Department of Spine Surgery and Orthopaedics, Xiangya Hospital, Central South University, Changsha, 410008, China.
Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha, 410008, China.
J Neuroinflammation. 2023 Jun 30;20(1):156. doi: 10.1186/s12974-023-02833-7.
Macrophage in the spinal cord injury (SCI) area imparts a chronic pro-inflammation effect that challenges the recovery of SCI. Previously, endothelial progenitor cell-produced exosomes (EPC-EXOs) have been noticed to facilitate revascularization and inflammation control after SCI. However, their effects on macrophage polarization remained unclear. This study aimed to investigate the EPC-EXOs' role in macrophage polarization and reveal its underlying mechanism.
We extracted the macrophages and EPC from the bone marrow suspension of C57BL/L mice by centrifugation. After cell identification, the EPC-EXOs were collected by ultra-high-speed centrifugation and exosome extraction kits and identified by transmission electron microscopy and nanoparticle tracking analysis. Then, macrophages were cultured with EPC-EXOs in different concentrations. We labeled the exosome to confirm its internalization by macrophage and detected the macrophage polarization marker level both in vitro and in vivo. We further estimated EPC-EXOs' protective effects on SCI by mice spinal cord tissue H&E staining and motor behavior evaluation. Finally, we performed RT-qPCR to identify the upregulated miRNA in EPC-EXOs and manipulate its expression to estimate its role in macrophage polarization, SOCS3/JAK2/STAT3 pathway activation, and motor behavior improvement.
We found that EPC-EXOs decreased the macrophages' pro-inflammatory marker expression and increased their anti-inflammatory marker expression on the 7 and 14 days after SCI. The spinal cord H&E staining results showed that EPC-EXOs raised the tissue-sparing area rate significantly after 28 days of SCI and the motor behavior evaluation indicated an increased BMS score and motor-evoked potential by EPC-EXOs treatment after SCI. The RT-qPCR assay identified that miR-222-3P upregulated in EPC-EXOs and its miRNA-mimic also decreased the pro-inflammatory macrophages and increased the anti-inflammatory macrophages. Additionally, miR-222-3P mimic activated the SOCS3/JAK2/STAT3 pathway, and SOCS3/JAK2/STAT3 pathway inhibition blocked miR-2223P's effects on macrophage polarization and mouse motor behavior.
Comprehensively, we discovered that EPC-EXOs-derived miR-222-3p affected macrophage polarization via SOCS3/JAK2/STAT3 pathway and promoted mouse functional repair after SCI, which reveals EPC-EXOs' role in modulation of macrophage phenotype and will provide a novel interventional strategy to induce post-SCI recovery.
脊髓损伤（SCI）区域中的巨噬细胞产生慢性促炎作用，这对 SCI 的恢复构成挑战。先前，已经注意到内皮祖细胞产生的外泌体（EPC-EXOs）有助于 SCI 后的再血管化和炎症控制。然而，它们对巨噬细胞极化的影响尚不清楚。本研究旨在探讨 EPC-EXOs 在巨噬细胞极化中的作用，并揭示其潜在机制。
我们通过离心从 C57BL/L 小鼠的骨髓悬浮液中提取巨噬细胞和 EPC。细胞鉴定后，通过超速离心和外泌体提取试剂盒收集 EPC-EXOs，并通过透射电子显微镜和纳米颗粒跟踪分析进行鉴定。然后，将巨噬细胞与不同浓度的 EPC-EXOs 共培养。我们标记外泌体以确认其被巨噬细胞内化，并在体外和体内检测巨噬细胞极化标志物水平。我们进一步通过小鼠脊髓组织 H&E 染色和运动行为评估来评估 EPC-EXOs 对 SCI 的保护作用。最后，我们进行 RT-qPCR 以鉴定 EPC-EXOs 中上调的 miRNA，并操纵其表达以评估其在巨噬细胞极化、SOCS3/JAK2/STAT3 通路激活和运动行为改善中的作用。
我们发现 EPC-EXOs 在 SCI 后 7 天和 14 天降低了巨噬细胞的促炎标志物表达，并增加了其抗炎标志物表达。脊髓 H&E 染色结果表明，EPC-EXOs 在 SCI 后 28 天显著提高了组织保存面积率，运动行为评估表明 EPC-EXOs 治疗后 SCI 的 BMS 评分和运动诱发电位增加。RT-qPCR 检测鉴定出 EPC-EXOs 中上调的 miR-222-3P，其 miRNA 模拟物也降低了促炎巨噬细胞，增加了抗炎巨噬细胞。此外，miR-222-3P 模拟物激活了 SOCS3/JAK2/STAT3 通路，而 SOCS3/JAK2/STAT3 通路抑制阻断了 miR-2223P 对巨噬细胞极化和小鼠运动行为的影响。
综上所述，我们发现 EPC-EXOs 衍生的 miR-222-3p 通过 SOCS3/JAK2/STAT3 通路影响巨噬细胞极化，并促进 SCI 后小鼠的功能修复，这揭示了 EPC-EXOs 在调节巨噬细胞表型中的作用，并为诱导 SCI 后恢复提供了一种新的干预策略。
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