Abstract：
BACKGROUND: Neuromedin B (Nmb) plays a pivotal role in the transmission of neuroinflammation, particularly during spinal cord ischemia-reperfusion injury (SCII). However, the detailed molecular mechanisms underlying this process remain elusive.
METHODS: The SCII model was established by clamping the abdominal aorta of male Sprague-Dawley (SD) rats for 60 min. The protein expression levels of Nmb, Cav3.2, and IL-1β were detected by Western blotting, while miR-214-3p expression was quantified by qRT-PCR. The targeted regulation between miR-214-3p and Nmb was investigated using a dual-luciferase reporter gene assay. The cellular localization of Nmb and Cav3.2 with cell-specific markers was visualized by immunofluorescence staining. The specific roles of miR-214-3p on the Nmb/Cav3.2 interactions in SCII-injured rats were explored by intrathecal injection of Cav3.2-siRNA, PD168368 (a specific NmbR inhibitor) and synthetic miR-214-3p agomir and antagomir in separate experiments. Additionally, hind-limb motor function was evaluated using the modified Tarlov scores.
RESULTS: Compared to the Sham group, the protein expression levels of Nmb, Cav3.2, and the proinflammatory factor Interleukin(IL)-1β were significantly elevated at 24 h post-SCII. Intrathecal injection of PD168368 and Cav3.2-siRNA significantly suppressed the expression of Cav3.2 and IL-1β compared to the SCII group. The miRDB database and dual-luciferase reporter gene assay identified Nmb as a direct target of miR-214-3p. As expected, in vivo overexpression of miR-214-3p by agomir-214-3p pretreatment significantly inhibited the increases in Nmb, Cav3.2 and IL-1β expression and improved lower limb motor function in SCII-injured rats, while antagomiR-214-3p pretreatment reversed these effects.
CONCLUSIONS: Nmb protein levels positively correlated with Cav3.2 expression in SCII rats. Upregulating miR-214-3p ameliorated hind-limb motor function and protected against neuroinflammation via inhibiting the aberrant Nmb/Cav3.2 interactions and downstream IL-1β release. These findings provide novel therapeutic targets for clinical prevention and treatment of SCII.
METHODS: The SCII model was established by clamping the abdominal aorta of male Sprague-Dawley (SD) rats for 60 min. The protein expression levels of Nmb, Cav3.2, and IL-1β were detected by Western blotting, while miR-214-3p expression was quantified by qRT-PCR. The targeted regulation between miR-214-3p and Nmb was investigated using a dual-luciferase reporter gene assay. The cellular localization of Nmb and Cav3.2 with cell-specific markers was visualized by immunofluorescence staining. The specific roles of miR-214-3p on the Nmb/Cav3.2 interactions in SCII-injured rats were explored by intrathecal injection of Cav3.2-siRNA, PD168368 (a specific NmbR inhibitor) and synthetic miR-214-3p agomir and antagomir in separate experiments. Additionally, hind-limb motor function was evaluated using the modified Tarlov scores.
RESULTS: Compared to the Sham group, the protein expression levels of Nmb, Cav3.2, and the proinflammatory factor Interleukin(IL)-1β were significantly elevated at 24 h post-SCII. Intrathecal injection of PD168368 and Cav3.2-siRNA significantly suppressed the expression of Cav3.2 and IL-1β compared to the SCII group. The miRDB database and dual-luciferase reporter gene assay identified Nmb as a direct target of miR-214-3p. As expected, in vivo overexpression of miR-214-3p by agomir-214-3p pretreatment significantly inhibited the increases in Nmb, Cav3.2 and IL-1β expression and improved lower limb motor function in SCII-injured rats, while antagomiR-214-3p pretreatment reversed these effects.
CONCLUSIONS: Nmb protein levels positively correlated with Cav3.2 expression in SCII rats. Upregulating miR-214-3p ameliorated hind-limb motor function and protected against neuroinflammation via inhibiting the aberrant Nmb/Cav3.2 interactions and downstream IL-1β release. These findings provide novel therapeutic targets for clinical prevention and treatment of SCII.
摘要：
背景：NeuromedinB（Nmb）在神经炎症的传播中起关键作用，特别是在脊髓缺血再灌注损伤（SCII）期间。然而,这个过程背后的详细分子机制仍然难以捉摸。
方法：雄性SD大鼠腹主动脉钳夹60min建立SCII模型。Nmb的蛋白表达水平,Cav3.2和IL-1β通过蛋白质印迹检测，而miR-214-3p表达通过qRT-PCR定量。使用双荧光素酶报告基因测定研究miR-214-3p和Nmb之间的靶向调节。通过免疫荧光染色观察具有细胞特异性标志物的Nmb和Cav3.2的细胞定位。通过鞘内注射Cav3.2-siRNA探讨miR-214-3p对SCII损伤大鼠Nmb/Cav3.2相互作用的具体作用,PD168368(特异性NmbR抑制剂)和合成的miR-214-3pagomir和antagomir在单独的实验中。此外,使用改良的Tarlov评分评估后肢运动功能。
结果：与Sham组相比，NMB的蛋白质表达水平，Cav3.2和促炎因子白细胞介素（IL）-1β在SCII后24h显着升高。与SCII组相比,鞘内注射PD168368和Cav3.2-siRNA显著抑制Cav3.2和IL-1β的表达。miRDB数据库和双荧光素酶报告基因测定将Nmb鉴定为miR-214-3p的直接靶标。不出所料,通过agomir-214-3p预处理体内过表达miR-214-3p显著抑制Nmb的增加，Cav3.2和IL-1β表达与改善SCII损伤大鼠下肢运动功能,而antagomiR-214-3p预处理逆转了这些作用。
结论：Nmb蛋白水平与SCII大鼠Cav3.2表达呈正相关。上调miR-214-3p通过抑制异常的Nmb/Cav3.2相互作用和下游IL-1β释放改善后肢运动功能并防止神经炎症。这些发现为SCII的临床预防和治疗提供了新的治疗靶标。
方法：雄性SD大鼠腹主动脉钳夹60min建立SCII模型。Nmb的蛋白表达水平,Cav3.2和IL-1β通过蛋白质印迹检测，而miR-214-3p表达通过qRT-PCR定量。使用双荧光素酶报告基因测定研究miR-214-3p和Nmb之间的靶向调节。通过免疫荧光染色观察具有细胞特异性标志物的Nmb和Cav3.2的细胞定位。通过鞘内注射Cav3.2-siRNA探讨miR-214-3p对SCII损伤大鼠Nmb/Cav3.2相互作用的具体作用,PD168368(特异性NmbR抑制剂)和合成的miR-214-3pagomir和antagomir在单独的实验中。此外,使用改良的Tarlov评分评估后肢运动功能。
结果：与Sham组相比，NMB的蛋白质表达水平，Cav3.2和促炎因子白细胞介素（IL）-1β在SCII后24h显着升高。与SCII组相比,鞘内注射PD168368和Cav3.2-siRNA显著抑制Cav3.2和IL-1β的表达。miRDB数据库和双荧光素酶报告基因测定将Nmb鉴定为miR-214-3p的直接靶标。不出所料,通过agomir-214-3p预处理体内过表达miR-214-3p显著抑制Nmb的增加，Cav3.2和IL-1β表达与改善SCII损伤大鼠下肢运动功能,而antagomiR-214-3p预处理逆转了这些作用。
结论：Nmb蛋白水平与SCII大鼠Cav3.2表达呈正相关。上调miR-214-3p通过抑制异常的Nmb/Cav3.2相互作用和下游IL-1β释放改善后肢运动功能并防止神经炎症。这些发现为SCII的临床预防和治疗提供了新的治疗靶标。
