Exosomes Secreted From Bone Marrow Mesenchymal Stem Cells Attenuate Oxygen-Glucose Deprivation/Reoxygenation-Induced Pyroptosis in PC12 Cells by Promoting AMPK-Dependent Autophagic Flux
Exosomes Secreted From Bone Marrow Mesenchymal Stem Cells Attenuate Oxygen-Glucose Deprivation/Reoxygenation-Induced Pyroptosis in PC12 Cells by Promoting AMPK-Dependent Autophagic Flux
复制标题
骨髓间充质干细胞分泌的外泌体通过促进 AMPK 依赖性自噬通量减轻 PC12 细胞中氧葡萄糖剥夺/复氧诱导的焦亡
DOI：
10.3389/fncel.2020.00182
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发表时间：
2020-07-17
影响因子：
5.3
通讯作者：
Huang, Guozhi
中科院分区：
文献类型：
--
作者：
Zeng, Qing;Zhou, Yuqing;Huang, Guozhi
背景：脑缺血再灌注损伤可导致严重的功能障碍，治疗难度大。据报道，核苷酸结合域和富含亮氨酸重复序列家族蛋白3(NLRP3)炎性小体介导的细胞下垂是脑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下垂。我们先前的研究发现，骨髓间充质干细胞对脑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分泌的外切体(BMSC-Exos)可能在BMSCs的有效生物学行为中发挥重要作用，BMSC-Exos的保护机制与激活自噬和减轻炎症有关，但在脑I/R损伤的研究中尚未见报道。我们旨在研究BMSC-Exos对脑I/R损伤的影响，并确定其机制是否与调节下垂和自噬通量有关。方法：采用缺氧-葡萄糖剥夺/复氧(OGD/R)诱导PC12细胞体外脑I/R，并与BMSC-Exos共培养。用CCK-8和乳酸脱氢酶(LDH)检测试剂盒检测细胞活力。采用扫描电子显微镜、Hoechst 33342/PI双重染色、2‘，7’-二氯二氢荧光素二乙酸酯分析、免疫荧光、Western印迹和酶联免疫吸附试验(ELISA)等方法检测细胞焦虑症。用透射电子显微镜、GFP-RFP-LC3腺病毒转染法和Western印迹法检测自噬通量及其对下垂的影响。最后，用免疫共沉淀法检测NLRP3与LC3的结合作用。结果：BMSC-Exos可提高OGD/R细胞活力，其抑制作用与NLRP3抑制剂MCC950相当，但可被NLRP3过表达所逆转。此外，BMSC-Exos通过AMPK/雷帕霉素途径的哺乳动物靶标促进自噬通量，而氯喹、AMPK沉默和化合物C阻断了对热下垂的抑制作用。结论：BMSC-Exos可通过促进AMPK依赖的自噬通量，减轻NLRP3炎性小体介导的热毒症，从而保护PC12细胞免受OGD/R损伤。
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/Rin vitroand 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 injuryviaattenuation of NLRP3 inflammasome-mediated pyroptosis by promoting AMPK-dependent autophagic flux.