Exosomes Secreted From Bone Marrow Mesenchymal Stem Cells Attenuate Oxygen-Glucose Deprivation/Reoxygenation-Induced Pyroptosis in PC12 Cells by Promoting AMPK-Dependent Autophagic Flux

Abstract

Background: Cerebral ischemia-reperfusion (I/R) injury can lead to severe dysfunction, and its treatment is difficult. It is reported that nucleotide-binding domain and leucine-rich repeat family protein 3 (NLRP3) inflammasome-mediated cell pyroptosis is an important part of cerebral I/R injury and the activation of autophagy can inhibit pyroptosis in some tissue injury. Our previous study found that the protective effects of bone marrow mesenchymal stem cells (BMSCs) in cerebral I/R injury may be associated with the regulation of autophagy. Recent studies have demonstrated that exosomes secreted from BMSCs (BMSC-Exos) may play an essential role in the effective biological performance of BMSCs and the protective mechanism of BMSC-Exos is associated with the activation of autophagy and the remission of inflammation, but it has not been reported in studies of cerebral I/R injury. We aimed to investigate the effects of BMSC-Exos on cerebral I/R injury and determine if the mechanism is associated with the regulation of pyroptosis and autophagic flux. Method: PC12 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to induce cerebral I/R in vitro and were cocultured with BMSC-Exos. Cell viability was determined with CCK-8 and lactate dehydrogenase (LDH) detection kits. Scanning electron microscopy (SEM), Hoechst 33342/propidium iodide (PI) double staining, 2',7'-dichlorodihydrofluorescein diacetate assay, immunofluorescence, Western blot, and Enzyme-linked immunosorbent assay (ELISA) were used to detect cell pyroptosis. Furthermore, transmission electron microscopy (TEM), GFP-RFP-LC3 adenovirus transfection, and Western blot were used to detect autophagic flux and its influence on pyroptosis. Finally, coimmunoprecipitation was used to detect the binding interaction between NLRP3 and LC3. Results: BMSC-Exos increased cell viability in OGD/R. The inhibitory effect of BMSC-Exos on pyroptosis was comparable to the NLRP3 inhibitor MCC950 and was reversed by NLRP3 overexpression. Furthermore, BMSC-Exos promoted autophagic flux through the AMP-activated kinase (AMPK)/mammalian target of the rapamycin pathway, whereas chloroquine, AMPK silencing, and compound C blocked the inhibitory effect on pyroptosis. Conclusions: BMSC-Exos can protect PC12 cells against OGD/R injury via attenuation of NLRP3 inflammasome-mediated pyroptosis by promoting AMPK-dependent autophagic flux.

Keywords: autophagy; cerebral ischemia/reperfusion; exosomes secreted from bone marrow mesenchymal stem cells; nucleotide-binding domain leucine-rich repeats family protein 3; pyroptosis.

Figure 1

Characterization of bone marrow mesenchymal stem cells (BMSCs) and exosomes from BMSCs (BMSC-Exos); BMSC-Exos increase cell viability following oxygen-glucose deprivation/reoxygenation (OGD/R). (A) Flow cytometry analyses indicated that BMSCs were positive for CD29, CD44, and CD90 but were negative for CD45. (B) Round morphologies of BMSC-Exos were demonstrated by tandem electron microscopy (TEM); scale bar = 100 nm. (C) Western blot analyses showed that the BMSC-Exos were positive for the specific exosome surface markers TSG101 and CD9. (D) NanoSight NTA analysis indicated that the diameters of BMSC-Exos were around 100 nm. (E) A significant reduction in cell viability was observed in OGD 6-h, 9-h, 12-h, and 24-h groups (n = 3). (F) BMSC-Exos enhanced cell viability in a dose-dependent manner (n = 3). (G) BMSC-Exos reduced lactate dehydrogenase (LDH) release following OGD/R (n = 3). *p < 0.05, ***p < 0.001 vs. control group; ^##p < 0.01 vs. OGD/R group.

Figure 2

BMSC-Exos reduce pyroptosis induced by OGD/R. (A) Morphological changes of PC12 cells were observed using scanning electron microscopy (SEM); scale bar = 20/10/5 μm. (B) Representative images of Hoechst 33342/propidium iodide (PI) staining; scale bar = 50 μm. (C) PI-stained cells (red puncta) were quantified as the percentage of red puncta/total puncta signals in merged images (n = 3). PI-stained areas were lower in the BMSC-Exos group than in the OGD/R group (n = 3). (D) Representative images of reactive oxygen species (ROS); scale bar = 10 μm. (E) ROS levels are presented relative to control levels (n = 3). ROS levels in the BMSC-Exos group were lower than in the OGD/R group. (F) Representative immunofluorescence images; scale bar = 75 μm. (G) Relative expression levels of N-terminal of gasdermin D (GSDMD-N) were lower in the BMSC-Exos group than in the OGD/R group (n = 3). **p < 0.01, ***p < 0.001 vs. control group, ^#p < 0.05; ^##p < 0.01 vs. OGD/R group.

Figure 3

BMSC-Exos reduce nucleotide-binding domain and leucine-rich repeat family protein 3 (NLRP3) inflammasome-mediated pyroptosis following OGD/R. (A) Representative Western blots of NLRP3, cleaved caspase-1, and GSDMD-N. (B–D) Expression levels of NLRP3, cleaved caspase-1, and GSDMD-N in the BMSC-Exos group were higher than in the OGD/R group, whereas no significant difference was observed between BMSC-Exos and MCC950 groups (n = 3). (E) Interleukin-1β (IL-1β) levels in the BMSC-Exos group were higher than in the OGD/R group, whereas no significant difference was observed between BMSC-Exos and MCC950 groups (n = 3). (F) No significant difference in cell viability was observed between BMSC-Exos and MCC950 groups (n = 3). (G) No significant difference in LDH release was observed between BMSC-Exos and MCC950 groups (n = 3). (H) Representative Western blots of cleaved caspase-1 and GSDMD-N. (I) Real-time quantitative polymerase chain reaction (RT-qPCR) showing the effects of pNLRP3 transfection (n = 3). (J,K) Expression levels of cleaved caspase-1 and GSDMD-N were higher in the BMSC-Exos+pNLRP3 group than in the BMSC-Exos group (n = 3). (L) IL-1β levels were higher in the BMSC-Exos+pNLRP3 group than in the BMSC-Exos group (n = 3). (M) Cell viability in the BMSC-Exos+pNLRP3 group was lower than in the BMSC-Exos group (n = 3). (N) LDH release in the BMSC-Exos+pNLRP3 group was higher than in the BMSC-Exos group (n = 3). **p < 0.01, ***p < 0.001 vs. control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. OGD/R group; ^$p < 0.05, ^$$p < 0.01, ^$$$ p < 0.001 vs. OGD/R+BMSC-Exos group; ^&p < 0.05, ^&&&p < 0.001 vs. OGD/R+pNLRP3 group.

Figure 4

BMSC-Exos promote autophagic flux through the AMP-activated kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway following OGD/R. (A) Autophagic flux was detected using TEM. Typical cytoplasms and nuclei (N) in the control group; double-membrane autophagosomes (AP) were observed in the OGD/R group. Autolysosomes were darkly stained, indicating that autolysosomes (ASS) are activated in the OGD/R+BMSC-Exos group; scale bar = 1 μm. (B) Quantitative analysis of numbers of autophagosomes and autolysosomes in each treatment group (n = 3). Autophagosomes and autolysosomes were more numerous in the OGD/R group than in the control group, whereas larger numbers of autolysosomes were detected in the BMSC-Exos group. (C) Representative images of GFP-RFP-LC3 staining; scale bar = 10 μm. (D) Autophagy was quantified as the ratio of red puncta (GFP-RFP+) to yellow puncta (GFP+RFP+) in each cell. This ratio was higher in the BMSC-Exos group than in control and OGD/R groups (n = 3). (E,F) The autophagy inhibitor Cq reversed cell viability and LDH release (n = 3). (G) Representative Western blots of p-AMPK, AMPK, p-mTOR, mTOR, LC3 II/I and P62. (H–K) LC3 II/I and p-AMPK/AMPK expression increased, although P62 and p-mTOR/mTOR expression levels decreased in the OGD/R group when compared with the control group (n = 3). LC3 II/I expression did not change significantly after BMSC-Exos treatment, whereas p-AMPK/AMPK expression increased further and p-mTOR/mTOR and P62 expression levels decreased (n = 3). (L,M) The AMPK inhibitor compound C reversed the effects of OGD/R on cell viability and LDH release (n = 3). *p < 0.05, ***p < 0.001 vs. control group; ^#p < 0.05, ^###p < 0.001 vs. OGD/R group; ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 vs.OGD/R+BMSC-Exos group.

Figure 5

AMPK knockdown negated the effects of BMSC-Exos on autophagic flux. (A–C) AMPK protein levels were reduced following gene silencing. AMPK knockdown reversed the effects of BMSC-Exos on (D–H) activation of p-AMPK/AMPK and LC3 II/I, inhibition of p-TOR/mTOR and P62, (I) increase in cell viability, and (J) reduction in LDH release (n = 3). (K,L) AMPK knockdown reversed the effects of BMSC-Exos on the ratio of red puncta to yellow puncta (n = 3). *p < 0.05, ***p < 0.001 vs. control group; ^#p < 0.05, ^###p < 0.001 vs. OGD/R group; ^$$p < 0.01, ^$$$p < 0.001 vs. OGD/R+BMSC-Exos group; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001 vs. OGD/R+BMSC-Exos+si-NC group.

Figure 6

Blocked autophagic flux reverses the protective effect of BMSC-Exos against OGD/R-induced pyroptosis. (A) Representative Western blots of NLRP3, cleaved caspase-1, and GSDMD-N. (B–D) NLRP3, cleaved caspase-1, and GSDMD-N expression levels in the BMSC-Exos+Cq group were higher than in the BMSC-Exos group (n = 3). (E) IL-1β expression was higher in the BMSC-Exos+Cq group than in the BMSC-Exos group (n = 3). (F) Representative Western blots of NLRP3, cleaved caspase-1, and GSDMD-N. (G–I) NLRP3, cleaved caspase-1, and GSDMD-N expression levels in the BMSC-Exos+compound C group were higher than in the BMSC-Exos group (n = 3). (J) IL-1β expression was higher in the BMSC-Exos+compound C group than in the BMSC-Exos group (n = 3). (K) Representative images of Hoechst 33342/PI staining; scale bar = 75 μm. (L) Proportions of PI stained areas were higher in the BMSC-Exos+compound C group than in the BMSC-Exos group (n = 3). (M) Representative immunofluorescence images; scale bar = 75 μm. (N) Relative expression levels of GSDMD-N were higher in the BMSC-Exos+compound C group than in the BMSC-Exos group (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. OGD/R group; ^$p < 0.05, ^$$p < 0.01, ^$$$ p < 0.001 vs. OGD/R+BMSC-Exos group.

Figure 7

AMPK knockdown negated the protective effect of BMSC-Exos against pyroptosis. (A–E) AMPK knockdown reversed the effect of BMSC-Exos on inhibition of NLRP3, cleaved caspase-1, GDDMD-N, and IL-1β (n = 3). (F,G) AMPK knockdown reversed the effects of BMSC-Exos on the proportions of PI stained areas (n = 3). (H,I) AMPK knockdown reversed the effects of BMSC-Exos on the relative expression levels of GSDMD-N (n = 3). ***p < 0.001 vs. control group; ^##p < 0.01, ^###p < 0.001 vs. OGD/R group; ^&p < 0.05, ^&&p < 0.01; ^&&& p < 0.001 vs. OGD/R+BMSC-Exos+si-NC group.

Figure 8

Coimmunoprecipitation of NLRP3 and LC3. (A) Cell lysates from PC12 cells under normal condition or OGD/R with or without BMSC-Exos were incubated with anti-LC3B antibody. NLRP3 and LC3 in precipitates were detected by Western blot. IgG was used as a negative control. (B) The NLRP3 was precipitated by anti-LC3B antibody, and the relative amount of NLRP3 was compared (n = 3 biological replicates per group). The direct interaction between NLRP3 and LC3 was significantly decreased under OGD/R, and BMSC-Exos could increase the interaction. *p < 0.05 vs. control group, ^#p < 0.05 vs. OGD/R group.

Figure 9

Graphical abstract demonstrating how BMSC-Exos inhibit NLRP3 inflammasome-mediated pyroptosis by promoting AMPK-dependent autophagic flux in OGD/R.