Purpose: Neuroinflammation, characterized by the increased expression of inflammatory proteins such as matrix metalloproteinases (MMPs), plays a critical role in neurodegenerative disorders. Lipopolysaccharide (LPS) has been shown to upregulate MMP-9 expression through the activation of various transcription factors, including activator protein 1 (AP-1) and forkhead box protein O1 (FoxO1). The flavonoid 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) has been demonstrated to possess antioxidant and anti-inflammatory properties in various types of cells. Here, we investigated the mechanisms underlying the inhibitory effect of galangin on LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells).
Methods: Pharmacological inhibitors and siRNAs were employed to explore the effects of galangin on LPS-challenged RBA-1 cells. Gelatin zymography, Western blotting, real-time PCR, and a luciferase reporter assay were used to detect MMP-9 activity, protein expression, mRNA levels, and promoter activity, respectively. The protein kinases involved in the LPS-induced MMP-9 expression were determined by Western blot. A chromatin immunoprecipitation (ChIP) assay was employed to evaluate the activity of c-Jun at the MMP-9 promoter.
Results: Galangin treatment attenuated the LPS-mediated induction of MMP-9 protein and mRNA expression, as well as the activity at the MMP-9 promoter. In addition, galangin exerted its inhibitory effects on MMP-9 expression through suppressing the LPS-stimulated activation of proline-rich tyrosine kinase (Pyk2), platelet-derived growth factor receptor beta (PDGFRβ), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPKs). Pretreatment with galangin attenuated the LPS-induced phosphorylation of c-Jun and FoxO1. LPS-induced cell migration was also suppressed by galangin pretreatment.
Conclusion: Galangin attenuates the LPS-induced inflammatory responses, including the induction of MMP-9 expression and cell migration, via inhibiting Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2 and p44/p42 MAPK cascade-dependent AP-1 and FoxO1 activities. These results provide new insights into the mechanisms through which galangin mitigates LPS-induced inflammatory responses, and suggest novel strategies for the management of LPS-related brain diseases.

Keywords: neuroinflammation, astrocytes, LPS, matrix metalloproteinase, protein kinases, Chinese herbal medicine


中文翻译：

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高良姜素通过抑制大鼠脑星形胶质细胞中蛋白激酶依赖性 AP-1 和 FoxO1 的激活来抑制 LPS 诱导的 MMP-9 表达

目的：以基质金属蛋白酶 (MMP) 等炎症蛋白表达增加为特征的神经炎症在神经退行性疾病中起关键作用。脂多糖 (LPS) 已显示通过激活各种转录因子上调 MMP-9 表达，包括激活蛋白 1 (AP-1) 和叉头盒蛋白 O1 (FoxO1)。黄酮类化合物 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) 已被证明在各种类型的细胞中具有抗氧化和抗炎特性。在这里，我们研究了高良姜素对 LPS 诱导的大鼠脑星形胶质细胞（RBA-1 细胞）中 MMP-9 表达的抑制作用的机制。
方法：使用药理抑制剂和 siRNA 来探索高良姜素对 LPS 攻击的 RBA-1 细胞的影响。明胶酶谱、蛋白质印迹、实时 PCR 和荧光素酶报告基因检测分别用于检测 MMP-9 活性、蛋白质表达、mRNA 水平和启动子活性。通过蛋白质印迹测定参与 LPS 诱导的 MMP-9 表达的蛋白激酶。染色质免疫沉淀 (ChIP) 测定用于评估 c-Jun 在MMP-9启动子处的活性。
结果：高良姜素治疗减弱了 LPS 介导的 MMP-9 蛋白和 mRNA 表达的诱导，以及MMP-9的活性发起人。此外，高良姜素通过抑制 LPS 刺激的富含脯氨酸的酪氨酸激酶 (Pyk2)、血小板衍生的生长因子受体 β (PDGFRβ)、磷酸肌醇 3-激酶 (PI3K)、蛋白质的激活来发挥其对 MMP-9 表达的抑制作用。激酶 B (Akt)、哺乳动物雷帕霉素靶蛋白 (mTOR) 和丝裂原活化蛋白激酶 (MAPKs)。用高良姜素预处理减弱了 LPS 诱导的 c-Jun 和 FoxO1 磷酸化。LPS 诱导的细胞迁移也受到高良姜素预处理的抑制。
结论：高良姜素通过抑制 Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2 和 p44/p42 MAPK 级联依赖性 AP-1 和 FoxO1 活性减弱 LPS 诱导的炎症反应，包括诱导 MMP-9 表达和细胞迁移. 这些结果为高良姜素减轻 LPS 诱导的炎症反应的机制提供了新的见解，并提出了治疗 LPS 相关脑部疾病的新策略。

关键词：神经炎症，星形胶质细胞，LPS，基质金属蛋白酶，蛋白激酶，中草药