A Brain-Protective Sterol from Soft Coral Inhibits Lipopolysaccharide-Induced Matrix Metalloproteinase-9-Mediated Astrocytic Migration.

Matrix metalloproteinases (MMPs), which are proteolytic enzymes, promote blood-brain barrier (BBB) disruption, leading to neuronal damage and neuroinflammation. Among them, MMP-9 upregulation serves as an inflammatory biomarker in the central nervous system (CNS). Currently, the development of marine organism-derived bioactive compounds or metabolites as anti-inflammatory drugs has received considerable attention. The 9,11-secosteroid, 3ß,11-dihydroxy-9,11-secogorgost-5-en-9-one (4p3f), is a novel sterol compound extracted from the soft coral Sinularia leptoclado with potential anti-inflammatory activity. However, the effect of and potential for brain protection of 4p3f on brain astrocytes remain unclear. Herein, we used rat brain astrocytes (RBAs) to investigate the effects and signaling mechanisms of 4p3f on lipopolysaccharide (LPS)-induced MMP-9 expression via zymographic, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, immunofluorescence staining, promoter-reporter, and cell migration analyses. We first found that 4p3f blocked LPS-induced MMP-9 expression in RBAs. Next, we demonstrated that LPS induced MMP-9 expression via the activation of ERK1/2, p38 MAPK, and JNK1/2, which is linked to the STAT3-mediated NF-κB signaling pathway. Finally, 4p3f effectively inhibited LPS-induced upregulation of MMP-9-triggered RBA cell migration. These data suggest that a novel sterol from soft coral, 4p3f, may have anti-inflammatory and brain-protective effects by attenuating these signaling pathways of MMP-9-mediated events in brain astrocytes. Accordingly, the soft coral-derived sterol 4p3f may emerge as a potential candidate for drug development or as a natural compound with neuroprotective properties.

Protective Effects of Sophoraflavanone G by Inhibiting TNF-α-Induced MMP-9-Mediated Events in Brain Microvascular Endothelial Cells.

The regulation of matrix metalloproteinases (MMPs), especially MMP-9, has a critical role in both physiological and pathological events in the central nervous system (CNS). MMP-9 is an indicator of inflammation that triggers several CNS disorders, including neurodegeneration. Tumor necrosis factor-α (TNF-α) has the ability to stimulate the production of different inflammatory factors, including MMP-9, in several conditions. Numerous phytochemicals are hypothesized to mitigate inflammation, including the CNS. Among them, a flavonoid compound, sophoraflavanone G (SG), found in Sophora flavescens has been found to possess several medicinal properties, including anti-bacterial and anti-inflammatory effects. In this study, mouse brain microvascular endothelial cells (bMECs) were used to explore TNF-α-induced MMP-9 signaling. The effects of SG on TNF-α-induced MMP-9 expression and its mechanisms were further evaluated. Our study revealed that the expression of MMP-9 in bMECs was stimulated by TNF-α through the activation of ERK1/2, p38 MAPK, and JNK1/2 via the TNF receptor (TNFR) with a connection to the NF-κB signaling pathway. Moreover, we found that SG can interact with the TNFR. The upregulation of MMP-9 by TNF-α may lead to the disruption of zonula occludens-1 (ZO-1), which can be mitigated by SG administration. These findings provide evidence that SG may possess neuroprotective properties by inhibiting the signaling pathways associated with TNFR-mediated MMP-9 expression and the subsequent disruption of tight junctions in brain microvascular endothelial cells.

Brain Protective Effect of Resveratrol via Ameliorating Interleukin-1ß-Induced MMP-9-Mediated Disruption of ZO-1 Arranged Integrity.

In the central nervous system (CNS), the matrix metalloproteinase-9 (MMP-9) is induced by several factors and contributes to CNS disorders, including inflammation and neurodegeneration. Thus, the upregulation of MMP-9 has been considered to be an indicator of inflammation. Interleukin-1ß (IL-1ß) is an important proinflammatory cytokine which can induce various inflammatory factors, such as MMP-9, in many inflammatory disorders. Several phytochemicals are believed to reduce the risk of several inflammatory disorders, including the CNS diseases. Among them, the resveratrol, a principal phenolic compound of the grape, blueberry, and mulberry peels and Cassia plants, has been shown to possess several medicinal properties, including antioxidative, anti-inflammatory, and antitumor function. Herein, we used mouse-brain microvascular endothelial cells (bMECs) to demonstrate the signaling mechanisms of IL-1ß-induced MMP-9 expression via zymographic, RT-PCR, Western blot, reactive oxygen species (ROS) detection, immunofluorescence stain, and promoter reporter analyses. Then we evaluated the effects of resveratrol on IL-1ß-induced MMP-9 expression in bMECs and its mechanism of action. We first demonstrated that IL-1ß induced MMP-9 expression in bMECs. Subsequently, IL-1ß induced MMP-9 expression via ROS-mediated c-Src-dependent transactivation of EGFR, and then activation of the ERK1/2, p38 MAPK, JNK1/2, and NF-κB signaling pathway. Finally, we determined that IL-1ß-induced upregulation of MMP-9 may cause the disruption of the arranged integrity of zonula occludens-1 (ZO-1), but this could be inhibited by resveratrol. These data indicated that resveratrol may have antioxidative and brain-protective activities by reducing these related pathways of ROS-mediated MMP-9 expression and tight junction disruption in brain microvascular endothelial cells.

Expression of tissue factor and TF-mediated integrin regulation in HTR-8/SVneo trophoblast cells.

Placenta is a crucial source of Tissue Factor (TF) to initiate coagulation. As far as the TF is concern, aberrant expression of TF has been reported to have a significant role in thrombosis, inflammation, cancer metastasis and atherosclerosis. It is evident that TF and TF-FVIIa complex has major roles in the disease process beyond hemostasis and thrombosis. On the other hand, TF-FVII-dependent signaling primarily activates PAR2 and inducing pro-angiogenic and immune-modulating cytokines in tumor environment. However, the role of TF has not been delineated in placental functions. Integrin typically binds to the extracellular matrix which in turn mediate cell-cell adhesion and cell behavior for migration. Dysregulation of integrin expression affects cell interaction, proliferation, and migration. Therefore, this study aims to ascertain the expression of TF in HTR-8/SVneo trophoblast cell line and its role in signal transduction of integrin (ITGα1, ITGα2, ITGß1) regulation concerning the invasion of trophoblasts. We have used RT-PCR and Western blot for the gene and protein expression analysis respectively. In addition, cell migration assays, MTT, and DAPI were performed to examine migration, cytotoxicity and apoptosis effect of FVIIa. The results suggest that the gene and protein level expressions of TF were predominant in HTR-8/SVneo cell line. Further, the cytotoxicity and apoptosis in HTR-8/SVneo cells were not observed when treated with FVIIa. The cells treated with FVIIa shown a dose-dependent up-regulation of integrin(s) (**p < 0.01, *p < 0.05) when compared to control. Migration of the HTR-8/SVneo cells was observed without any apoptosis in FVIIa-treated cells when compared to that of control. On the whole, these observations delineated the TF-FVIIa interaction in modulating the TF-dependent integrin signal transduction in HTR-8/SVneo trophoblast cell line.

Bradykinin, as a Reprogramming Factor, Induces Transdifferentiation of Brain Astrocytes into Neuron-like Cells.

Kinins are endogenous, biologically active peptides released into the plasma and tissues via the kallikrein-kinin system in several pathophysiological events. Among kinins, bradykinin (BK) is widely distributed in the periphery and brain. Several studies on the neuro-modulatory actions of BK by the B2BK receptor (B2BKR) indicate that this neuropeptide also functions during neural fate determination. Previously, BK has been shown to induce differentiation of nerve-related stem cells into neuron cells, but the response in mature brain astrocytes is unknown. Herein, we used rat brain astrocyte (RBA) to investigate the effect of BK on cell transdifferentiation into a neuron-like cell morphology. Moreover, the signaling mechanisms were explored by zymographic, RT-PCR, Western blot, and immunofluorescence staining analyses. We first observed that BK induced RBA transdifferentiation into neuron-like cells. Subsequently, we demonstrated that BK-induced RBA transdifferentiation is mediated through B2BKR, PKC-δ, ERK1/2, and MMP-9. Finally, we found that BK downregulated the astrocytic marker glial fibrillary acidic protein (GFAP) and upregulated the neuronal marker neuron-specific enolase (NSE) via the B2BKR/PKC-δ/ERK pathway in the event. Therefore, BK may be a reprogramming factor promoting brain astrocytic transdifferentiation into a neuron-like cell, including downregulation of GFAP and upregulation of NSE and MMP-9 via the B2BKR/PKC-δ/ERK cascade. Here, we also confirmed the transdifferentiative event by observing the upregulated neuronal nuclear protein (NeuN). However, the electrophysiological properties of the cells after BK treatment should be investigated in the future to confirm their phenotype.