外泌体源性microRNA-124对急性创伤性脊髓损伤大鼠脊髓小胶质细胞活化及炎症反应的影响

Effects of exosomal miR-124 on microglial activation and inflammation of spinal cord in rats with acute traumatic spinal cord injury

摘要:

目的 观察外泌体（Exos）源性microRNA-124（miR-124）对急性创伤性脊髓损伤（ASCI）大鼠脊髓小胶质细胞（MG）活化及炎症反应的影响。方法 取对数期HEK239细胞，分为空白组、转染组。空白组不处理，转染组转染miR-124-mimics质粒。从35只大鼠中随机选取10只仅咬除椎板显露脊髓，不进行损伤处理，设为假手术组；其余25只复制ASCI模型，模型复制成功20只，随机分为ASCI组、实验组，每组10只。分离含有miR-124的Exos，鉴定后用于后续实验。模型复制成功后24 h，实验组经尾静脉注射Exos 3×10⁹个微粒，假手术组及ASCI组注射等量PBS溶液。实时荧光定量聚合酶链反应检测脊髓组织miR-124的表达；流式细胞术检测脊髓组织MG占比；酶联免疫吸附试验检测脊髓组织肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）水平；Western bloting检测脊髓组织核苷酸结合寡聚化结构域样受体3（NLRP3）、半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）、P2X7蛋白的表达。结果 转染组miR-124相对表达量高于空白组（P <0.05）。与假手术组、ASCI组比较，实验组脊髓组织miR-124相对表达量升高（P <0.05）。与假手术组比较，ASCI组脊髓组织Iba-1^＋/CD32^＋、Iba-1^＋/CD206^＋占比均升高（P <0.05）；与ASCI组比较，实验组脊髓组织Iba-1^＋/CD32^＋占比降低（P <0.05），Iba-1^＋/CD206^＋占比升高（P <0.05）。与假手术组比较，ASCI组脊髓组织TNF-α、IL-6、IL-8升高（P <0.05）；与ASCI组比较，实验组脊髓组织TNF-α、IL-6、IL-8降低（P <0.05）。与假手术组比较，ASCI组脊髓组织NLRP3、Caspase-1、P2X7蛋白相对表达量升高（P <0.05）；与ASCI组比较，实验组脊髓组织NLRP3、Caspase-1、P2X7蛋白相对表达量降低（P <0.05）。结论 Exos源性miR-124可促进ASCI大鼠脊髓M1型MG转化为M2型，减轻炎症反应，推测其作用机制与抑制P2X7-NLRP3/Caspase-1信号通路活性有关。

Abstract:

Objective To observe the effects of exosomal microRNA (miR)-124 on microglial activation and inflammation of spinal cord in rats with acute traumatic spinal cord injury (ASCI).Methods Ten of the 35 rats were randomly selected as the sham operation group, where excision of vertebral plates was performed to expose the spinal cord. The rest 25 rats were used to establish the ASCI models, and the 20 rats successfully modeled were randomly divided into ASCI group and experimental group, each with 10 rats. Exosomes containing miR-124 were isolated and used for subsequent experiments after identification. Twenty-four hours after modeling, the experimental group was injected with 3×10⁹ particles of exosomes via the tail vein, and the sham group and the ASCI group were injected with the same amount of PBS solution. The expression of miR-124 in spinal cord tissues was detected by quantitative real-time polymerase chain reaction. The proportion of microglia in spinal cord tissues was detected by flow cytometry. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) in spinal cord tissues were detected by ELISA. The protein expression levels of nucleotide-binding oligomerization domain-like receptor 3 (NLRP3), Caspase-1, and P2X7 in the spinal cord tissues were detected by Western blotting.Results The relative expression of miR-124 was higher in the transfection group than that in the blank group (P < 0.05). Compared with the sham group and the ASCI group, the expression of miR-124 in the spinal cord of the experimental group was increased (P < 0.05). Compared with the sham group, the proportions of Iba-1⁺/CD32⁺ and Iba-1⁺ /CD206⁺ cells in the spinal cord tissues of the ASCI group were increased (P < 0.05). Compared with the ASCI group, the proportion of Iba-1⁺/CD32⁺ cells in the spinal cord tissues was decreased (P < 0.05), while the proportion of Iba-1⁺/CD206⁺ cells in the spinal cord tissues was increased in the experimental group (P < 0.05). Compared with the sham group, the levels of TNF-α, IL-6, and IL-8 in the spinal cord of the ASCI group were increased (P < 0.05). Compared with the ASCI group, the levels of TNF-α, IL-6, and IL-8 in the spinal cord of the experimental group were decreased (P < 0.05). Compared with the sham group, the expressions of NLRP3, Caspase-1 and P2X7 in the spinal cord of the ASCI group were increased (P < 0.05). Compared with the ASCI group, the expressions of NLRP3, Caspase-1, and P2X7 in the spinal cord of the experimental group were decreased (P < 0.05).Conclusions Exosomal miR-124 can promote the conversion of M1 microglia to M2 microglia and ameliorate the inflammation in the spinal cord of ASCI rats. It is speculated that its mechanism is related to the inhibition of P2X7-NLRP3/Caspase-1 signaling pathway.