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Published on: August 11, 2023
Pristimerin Inhibits MMP-9 Expression and Cell Migration Through Attenuating NOX/ROS-Dependent NF-κB Activation in
Chien-Chung Yang1,2, Li-Der Hsiao3, Hui-Ching Tseng3
1Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Tao-Yuan 33302, Taiwan.
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. 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.
Results:
Our 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.
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.
Insights
Lipopolysaccharide (LPS) induces matrix metalloproteinase-9 (MMP-9) via reactive oxygen species (ROS) and nuclear factor-kappaB (NF-κB) signaling in astrocytes. Pristimerin inhibits these inflammatory responses, offering therapeutic potential for neuroinflammation.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Neuroinflammation is a key factor in neurodegenerative diseases.
- Matrix metalloproteinases (MMPs), particularly MMP-9, are markers of neuroinflammation.
- Lipopolysaccharide (LPS) is known to induce MMP-9 expression, but the underlying mechanisms in astrocytes are not fully understood.
Purpose of the Study:
- To investigate the roles of reactive oxygen species (ROS) and nuclear factor-kappaB (NF-κB) in LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1).
- To evaluate the potential of pristimerin, an agent with known antioxidant and anti-inflammatory effects, in modulating LPS-induced inflammatory responses.
Main Methods:
- Utilized RBA-1 cells and employed pharmacological inhibitors and siRNAs to dissect signaling pathways.
- Assessed protein and mRNA expression of MMP-9 using Western blotting, gelatin zymography, and real-time PCR.
- Measured ROS generation via H2DCF-DA and DHE staining, and NF-κB p65 activation using immunofluorescence, promoter-reporter assays, and ChIP assays.
Main Results:
- LPS exposure increased ROS generation, which was reduced by antioxidants like edaravone, apocynin (APO), diphenyleneiodonium (DPI), and pristimerin.
- Pretreatment with APO, DPI, edaravone, Bay11-7082, and pristimerin suppressed NF-κB p65 phosphorylation, nuclear translocation, and promoter binding activity.
- Inhibition of these pathways also reduced LPS-induced MMP-9 upregulation and subsequent cell migration.
Conclusions:
- LPS upregulates MMP-9 expression in astrocytes through a pathway involving nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), ROS, and NF-κB activation.
- Pristimerin effectively attenuates LPS-induced MMP-9 expression and neuroinflammatory responses, highlighting its therapeutic potential.
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