Purpose: Neuroinflammation plays a crucial role in neurodegenerative diseases. Matrix metalloproteinases (MMPs) are a landmark of neuroinflammation. Lipopolysaccharide (LPS) has been demonstrated to induce MMP-9 expression. The mechanisms underlying LPS-induced MMP-9 expression have not been completely elucidated in astrocytes. Nuclear factor-kappaB (NF-κB) is well known as one of the crucial transcription factors in MMP-9 induction. Moreover, reactive oxygen species (ROS) could be an important mediator of neuroinflammation. Here, we differentiated whether ROS and NF-κB contributed to LPS-mediated MMP-9 expression in rat brain astrocytes (RBA-1). Besides, pristimerin has been revealed to possess antioxidant and anti-inflammatory effects. We also evaluated the effects of pristimerin on LPS-induced inflammatory responses.
Methods: RBA-1 cells were used for analyses. Pharmacological inhibitors and siRNAs were used to evaluate the signaling pathway. Western blotting and gelatin zymography were conducted to evaluate protein and MMP-9 expression, respectively. Real-time PCR was for mRNA expression. Wound healing assay was for cell migration. 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H₂DCF-DA) and dihydroethidium (DHE) staining were for ROS generation. Immunofluorescence staining was conducted to assess NF-κB p65. Promoter-reporter gene assay and chromatin immunoprecipitation (ChIP) assay were used to detect promoter activity and the association of nuclear proteins with the promoter.
Results: Our results showed that the increased level of ROS generation was attenuated by edaravone (a ROS scavenger), apocynin (APO; an inhibitor of p47^Phox), diphenyleneiodonium (DPI; an inhibitor of NOX), and pristimerin in RBA-1 cells exposed to LPS. Besides, pretreatment with APO, DPI, edaravone, Bay11-7082, and pristimerin also inhibited the phosphorylation, nuclear translocation, promoter binding activity of NF-κB p65 as well as upregulation of MMP-9 expression-mediated cell migration in RBA-1 cells challenged with LPS.
Conclusion: These results suggested that LPS enhances the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)/ROS-dependent NF-κB activity. These results also provide new insights into the mechanisms by which pristimerin attenuates LPS-mediated MMP-9 expression and neuroinflammatory responses.

Keywords: neuroinflammation, NOX, ROS, LPS, MMP-9, pristimerin, astrocytes


中文翻译：

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Pristimerin 通过减弱受到 LPS 攻击的大鼠脑星形胶质细胞中的 NOX/ROS 依赖性 NF-κB 活化来抑制 MMP-9 表达和细胞迁移。

目的：神经炎症在神经退行性疾病中起着至关重要的作用。基质金属蛋白酶 (MMP) 是神经炎症的标志物。已证明脂多糖 (LPS) 可诱导 MMP-9 表达。LPS 诱导的 MMP-9 表达的机制尚未在星形胶质细胞中完全阐明。核因子-kappaB (NF-κB) 是众所周知的 MMP-9 诱导的关键转录因子之一。此外，活性氧（ROS）可能是神经炎症的重要介质。在这里，我们区分了 ROS 和 NF-κB 是否有助于 LPS 介导的大鼠脑星形胶质细胞 (RBA-1) 中的 MMP-9 表达。此外，pristimerin 已被证明具有抗氧化和抗炎作用。我们还评估了 pristimerin 对 LPS 诱导的炎症反应的影响。
方法： RBA-1 细胞用于分析。药理抑制剂和siRNA用于评估信号通路。进行蛋白质印迹和明胶酶谱法以分别评估蛋白质和 MMP-9 的表达。实时 PCR 用于 mRNA 表达。伤口愈合试验用于细胞迁移。2′,7′-二氯二氢荧光素二乙酸酯 (H ₂ DCF-DA) 和二氢乙锭 (DHE) 染色用于产生 ROS。进行免疫荧光染色以评估 NF-κB p65。启动子-报告基因测定和染色质免疫沉淀 (ChIP) 测定用于检测启动子活性和核蛋白与启动子的关联。
结果：我们的研究结果表明，依达拉奉（一种 ROS 清除剂）、夹竹桃麻素（APO；一种 p47 ^Phox的抑制剂）、二苯碘鎓（DPI；一种 NOX 的抑制剂）和 pristimerin 在 RBA-1 细胞暴露于脂多糖。此外，用 APO、DPI、依达拉奉、Bay11-7082 和 pristimerin 预处理还抑制了 NF-κB p65 的磷酸化、核易位、启动子结合活性以及 RBA-1 细胞中 MMP-9 表达介导的细胞迁移的上调受到 LPS 的挑战。
结论：这些结果表明，LPS 通过烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 氧化酶 (NOX)/ROS 依赖性 NF-κB 活性增强 MMP-9 的上调。这些结果还提供了关于pristimerin减弱LPS介导的MMP-9表达和神经炎症反应的机制的新见解。

关键词：神经炎症，NOX，ROS，LPS，MMP-9，pristimerin，星形胶质细胞