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Melatonin protects blood-brain barrier integrity and permeability by inhibiting matrix metalloproteinase-9 via the NOTCH3/NF-κB pathway.

ABSTRACT: The pathophysiological mechanism of white matter hyperintensities of cerebral small vessel disease (CSVD) includes an impaired blood-brain barrier (BBB) with increased permeability. Neuroinflammation likely contributes to the disruption of the BBB in CSVD. Therefore, understanding the molecular mechanism of how neuroinflammation causes BBB damage is essential to preventing BBB disruption in CSVD. Matrix metalloproteinase 9 (MMP-9) contributes to BBB damage in neuroinflammatory diseases. In this study, we observed that interleukin-1β (IL-1β)-induced MMP-9 secretion in pericytes increased BBB permeability to sodium fluorescein (Na-F) by damaging the disruption of VE-cadherin, occludin, claudin-5, and zonula occludin-1 (ZO-1). Melatonin reduced BBB permeability to Na-F and inhibited the disruption of the adherens and tight junction proteins. Melatonin also downregulated MMP-9 and upregulated tissue inhibitor of metalloproteinases 1 (TIMP-1) gene expression, which decreased the MMP-9/TIMP-1 ratio. In addition, nuclear translocation of NF-κB/p65 induced by IL-1β in pericytes upregulated MMP-9 expression, which was inhibited by the NF-κB inhibitor PDTC. However, the NOTCH3 inhibitor DAPT significantly inhibited NF-κB/p65 translocation to the nucleus, while melatonin in combination with DAPT significantly prevented NF-κB/p65 translocation than DAPT alone. Our results suggest that melatonin reduced MMP-9-induced permeability of the BBB. Melatonin reduced MMP-9 expression and activity, which was induced by IL-1β through the regulation of the NOTCH3/NF-κB signaling pathway in pericytes, suggesting that pericytes regulate BBB integrity and function.

SUBMITTER: Qin W

PROVIDER: S-EPMC6932927 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Microvascular hyperpermeability that occurs at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema and elevated intracranial pressure following traumatic brain injury (TBI). At a cellular level, tight junction proteins (TJPs) between neighboring endothelial cells maintain the integrity of the BBB via TJ associated proteins particularly, zonula occludens-1 (ZO-1) that binds to the transmembrane TJPs and actin cytoskeleton intracellularly. The pro-inflammatory cytokine, interleukin-1β (IL-1β) as well as the proteolytic enzymes, matrix metalloproteinase-9 (MMP-9) are key mediators of trauma-associated brain edema. Recent studies indicate that melatonin a pineal hormone directly binds to MMP-9 and also might act as its endogenous inhibitor. We hypothesized that melatonin treatment will provide protection against TBI-induced BBB hyperpermeability via MMP-9 inhibition. Rat brain microvascular endothelial cells grown as monolayers were used as an in vitro model of the BBB and a mouse model of TBI using a controlled cortical impactor was used for all in vivo studies. IL-1β (10 ng/mL; 2 hours)-induced endothelial monolayer hyperpermeability was significantly attenuated by melatonin (10 μg/mL; 1 hour), GM6001 (broad spectrum MMP inhibitor; 10 μM; 1 hour), MMP-9 inhibitor-1 (MMP-9 specific inhibitor; 5 nM; 1 hour) or MMP-9 siRNA transfection (48 hours) in vitro. Melatonin and MMP-9 inhibitor-1 pretreatment attenuated IL-1β-induced MMP-9 activity, loss of ZO-1 junctional integrity and f-actin stress fiber formation. IL-1β treatment neither affected ZO-1 protein or mRNA expression or cell viability. Acute melatonin treatment attenuated BBB hyperpermeability in a mouse controlled cortical impact model of TBI in vivo. In conclusion, one of the protective effects of melatonin against BBB hyperpermeability occurs due to enhanced BBB integrity via MMP-9 inhibition. In addition, acute melatonin treatment provides protection against BBB hyperpermeability in a mouse model of TBI indicating its potential as a therapeutic agent for brain edema when established in humans.

Project description:BackgroundAdiponectin is an adipocytokine that plays a key regulatory role in glucose and lipid metabolism in obesity. The prevalence of obesity has led to an increase in the incidence of obesity-related glomerulopathy (ORG). This study aimed to identify the protective role of adiponectin in ORG.MethodsSmall-interfering RNA (siRNA) against the gene encoding adiponectin was transfected into podocytes. The oxidative stress level was determined using a fluorometric assay. Apoptosis was analyzed by flow cytometry. The expressions of podocyte markers and pyrin domain containing protein 3 (NLRP3) inflammasome-related proteins were measured by qRT-PCR, immunohistochemistry, and Western blot.ResultsPodocytes treated with palmitic acid (PA) showed downregulated expressions of podocyte markers, increased apoptosis, upregulated levels of NLRP3 inflammasome-related proteins, increased production of inflammatory cytokines (IL-18 and IL-1β), and induced activation of NF-κB as compared to the vehicle-treated controls. Decreased adiponectin expression was observed in the serum samples from high fat diet (HFD)-fed mice. Decreased podocin expression and upregulated NLRP3 expression were observed in the kidney samples from high fat diet (HFD)-fed mice. Treatment with adiponectin or the NLRP3 inflammasome inhibitor, MCC950, protected cultured podocytes against podocyte apoptosis and inflammation. Treatment with adiponectin protected mouse kidney tissues against decreased podocin expression and upregulated NLRP3 expression. The knockout of adiponectin gene by siRNA increased ROS production, resulting in the activation of NLRP3 inflammasome and the phosphorylation of NF-κB in podocytes. Pyrrolidine dithiocarbamate, an NF-κB inhibitor, prevented adiponectin from ameliorating FFA-induced podocyte injury and NLRP3 activation.ConclusionsOur study showed that adiponectin ameliorated PA-induced podocyte injury in vitro and HFD-induced injury in vivo via inhibiting the ROS/NF-κB/NLRP3 pathway. These data suggest the potential use of adiponectin for the prevention and treatment of ORG.

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Melatonin protects blood-brain barrier integrity and permeability by inhibiting matrix metalloproteinase-9 via the NOTCH3/NF-κB pathway.

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