Bone marrow mesenchymal stem cell-derived exosomes attenuate cerebral ischemia-reperfusion injury-induced neuroinflammation and pyroptosis by modulating microglia M1/M2 phenotypes.

Pyroptosis mediated by NLRP3 inflammasome plays a critical role in the pathogenesis of cerebral ischemia-reperfusion (I/R) injury. Mounting evidences have verified the efficacy of exosomes by relieving the inflammatory response during cerebral I/R injury, but the specific mechanism has not been well elucidated. This study aimed to clarify whether the neuroprotective effects of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) are associated with the attenuation of NLPR3-mediated neuron pyroptosis by modulating microglial polarization. Rats were initially subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion. Then, BMSC-Exos were administered intravenously 2 h after MCAO. The neuroprotective effects were measured using a modified neurological severity score(mNSS), triphenyltetrazolium chloride (TTC) staining, brain water content, Morris water maze,and CatWalk system. Western blotting and immunofluorescence staining were applied to detect NLRP3 inflammasome and pyroptosis. Microglial polarization was determined by real-time polymerase chain reaction (RT-PCR) and immunofluorescence staining. To mimic cerebral I/R injury in vitro, BV2 and PC12 cells were exposed to oxygen-glucose deprivation/reoxygenation. After treatment with PBS, BMSC-Exos, IL-4, or LPS, BV2 cells were co-cultured with PC12 cells in a Transwell system. BMSC-Exos reduced the brain infarct area and brain water content at 24 h dose dependently and improved the neurological function up to 5 weeks after stroke. In vivo, NLRP3 inflammasome- and pyroptosis-related proteins were mainly expressed on neurons and downregulated by BMSC-Exos. Furthermore, cerebral I/R injury-induced M1-polarized microglia could be shifted toward M2 phenotype by BMSC-Exos. In vitro, BMSC-Exos alleviated the neuron pyroptosis partially by modulating microglial polarization. BMSC-Exos could ameliorate cerebral I/R injury via suppression of NLRP3 inflammasome-mediated inflammation and pyroptosis by modulating microglial polarization.

Liu X ，Zhang M ，Liu H ，Zhu R ，He H ，Zhou Y ，Zhang Y ，Li C ，Liang D ，Zeng Q ，Huang G ... -  《-》

Salvianolic Acids for Injection alleviates cerebral ischemia/reperfusion injury by switching M1/M2 phenotypes and inhibiting NLRP3 inflammasome/pyroptosis axis in microglia in vivo and in vitro.

Ma DC ，Zhang NN ，Zhang YN ，Chen HS ... -  《-》

Aloe-emodin alleviates cerebral ischemia-reperfusion injury by regulating microglial polarization and pyroptosis through inhibition of NLRP3 inflammasome activation.

Li X ，Yao M ，Li L ，Ma H ，Sun Y ，Lu X ，Jing W ，Nie S ... -  《-》

BMSC-Exosomes attenuate ALP dysfunction by restoring lysosomal function via the mTOR/TFEB Axis to reduce cerebral ischemia-reperfusion injury.

The complex pathophysiological changes following cerebral ischemia-reperfusion injury (CIRI) include the accumulation of defective proteins and damaged organelles, which cause massive neuron demise. To preserve cellular homeostasis, the autophagy-lysosomal pathway (ALP) is crucial for neurons to dispose of these substances. Many studies have shown that bone mesenchymal stem cell exosomes (BMSC-Exos) can reduce CIRI. However, the specific mechanisms have not been well elucidated, a fact that limits its widespread clinical use. This study aimed to clarify whether BMSC-Exos could attenuate ALP dysfunction by restoring lysosomal function after CIRI via inhibiting mTOR and then activating TFEB nucleus translocation. In this study, Flow cytometry, Nanoparticle tracking analysis (NTA), Transmission electron microscope (TEM), and Western blot were used to identify the BMSCs and BMSC-Exos used in this experiment as conforming to the requirements. In vivo experiments, SD rats were modeled with temporary middle cerebral artery occlusion (tMCAO), and BMSC-Exos was injected into the tail vein 2 h after modeling. Triphenyl tetrazolium chloride (TTC) staining, modified neurological severity scores (mNSS), corner turn test, and rotating rod test were used to detect neurological deficits in rats after BMSC-Exos intervention. Western blot and Immunofluorescence were used to detect ALP, transcription factor EB(TFEB) nucleus translocation, and mammalian target of rapamycin (mTOR) change at different time points after modeling and after BMSC-Exos intervention. In vitro experiments, pheochromocytoma cells (PC12) cells were subjected to oxygen-glucose deprivation and reperfusion (OGD/R) modeling to mimic CIRI, and were respectively intervened with BMSC-Exos, BMSC-Exos + MHY 1485 (the mTOR agonist), Rapamycin (the mTOR inhibitor). CCK8, Western blot, and Immunofluorescence were used to detect PC12 cell survival, TFEB nucleus translocation, and cathepsin B(CTSB) Immunofluorescence intensity. We found that ALP dysfunction occurred 72 h after tMCAO, and BMSC-Exos can attenuate ALP dysfunction by restoring lysosomal function. Next, we examined TFEB nucleus translocation and the expression of mTOR, a key regulator of translocation. We found that BMSC-Exos could inhibit mTOR and activate TFEB nucleus translocation. Additional in vitro tests revealed that BMSC-Exos could increase PC12 cell survival after OGD/R, activating TFEB nucleus translocation and enhancing the fluorescence intensity of CTSB, which in turn could be reversed by the mTOR agonist, MHY1485. This effect was similar to another mTOR inhibitor, Rapamycin. BMSC-Exos could attenuate ALP dysfunction by restoring lysosomal function after CIRI by inhibiting mTOR and then promoting TFEB nucleus translocation.

Liu H ，Li C ，Zhang X ，Chen H ，Zhang Q ，Zeng Y ，Zheng S ，Zou J ，Zhao Y ，Zheng X ，Huang G ，Zeng Q ... -  《-》

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

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.

Zeng Q ，Zhou Y ，Liang D ，He H ，Liu X ，Zhu R ，Zhang M ，Luo X ，Wang Y ，Huang G ... -  《-》