by increasing anti‐inflammatory cytokines and decreasing pro‐inflammatory cytokines.
FNDC5-MSCs-Exo dramatically increased M2 macrophage polarization and decrease M1 macrophage polarization in vitro
To explore macrophage polarization, we detected macrophages markers CD11b, CXCL10 (M1 macrophages) and CD206, ArgI (M2 macrophages). Western blot analysis indicated that MSCs-Exo, FNDC5-MSCs-Exo significantly increased the expression level of CD206, while markedly reduced the expression level of CD11b compared with LPS group (Fig. 4A-C, P < .05). Furthermore, the RT‐qPCR results showed that the mRNA level of CXCL10 remarkably reduced and Arg1 was significantly elevated in FNDC5-MSCs-Exo group (Fig. 4D-4E, P < 0.05).
Taken together, these results showed that FNDC5-MSCs-Exo markedly promoted M2 macrophage polarization while reduced M1 macrophage polarization.
FNDC5-MSCs-Exo reduced the inflammation by suppressing the NF‐κB signaling pathway and upregulating Nrf2/HO-1 Axis
To further understand the molecular mechanism of inflammation regulated by exosomes, we evaluated the effects of MSCs-Exo and FNDC5-MSCs‐Exo on LPS‐dependent NF‐κB signaling pathway. The Western blot results showed that compared with LPS group, total protein of IκBα was increased while p-IκBα was decreased in both MSCs‐Exo and FNDC5-MSCs‐Exo group (Fig. 5A-5C, P < 0 .05). Furthermore, NF‐κB p65 translocation from the cytoplasm to nucleus was suppressed (Fig. 5D-5E, P < 0 .05). We also found that compared with MSCs-Exo group, FNDC5-MSCs‐Exo prominently suppressed the nuclear translocation of NF‐κB p65 (Fig. 5F-5G, P < 0 .05). Meanwhile, we focused on the effects of MSCs‐Exo and FNDC5-MSCs‐Exo on the Nrf2/HO-1 pathway by immunostaining and Western blot analysis. As indicated in Fig. 6A, FNDC5-MSCs‐Exo obviously induced the nuclear translocation of Nrf2. Then, Western blot analysis also suggested that FNDC5-MSCs‐Exo increased HO-1 expression (Fig. 6B-6C, P < 0 .05).
To clarify the impact of HO-1 on the anti-inflammatory activities of FNDC5-MSCs-Exo, HO-1 specific inhibitor Sn (IV) protoporphyrin IX dichloride (SnPP, 20 µM) was administrated to block the enzyme activity of HO-1. As shown in Fig. 6D and 6E, SnPP attenuated the inhibitory effect of FNDC5-MSCs‐Exo on nucleus translocation of NF-ĸB p65 (P < 0 .05). ELISA analysis suggested that HO-1 inhibitor dramatically recovered secretion of pro-inflammatory cytokines such as TNF‐α, IL‐6, and IL-1β (Fig. 6F-6H, P < 0 .05). Furthermore, the RT‐qPCR results showed that HO-1 inhibitor significantly elevated the mRNA level of CXCL10 while reduced that of Arg1 compared with FNDC5-MSCs-Exo group (Fig. 6I-6J, P < 0.05).