Ferroptosis is a regulated form of necrotic cell death caused by iron-dependent accumulation of oxidized phospholipids in cellular membranes, culminating in plasma membrane rupture (PMR) and cell ...lysis. PMR is also a hallmark of other types of programmed necrosis, such as pyroptosis and necroptosis, where it is initiated by dedicated pore-forming cell death-executing factors. However, whether ferroptosis-associated PMR is also actively executed by proteins or driven by osmotic pressure remains unknown. Here, we investigate a potential ferroptosis role of ninjurin-1 (NINJ1), a recently identified executor of pyroptosis-associated PMR. We report that NINJ1 oligomerizes during ferroptosis, and that
Ninj1
-deficiency protects macrophages and fibroblasts from ferroptosis-associated PMR. Mechanistically, we find that NINJ1 is dispensable for the initial steps of ferroptosis, such as lipid peroxidation, channel-mediated calcium influx, and cell swelling. In contrast, NINJ1 is required for early loss of plasma membrane integrity, which precedes complete PMR. Furthermore, NINJ1 mediates the release of cytosolic proteins and danger-associated molecular pattern (DAMP) molecules from ferroptotic cells, suggesting that targeting NINJ1 could be a therapeutic option to reduce ferroptosis-associated inflammation.
Synopsis
Induction of ferroptosis by lipid peroxidation can lead to plasma membrane rupture, but the mediators of this rupture are unknown. This study identifies Ninjurin-1 (NINJ1) as the executor of plasma membrane permeabilization in ferroptotic macrophages and fibroblasts.
Ferroptosis induction leads to NINJ1 activation and oligomerization in macrophages and fibroblasts.
NINJ1 activation occurs downstream of lipid peroxidation and calcium influx, the first steps of ferroptosis.
NINJ1 activation triggers the last steps of ferroptosis, i.e., loss of membrane integrity and rupture.
NINJ1 controls the release of cytosolic proteins and danger-associated molecular pattern molecules from ferroptotic cells.
Ninjurin-1 (NINJ1) oligomerizes and mediates loss of plasma membrane integrity as well as plasma membrane rupture in ferroptotic cells.
Previous studies have shown that Ninjurin-1 participates in cell trafficking and axonal growth following central and peripheral nervous system neuroinflammation. But its precise roles in these ...processes and involvement in spinal cord injury pathophysiology remain unclear. Western blot assay revealed that Ninjurin-1 levels in rats with spinal cord injury exhibited an upregulation until day 4 post-injury and slightly decreased thereafter compared with sham controls. Immunohistochemistry analysis revealed that Ninjurin-1 immunoreactivity in rats with spinal cord injury sharply increased on days 1 and 4 post-injury and slightly decreased on days 7 and 21 post-injury compared with sham controls. Ninjurin-1 immunostaining was weak in vascular endothelial cells, ependymal cells, and some glial cells in sham controls while it was relatively strong in macrophages, microglia, and reactive astrocytes. These findings suggest that a variety of cells, including vascular endothelial cells, macrophages, and microglia, secrete Ninjurin-1 and they participate in the pathophysiology of compression-induced spinal cord injury. All experimental procedures were approved by the Care and Use of Laboratory Animals of Jeju National University (approval No. 2018-0029) on July 6, 2018.
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•GEx diminishes MCP-1, VCAM-1 and monocyte adhesion in TNFα-exposed HEC.•GEx anti-inflammatory action was correlated with the decrease of Ninj-1 and TNFR1.•GEx decreases intracellular ...RAGE expression and increases sRAGE levels.•GEx decrease ROS by reducing p22phox and NOX4 and activating Nrf2/HO-1 axis.
Dysfunction of endothelial cells (EC) is important for atherosclerosis progression. The aim of this study was to evaluate the potential of ginger extract (GEx), 6-gingerol (6-G) and 6-shogaol (6-Sh) to reverse EC dysfunction and to investigate its mechanism of action, using cultured human EC incubated with TNFα. The results showed that GEx decreases monocyte chemoattractant protein-1, vascular cell adhesion molecule-1 and monocyte adhesion. This decrease was associated with the: (1) decrease of ninjurin-1 expression; (2) reduction of TNFα receptor1 and of the receptor for advanced glycation end-products expression (RAGE), in parallel with the increase of soluble RAGE; (3) decrease of NADPH oxidase subunits expression; (4) activation of antioxidant Nrf2 and heme oxygenase-1, and (5) inhibition of NF-kB. The benefic effects of 6-G and 6-Sh were weaker than those of GEx (GEx > 6-Sh > 6-G). In conclusion, GEx might be a promising alternative to ameliorate disorders in which oxidative stress and inflammation are important.
Nerve injury-induced protein (Ninjurin)-1 is a cell adhesion molecule that is upregulated in neurons and Schwann cells after transection injury in rats. In this study, we investigated the ...localization of Ninjurin-1 in various tissues, including the cerebrum, sciatic nerve, spleen, lung, stomach, ileum, colon, liver, pancreas, kidney, testis, and skin in C57BL/6 mice, using Western blotting and immunohistochemistry. Western blot analysis showed that Ninjurin-1 was differentially expressed among organs. Ninjurin-1 was abundant in skin and ileum, weakly expressed in cerebrum, and moderately expressed in the other organs studied. Immunohistochemical analysis largely confirmed the results of the western blot analysis with often localization of Ninjurin-1 in the regions with abundant connective tissues. In addition, Ninjurin-1 was differentially expressed in various cell types in the tissues under the investigation. These findings suggest that Ninjurin-1 may play organ-specific roles in development and homeostasis of many organs.
Objective
The aim of this study was to clarify the expression of Ninj1 in endometriosis and adenomyosis lesions, and its inductive factor in human endometriotic stromal cells (ESCs).
Background
Nerve ...injury-induced protein 1 (Ninj1) is a molecule originally identified in dorsal root ganglion neurons and Schwann cells after nerve injury and promotes neurite outgrowth. The aim of this study was to clarify the expression of Ninj1 in endometriosis and adenomyosis lesions, and its inductive factor in human endometriotic stromal cells (ESCs).
Materials and Methods
Tissues were obtained with consent from patients diagnosed with ovarian endometrioma (n - 15 in total), peritoneal endometriosis (n - 5), adenomyosis (n - 5), and other gynecological disorders (n - 5, control) during surgery. Immunohistochemistry was conducted in order to detect Ninj1 protein expression in the lesion of endometriosis, adenomyosis, and eutopic endometrium. Nerve fibers in the ovarian endometrioma were detected by positive staining of PGP-9.5. To evaluate the effects of IL-1β on Ninj1 gene expression in endometriosis, ESCs isolated from ovarian endometrioma (n - 5) were treated with IL-1β (5 ng/mL) for 3 or 6 hours. Messenger RNA (mRNA) expression for Ninj1 was examined using quantitative RT-PCR.
Results
The Ninj1 protein was expressed by ovarian endometrioma, peritoneal endometriotic, and adenomyotic tissue. Nerve fibers were found in the areas of positive staining for Ninj1 in ovarian endometrioma. IL-1β, an indicator of inflammation in endometriosis, significantly increased Ninj1 mRNA expression by ESC.
Conclusion
Our study demonstrates that Ninj1 is expressed in endometriosis and adenomyosis and is induced by the inflammatory stimuli. Given the neurogenetic property of Ninj1, our results imply that Ninj1, induced by inflammation in endometriosis lesion, may contribute to the pathogenesis of pain symptoms characteristic of endometriosis.
Ninjurin-1 is a novel adhesion molecule which is involved in many inflammatory diseases. Functional blockage of Ninjurin-1 has exerted an atheroprotective effect. The aim of the study is to explore ...the association between serum Ninjurin-1 and the risk of large artery atherosclerotic acute ischemic stroke. From August 2020 through December 2021, patients with large artery atherosclerotic acute ischemic stroke (LAA-AIS) admitted to the First Hospital Affiliated to Soochow University, and age- and sex-matched controls free of ischemic stroke were recruited. Serum Ninj1 was measured with an enzyme-linked immunosorbent assay. Multivariable logistic regression models were used to calculate the odds ratios and 95% confidence intervals of LAA-AIS associated with serum Ninj1 levels, and receiver operating characteristic (ROC) curves were performed to assess the improvement value of Ninj1 for the prediction of LAA-AIS after adding Ninj1 to established risk factors. Of the 110 patients and 110 age- and sex-matched controls free of ischemic stroke enrolled, serum Ninj1 levels in LAA-AIS patients were significantly higher than that in control group (142.70 ng/ml IQR: 110.41–163.44 vs 101.62 ng/ml IQR: 86.63–120.86,
p
< 0.001). In multivariable analysis, Ninj1 levels were expressed as continuous variable and ordinal variable (tertiles), and it turned out that Ninj1 levels were positively associated with increased risk of LAA-AIS, especially in tertile3 compared with tertile1 (adjusted
OR
= 12.567, 95%CI: 5.148–30.678,
p
< 0.001), and the adjusted odds OR per 10 ng/ml increment was 1.541, 95%CI: 1.348–1.763,
p
< 0.001. Furthermore, adding Ninj1 to a multivariate logistic model including conventional risk factors associated LAA-AIS improved the area under ROC curves from 0.787 to 0.874. Elevated circulating levels of Ninj1 were associated with increased risk of LAA-AIS, indicating that serum Ninj1 may act as a predictor independent of established conventional risk factors.
Clinical data implicate fluctuations of high levels of plasma glucose in cardiovascular diseases. Endothelial cells (EC) are the first cells of the vessel wall exposed to them. Our aim was to ...evaluate the effects of oscillating glucose (OG) on EC function and to decipher new molecular mechanisms involved. Cultured human ECs (EA.hy926 line and primary cells) were exposed to OG (5/25 mM alternatively at 3 h), constant HG (25 mM) or physiological concentration (5 mM, NG) for 72 h. Markers of inflammation (Ninj-1, MCP-1, RAGE, TNFR1, NF-kB, and p38 MAPK), oxidative stress (ROS, VPO1, and HO-1), and transendothelial transport proteins (SR-BI, caveolin-1, and VAMP-3) were assessed. Inhibitors of ROS (NAC), NF-kB (Bay 11-7085), and Ninj-1 silencing were used to identify the mechanisms of OG-induced EC dysfunction. The results revealed that OG determined an increased expression of Ninj-1, MCP-1, RAGE, TNFR1, SR-B1, and VAMP-3 andstimulated monocyte adhesion. All of these effects were induced bymechanisms involving ROS production or NF-kB activation.
silencing inhibited the upregulation of caveolin-1 and VAMP-3 induced by OG in EC. In conclusion, OG induces increased inflammatory stress, ROS production, and NF-kB activation and stimulates transendothelial transport. To this end, we propose a novel mechanism linking Ninj-1 up-regulation to increased expression of transendothelial transport proteins.
To investigate the regulatory effects of ninjurin-1 on adhesion of myeloid cells in the retina at the early stage of diabetic rats.
Experimental study. The rat diabetic model was induced by ...intraperitoneal injection of streptozotocin. After 2 months of diabetes induction, 27 diabetic rats were randomly chosen and assigned to 3 groups, including diabetes and phosphate buffered saline (PBS) injection group (group B), diabetes and anti-Ninj-1 injection group (group C) and diabetes and anti-IgG injection group (group D), with 9 rats in each group. Nine age matched health rats were chosen as control group (group A). Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Retinal myeloid cell infiltration activity was measured by enzyme linked immunosorbent assay of myeloperoxidase (MPO). The differences of the mean values among the four groups were analyzed by one-factor analysis of variance. The multiple comparisons of the mean values among the four groups were analyzed by LSD-t analysis.
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