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.

背景：脑缺血再灌注（I / R）损伤可导致严重功能障碍，并且治疗困难。据报道，核苷酸结合结构域和富亮氨酸重复家族蛋白3（NLRP3）炎性小体介导的细胞凋亡是脑I / R损伤的重要组成部分，自噬的激活可以抑制某些组织损伤中的凋亡。我们先前的研究发现，骨髓间充质干细胞（BMSCs）对脑I / R损伤的保护作用可能与自噬的调节有关。最近的研究表明，BMSCs分泌的外泌体（BMSC-Exos）可能在BMSCs的有效生物学性能中起重要作用，并且BMSC-Exos的保护机制与自噬的激活和炎症的缓解有关，但在脑I / R损伤研究中尚未报道。我们旨在研究BMSC-Exos对脑I / R损伤的影响，并确定该机制是否与调节细胞凋亡和自噬通量有关。

方法：对PC12细胞进行氧葡萄糖剥夺/复氧（OGD / R）诱导脑I / R 体外并与BMSC-Exos共培养。用CCK-8和乳酸脱氢酶（LDH）检测试剂盒确定细胞活力。扫描电子显微镜（SEM），Hoechst 33342 /碘化丙啶（PI）双重染色，2'，7'-二氯二氢荧光素双乙酸盐测定，免疫荧光，蛋白质印迹和酶联免疫吸附测定（ELISA）用于检测细胞的热解。此外，使用透射电镜（TEM），GFP-RFP-LC3腺病毒转染和Western印迹检测自噬通量及其对细胞凋亡的影响。最后，使用共免疫沉淀法检测NLRP3和LC3之间的结合相互作用。

结果：BMSC-Exos增加了OGD / R细胞的活力。BMSC-Exos对发烧的抑制作用与NLRP3抑制剂MCC950相当，并且被NLRP3过表达逆转。此外，BMSC-Exos通过雷帕霉素途径的AMP激活激酶（AMPK）/哺乳动物靶标促进了自噬通量，而氯喹，AMPK沉默和化合物C阻止了对凋亡的抑制作用。

结论：BMSC-Exos可以保护PC12细胞免受OGD / R损伤 通过 通过促进AMPK依赖的自噬通量来减轻NLRP3炎性体介导的细胞凋亡。