Abstract Context: Matrine is a well-known anti-inflammatory quinolizidine alkaloid derived from leguminous plant Sophora flavescens Ait. (Leguminosae). Objective: This study was designed to uncover the potential application of matrine in treating spinal cord injury (SCI). Materials and methods: Neuron-like PC12 cells in experimental groups were pre-treated with/without matrine (200 μM) for 24 h and then stimulated by lipopolysaccharide (LPS, 5 μg/mL) for 12 h. PC12 cells in control group were cultured in complete medium. CCK-8 assay, flow cytometry, qRT-PCR, western blot and ELISA were performed to evaluate cell damage. Moreover, after cells were transfected with miR-9 inhibitor for 48 h, above indicators were tested again. qRT-PCR and western blot were also conducted to uncover the downstream effectors and signalling pathways for matrine. Results: LPS (5 μg/mL) decreased cell viability about 50%. Matrine (200 μM) decreased cell viability about 0%, 13.8% and 30% at 24 h, 48 h and 72 h, respectively. The loss of viability, stimulation of apoptosis, and release of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) evoked by LPS were attenuated by the pre-treatment of matrine partly. Meanwhile, LPS reduced miR-9 expression about 60%, but matrine completely reversed LPS-decreased miR-9 level. By silencing miR-9 expression, the protective properties of matrine towards PC12 cells were impeded. Besides, matrine inhibited the activation of JNK and NF–κB pathways even under the condition of LPS. And the impact of matrine on the signalling were attenuated by miR-9 silencing. Discussion and Conclusion: This paper provided in vitro evidence that matrine was able to protect PC12 cells against LPS-evoked damage. The neuroprotective properties of matrine may be due to its regulation of miR-9 expression as well as JNK and NF–κB pathways.

中文翻译：

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苦参碱通过上调 miR-9 保护 PC12 细胞免受脂多糖诱发的炎症损伤

摘要背景：苦参碱是一种著名的抗炎喹唑啉生物碱，来源于豆科植物苦参。（豆科）。目的：本研究旨在揭示苦参碱在治疗脊髓损伤 (SCI) 中的潜在应用。材料与方法：实验组神经元样PC12细胞用/不用苦参碱（200 μM）预处理24 h，然后用脂多糖（LPS，5 μg/mL）刺激12 h。对照组PC12细胞在完全培养基中培养。进行CCK-8测定、流式细胞术、qRT-PCR、蛋白质印迹和ELISA以评估细胞损伤。此外，细胞转染miR-9抑制剂48小时后，再次检测上述指标。还进行了 qRT-PCR 和蛋白质印迹以揭示苦参碱的下游效应子和信号通路。结果：LPS (5 μg/mL) 降低细胞活力约 50%。苦参碱 (200 μM) 在 24 小时、48 小时和 72 小时时分别使细胞活力降低约 0%、13.8% 和 30%。LPS 引起的活力丧失、细胞凋亡刺激和促炎细胞因子（IL-1β、IL-6 和 TNF-α）的释放通过苦参碱的预处理部分减弱。同时，LPS 降低了 miR-9 表达约 60%，但苦参碱完全逆转了 LPS 降低的 miR-9 水平。通过沉默 miR-9 表达，苦参碱对 PC12 细胞的保护特性受到阻碍。此外，苦参碱即使在 LPS 条件下也能抑制 JNK 和 NF-κB 通路的激活。并且苦参碱对信号传导的影响被 miR-9 沉默减弱。讨论和结论：本文提供的体外证据表明苦参碱能够保护 PC12 细胞免受 LPS 诱发的损伤。苦参碱的神经保护特性可能是由于其对 miR-9 表达以及 JNK 和 NF-κB 通路的调节。