Anti-Inflammatory Effects of Rhamnetin on Bradykinin-Induced Matrix Metalloproteinase-9 Expression and Cell Migration in Rat Brain Astrocytes

Abstract

Bradykinin (BK) has been shown to induce matrix metalloproteinase (MMP)-9 expression and participate in neuroinflammation. The BK/MMP-9 axis can be a target for managing neuroinflammation. Our previous reports have indicated that reactive oxygen species (ROS)-mediated nuclear factor-kappaB (NF-κB) activity is involved in BK-induced MMP-9 expression in rat brain astrocytes (RBA-1). Rhamnetin (RNT), a flavonoid compound, possesses antioxidant and anti-inflammatory effects. Thus, we proposed RNT could attenuate BK-induced response in RBA-1. This study aims to approach mechanisms underlying RNT regulating BK-stimulated MMP-9 expression, especially ROS and NF-κB. We used pharmacological inhibitors and siRNAs to dissect molecular mechanisms. Western blotting and gelatin zymography were used to evaluate protein and MMP-9 expression. Real-time PCR was used for gene expression. Wound healing assay was applied for cell migration. 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H2DCF-DA) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) were used for ROS generation and NOX activity, respectively. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay were applied to detect gene transcription. Our results showed that RNT inhibits BK-induced MMP-9 protein and mRNA expression, promoter activity, and cell migration in RBA-1 cells. Besides, the levels of phospho-PKCδ, NOX activity, ROS, phospho-ERK1/2, phospho-p65, and NF-κB p65 binding to MMP-9 promoter were attenuated by RNT. In summary, RNT attenuates BK-enhanced MMP-9 upregulation through inhibiting PKCδ/NOX/ROS/ERK1/2-dependent NF-κB activity in RBA-1.

Induction of Heme Oxygenase-1 by 15d-Prostaglandin J2 Mediated via a ROS-Dependent Sp1 and AP-1 Cascade Suppresses Lipopolysaccharide-Triggered Interleukin-6 Expression in Mouse Brain Microvascular Endothelial Cells

“…The lysates were dissolved in 1.25× sample buffer and heated at 95 • C for 5 min. The denatured proteins were centrifuged at 13,000× g for 1 min, as described previously [30]. The mixed samples (15 µL) were subjected to SDS-PAGE using a 10% running gel.…”

“…c-Jun siRNA (sc-29224, NM_010591.2) and JNK2 siRNA (sc-39102, NM_001163671.1) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Briefly, transient transfection of siRNAs was performed by using Genmute reagent and Opti-MEM, as described previously [30]. The transfection complex (Genmute reagent 2.5 µL, Opti-MEM 100 µL, and siRNA 100 nM) was directly administrated to the cells and incubated for 5 h. Transfection complex medium was replaced with DMEM/F-12 medium containing 10% FBS overnight.…”

“…The levels of RNA were spectrophotometrically measured at 260 nm. mRNA was reverse-transcribed into cDNA and determined by real-time RT-PCR, as described previously [30]. Real-time PCR was performed using Kapa Probe Fast qPCR Kit Master Mix (2X) Universal (KK4705; KAPA Biosystems, Wilmington, MA, USA), a StepOnePlusTM real-time qPCR system (ThermoScientific-Applied Biosystems, Foster City, CA, USA), and primers specific for HO-1 (NM_010442.2): 5 -CACGCATATACCCGCTACCT-3 (sense); 5 -TCT GTCACCCTGTGCTTGAC-3 (antisense) and for interleukin-6 (IL-6; NM_001314054.1): 5 -ACAACCACGGCCTTCCCTACTT-3 (sense), 5 -CACGATTTCCCAGAGAACATGTG-3 (antisense).…”

“…This method is based on detecting fluorescent 2 -7 dichlorofluorescein (DCF), oxidatively converted from non-fluorescent 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCF-DA) by H 2 O 2 , as described previously [30]. The intracellular H 2 O 2 levels were determined by measuring the fluorescent intensity of DCF.…”

“…The assay was performed by the method with modifications, as described previously [30]. In short, bEnd.3 cells stimulated with 10 µM 15d-PGJ 2 for the indicated time points were cross-linked with 1% formaldehyde for 10 min at 37 • C and washed thrice with ice-cold PBS containing 1% aprotinin and 1 mM phenylmethyl-sulfonyl fluoride.…”