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Melatonin attenuates low shear stress-induced pyroptosis and endothelial cell dysfunction via the RORα/miR-223/STAT-3 signalling pathway.

ABSTRACT: Endothelial cells sense changes in blood flow shear stress and affect the progression of atherosclerotic plaques. Pyroptosis is an inflammatory form of cell death and has been implicated in cardiovascular diseases. Melatonin and its nuclear receptor retinoid-related orphan receptor α (RORα) have protective effects on the development of atherosclerosis. To date, whether melatonin can prevent endothelial cell pyroptosis and dysfunction in pathological shear stress remains unclear. In the present study, human umbilical vein endothelial cells (ECs) were cultured under low shear stress conditions (5 dyne/cm²) for 24 h and treated with or without melatonin (2 µmol/l). The binding sites of the microRNA (miR)-223 promoter and RORα were predicted using the JASPAR website. Expression of pyroptosis-related proteins, including cleaved N-terminal gasdermin D, caspase-1, intercellular adhesion molecule 1 (ICAM-1) and nitric oxide (NO) were assessed. The results indicated that low shear stress increased pyroptosis and ICAM-1 expression, whereas it decreased NO levels. Melatonin alleviated pyroptosis and ICAM-1 expression and increased the production of NO in ECs. Further assessment revealed that low-level shear stress decreased RORα protein and mRNA expression, whereas melatonin would bind to RORα and thereby promoted miR-223 transcription in ECs. The present study also identified signal transducer and activator of transcription 3 (STAT-3) as a potential target gene of miR-223-3p. When transfected with miR-223 inhibitor, ECs up-regulated the expression of pyroptosis-related proteins and ICAM-1, and down-regulated NO levels. By contrast, silencing STAT-3 expression diminished the protective effect of miR-223. These results indicated that melatonin prevented ECs from undergoing pyroptosis and alleviated dysfunction via the RORα/miR-223/STAT-3 signalling pathway. This information could aid in the development of novel therapeutic approaches and provide new insights into atherosclerosis.

SUBMITTER: Yi S

PROVIDER: S-EPMC8506941 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEndothelial injury is one of the predominant pathophysiological characteristics of sepsis and is the major cause of sepsis-induced multiple organ failure. Endothelial pyroptosis is a fatal mechanism of endothelial injury in sepsis, and specific, effective therapies are lacking. Although hepatocyte growth factor (HGF) has been shown to have anti-apoptotic and anti-necrotic effects, whether it prevents pyroptosis to improve endothelial injury in sepsis remains unclear.MethodsRecombinant HGF was intravenously injected into mice with sepsis caused by caecal ligation puncture (CLP). Histopathological examination and transmission electron microscopy (TEM) were used to measure lung vascular endothelial injury. Lipopolysaccharide (LPS) was transfected into EA.hy926 cells to induce endothelial pyroptosis, and the cells were treated with HGF in the presence of inhibitors of c-Met and mTOR, namely, PHA-665752 and rapamycin, respectively. The mTOR signalling pathway and mitochondrial physiology were assessed using Western blot and flow cytometry.ResultsIntravenous HGF effectively alleviated pulmonary vascular endothelial injury and acute lung injury in the septic mice. The TEM results of lung tissue revealed that HGF attenuated pulmonary vascular endothelial pyroptosis, which was confirmed in vitro. Transfected LPS induced the pyroptosis of EA.hy926 cells and damaged their paracellular permeability, and these effects were ameliorated by treating the cells with recombinant HGF. The protective effect of HGF against pyroptosis was dependent on c-Met/mTOR signalling. mTOR activation effectively protected mitochondrial physiology and decreased reactive oxygen species (ROS) production in EA.hy926 cells in vitro.ConclusionsThese results demonstrated that HGF protected mitochondrial physiology by activating mTOR signalling to partially ameliorate endothelial pyroptosis and attenuate vascular endothelial injury and acute lung injury in sepsis animal model.

Project description:BackgroundAtherosclerosis (AS) is the primary cause of cardiovascular disease and the incidence is extremely common; however, there are currently few drugs that can effectively treat AS. Although oridonin has been widely used to treat inflammation and cancer for numerous years, to the best of our knowledge, its protective effect against AS has not been reported. Therefore, the present study aimed to investigate whether oridonin attenuated AS.MethodsBy using text mining, chemometric and chemogenomic methods, oridonin was predicted to be a beneficial agent for the treatment of AS. A parallel flow chamber was used to establish a low shear stress (LSS)-induced endothelial cell (EC) dysfunction model. Briefly, ECs were exposed to 3 dyn/cm2 LSS for 30 min and subsequently treated with oridonin or transfected with a small interfering RNA (siRNA) targeting nuclear factor erythroid 2-related factor 2 (NRF2). Reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and glutathione disulfide (GSSG) in EA.hy926 cells were analyzed to determine the level of oxidative stress. The nitric oxide (NO) levels and mRNA expression levels of endothelial NO synthase (eNOS), endothelin-1 (ET-1) and prostaglandin synthase (PGIS) in EA.hy926 cells were analyzed to determine EC dysfunction. Furthermore, the mRNA and protein expression levels of NRF2 were analyzed using reverse transcription-quantitative PCR and western blot. In addition, zebrafish were fed with a high-cholesterol diet to establish a zebrafish AS model, which was used to observe lipid accumulation and inflammation under a fluorescence microscope.ResultsWe found LSS led to oxidative stress and EC dysfunction; this was primarily indicated through the significantly decreased SOD and GSH content, the significantly increased MDA, GSSG and ROS content, the upregulated mRNA expression levels of ET-1, and the downregulated NO levels and mRNA expression levels of eNOS and PGIS in ECs. Notably, oridonin could improve LSS-induced oxidative stress and EC dysfunction, and the effects of oridonin were reversed by the transfection with NRF2 siRNA. Oridonin also attenuated lipid accumulation and neutrophil recruitment at the LSS regions in the zebrafish AS model.ConclusionsIn conclusion, the results of the present study suggested that oridonin may ameliorate LSS-induced EC dysfunction and oxidative stress by activating NRF2, thereby attenuating AS.

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Melatonin attenuates low shear stress-induced pyroptosis and endothelial cell dysfunction via the RORα/miR-223/STAT-3 signalling pathway.

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