Department of pharmacy, The Second Affiliated Hospital of Jiaxing University, Jiaxing, China.
Department of Ultrasonography, The Second Affiliated Hospital of Jiaxing University, Jiaxing, China.
J Cell Mol Med. 2021 Nov;25(21):10268-10278. doi: 10.1111/jcmm.16965. Epub 2021 Oct 5.
Our previous research has found that miRNA-22 can inhibit the occurrence of pyroptosis by targeting GSDMD and decrease the production and release of inflammatory factors. In consideration of the therapeutic effects of mesenchymal stem cells (MSCs), MSCs-EV were loaded with miRNA-22 (EV-miRNA-22) to investigate the inhibitory effect of EV-miRNA-22 on the inflammatory response in SCI in rats in this study. LPS/Nigericin (LPS/NG) was used to induce pyroptosis in rat microglia in vitro. Propidium iodide (PI) staining was performed to observe cell permeability, lactate dehydrogenase (LDH) release assay was adopted to detect cytotoxicity, flow cytometry was conducted to detect pyroptosis level, immunofluorescence (IF) staining was utilized to observe the expression level of GSDMD (a key protein of pyroptosis), Western blot was performed to detect the expression of key proteins. For animal experiments, the T10 spinal cord of rats was clamped by aneurysm clip to construct the SCI model. BBB score, somatosensory evoked potential (SEP) and motor evoked potential (MEP) were performed to detect nerve function. HE staining and Nissl staining were used to detect spinal cord histopathology and nerve cell damage. EV-miRNA-22 could inhibit the occurrence of pyroptosis in microglia, suppress the cell membrane pore opening, and inhibit the release of inflammatory factors and the expression of GSDMD. In addition, EV-miRNA-22 showed higher pyroptosis-inhibiting ability than EV. Consequently, EV-miRNA-22 could inhibit the nerve function injury after SCI in rats, inhibit the level of inflammatory factors in the tissue and the activation of microglia. In this study, we found that miRNA-22-loaded MSCs-EV (EV-miRNA-22) could cooperate with EV to inhibit inflammatory response and nerve function repair after SCI.
我们之前的研究发现，miRNA-22 可以通过靶向 GSDMD 抑制细胞焦亡的发生，减少炎症因子的产生和释放。考虑到间充质干细胞（MSCs）的治疗效果，本研究将 MSCs-EV 负载 miRNA-22（EV-miRNA-22），以探讨 EV-miRNA-22 对大鼠脊髓损伤（SCI）中炎症反应的抑制作用。LPS/Nigericin（LPS/NG）被用于体外诱导大鼠小胶质细胞发生细胞焦亡。采用碘化丙啶（PI）染色观察细胞通透性，采用乳酸脱氢酶（LDH）释放实验检测细胞毒性，采用流式细胞术检测细胞焦亡水平，免疫荧光（IF）染色观察 GSDMD（细胞焦亡的关键蛋白）的表达水平，Western blot 检测关键蛋白的表达。在动物实验中，使用动脉瘤夹夹大鼠 T10 脊髓以构建 SCI 模型。采用 BBB 评分、体感诱发电位（SEP）和运动诱发电位（MEP）检测神经功能。采用 HE 染色和尼氏染色检测脊髓组织病理学和神经细胞损伤。EV-miRNA-22 可以抑制小胶质细胞发生细胞焦亡，抑制细胞膜孔的打开，抑制炎症因子的释放和 GSDMD 的表达。此外，EV-miRNA-22 表现出比 EV 更高的细胞焦亡抑制能力。因此，EV-miRNA-22 可以抑制大鼠 SCI 后的神经功能损伤，抑制组织中炎症因子的水平和小胶质细胞的激活。在这项研究中，我们发现负载 miRNA-22 的 MSCs-EV（EV-miRNA-22）可以与 EV 协同抑制 SCI 后的炎症反应和神经功能修复。
