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Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

ABSTRACT: 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 (PDGFR?), 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/PDGFR?/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.

SUBMITTER: Yang CC

PROVIDER: S-EPMC7685391 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

Yang Chien-Chung CC Hsiao Li-Der LD Yang Chuen-Mao CM

Journal of inflammation research 20201120

<h4>Purpose</h4>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 po ...[more]

PMID: 33244253

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Project description:Neuroinflammation is characterized by the elevated expression of various inflammatory proteins, including matrix metalloproteinases (MMPs), induced by various pro-inflammatory mediators, which play a critical role in neurodegenerative disorders. Interleukin-1β (IL-1β) has been shown to induce the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-reactive oxygen species (ROS)-dependent signaling pathways. N-(2-cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2-2-difluoropropanamide (RTA 408), a novel synthetic triterpenoid, has been shown to possess anti-oxidant and anti-inflammatory properties in various types of cells. Here, we evaluated the effects of RTA 408 on IL-1β-induced inflammatory responses by suppressing MMP-9 expression in a rat brain astrocyte (RBA-1) line. IL-1β-induced MMP-9 protein and mRNA expression, and promoter activity were attenuated by RTA 408. The increased level of ROS generation in RBA-1 cells exposed to IL-1β was attenuated by RTA 408, as determined by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and CellROX. In addition, the inhibitory effects of RTA 408 on MMP-9 expression resulted from the suppression of the IL-1β-stimulated activation of Pyk2 (proline-rich tyrosine kinase), platelet-derived growth factor receptor β (PDGFRβ), Akt, ROS, and mitogen-activated protein kinases (MAPKs). Pretreatment with RTA 408 attenuated the IL-1β-induced c-Jun phosphorylation, mRNA expression, and promoter activity. IL-1β-stimulated nuclear factor-κB (NF-κB) p65 phosphorylation, translocation, and promoter activity were also attenuated by RTA 408. Furthermore, IL-1β-induced glial fibrillary acidic protein (GFAP) protein and mRNA expression, and cell migration were attenuated by pretreatment with RTA 408. These results provide new insights into the mechanisms by which RTA 408 attenuates IL-1β-mediated inflammatory responses and exerts beneficial effects for the management of brain diseases.

Project description:PurposeNeuroinflammation 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.MethodsRBA-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. 2',7'-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.ResultsOur results showed that the increased level of ROS generation was attenuated by edaravone (a ROS scavenger), apocynin (APO; an inhibitor of p47Phox), 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.ConclusionThese 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.

Project description:Clara cell protein (CC16) is a well-known anti-inflammatory protein secreted by the epithelial Clara cells of the airways. It is involved in the development of airway inflammatory diseases such as chronic obstructive pulmonary disease and asthma. Previous studies suggest that CC16 gene transfer suppresses expression of interleukin (IL)-8 in bronchial epithelial cells. However, its role in the function of these cells during inflammation is not well understood. In this study, we evaluated the effect of CC16 on the expression of matrix metalloproteinase (MMP)-9 in lipopolysaccharide (LPS)-stimulated rat tracheal epithelial cells and its underlying molecular mechanisms. We generated recombinant rat CC16 protein (rCC16) which was bioactive in inhibiting the activity of phospholipase A2. rCC16 inhibited LPS-induced MMP-9 expression at both mRNA and protein levels in a concentration-dependent (0-2 µg/mL) manner, as demonstrated by real time RT-PCR, ELISA, and zymography assays. Gene transcription and DNA binding studies demonstrated that rCC16 suppressed LPS-induced NF-κB activation and its binding of gene promoters as identified by luciferase reporter and gel mobility shift assays, respectively. Western blotting and immunofluorescence staining analyses further revealed that rCC16 concentration dependently inhibited the effects of LPS on nuclear increase and cytosol reduction of NF-κB, on the phosphorylation and reduction of NF-κB inhibitory IκBα, and on p38 MAPK-dependent NF-κB activation by phosphorylation at Ser276 of its p65 subunit. These data indicate that inhibition of LPS-mediated NF-κB activation by rCC16 involves both translocation- and phosphorylation-dependent signaling pathways. When the tracheal epithelial cells were pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, cellular uptake of rCC16 and its inhibition of LPS-induced NF-κB nuclear translocation and also MMP-9 production were significantly abolished. Taken together, our data suggest that clathrin-mediated uptake of rCC16 suppresses LPS-mediated inflammatory MMP-9 production through inactivation of NF-κB and p38 MAPK pathways in tracheal epithelial cells.

Dataset Information

Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

Publications

Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

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