OBJECTIVE: Polydopamine nanoparticles (PDA NPs) are a promising topic in the fields of drug delivery, tissue engineering, bioimaging, etc. The present study aims to explore the impact of PDA NPs carrying ferroptosis inhibitor ferstatin-1 (Fer-1) on myocardial ischemia-reperfusion injury (MIRI).
METHODS: After establishment of a rat model of MIRI and PDA NPs, the rats were divided into 4 groups: model group, sham operation group, Fer-1 group, and nano+Fer-1 group (n=8). To detect the effect of PDA NPs encapsulating Fer-1 on ferroptosis in MIRI rats, we further set up NOX4 overexpression group (pc-NOX4 group), NOX4 inhibitor group (Fulvene-5 group), nano+Fer-1+pc-NOX4 group, and nano+Fer-1+Fulvene-5 group (n=8). A CCK-8 assay was conducted to assess cell viability and staining to detect cardiomyocyte apoptosis and observe myocardial infraction.
RESULTS: PDA NPs loaded with Fer-1 were successfully prepared with good safety and biocompatibility. Administration of PDA NPs carrying Fer-1 notably alleviated myocardial injury and hindered the process of ferroptosis in MIRI rats when inducing downregulation of NOX4 expression. Additionally, overexpression of GPX4 significantly attenuated myocardial injury in MIRI rats. While Fer-1 was shown to inhibit the expression of NOX4, the NOX4 inhibitor Fulvene-5 greatly elevated GPX4 and FTH1 expression in cardiomyocytes, and down-regulated the content of Fe2+, especially in the nanometer+Fer-1+Fulvene-5 group.
CONCLUSIONS: With promising safety and biocompatibility, PDA NPs encapsulated Fer-1 decrease GPX4 and FTH1 expression by inhibiting the level of NOX4 in myocardial cells of MIRI rats, thereby suppressing ferroptosis of cardiomyocytes and alleviating myocardial injury.

BACKGROUND: Metabolic reprogramming plays a key role in cancer progression and clinical outcomes; however, the patterns and primary regulators of metabolic reprogramming in colorectal cancer (CRC) are not well understood.
OBJECTIVE: To explore the role of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in promoting progression of CRC.
METHODS: We evaluated the expression and function of dysregulated and survival-related metabolic genes using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Consensus clustering was used to cluster CRC based on dysregulated metabolic genes. A prediction model was constructed based on survival-related metabolic genes. Sphere formation, migration, invasion, proliferation, apoptosis and clone formation was used to evaluate the biological function of NOX4 in CRC. mRNA sequencing was utilized to explore the alterations of gene expression NOX4 over-expression tumor cells. In vivo subcutaneous and lung metastasis mouse tumor model was used to explore the effect of NOX4 on tumor growth.
RESULTS: We comprehensively analyzed 3341 metabolic genes in CRC and identified three clusters based on dysregulated metabolic genes. Among these genes, NOX4 was highly expressed in tumor tissues and correlated with worse survival. In vitro, NOX4 overexpression induced clone formation, migration, invasion, and stemness in CRC cells. Furthermore, RNA-sequencing analysis revealed that NOX4 overexpression activated the mitogen-activated protein kinase-MEK1/2-ERK1/2 signaling pathway. Trametinib, a MEK1/2 inhibitor, abolished the NOX4-mediated tumor progression. In vivo, NOX4 overexpression promoted subcutaneous tumor growth and lung metastasis, whereas trametinib treatment can reversed the metastasis.
CONCLUSIONS: Our study comprehensively analyzed metabolic gene expression and highlighted the importance of NOX4 in promoting CRC metastasis, suggesting that trametinib could be a potential therapeutic drugs of CRC clinical therapy targeting NOX4.

Endothelial malfunction is a major contributor to early or delayed vasospasm after subarachnoid hemorrhage (SAH). As a representative form of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) uncoupling leads to a reduction in nitric oxide (NO) generated by endothelial cells. In this study, we investigated how the interaction between endothelial NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4) and DHFR (dihydrofolate reductase) contributes to eNOS uncoupling after SAH. Setanaxib and the adeno-associated virus (AAV) targeting brain vascular endothelia were injected through the tail vein and the expression and localization of proteins were examined by western blot and immunofluorescence staining. The NO content was measured using the NO assay kit, and laser speckle contrast imaging was used to assess cortical perfusion. ROS (reactive oxygen species) level was detected by DHE (dihydroethidium) staining, DCFH-DA (2\',7\'-dichlorofluorescin diacetate) staining and H2O2 (hydrogen peroxide) measurement. The Garcia score was employed to examine neurological function. Setanaxib is widely used for its preferential inhibition for NOX1/4 over other NOX isoforms. After endothelial NOX4 was inhibited by Setanaxib in a mouse model of SAH, the endothelial DHFR level was significantly elevated, which attenuated eNOS uncoupling, increased cortical perfusion, and improved the neurological function. The protective role of inhibiting endothelial NOX4, however, disappeared after knocking down endothelial DHFR. Our results suggest that endothelial DHFR decreased significantly because of the elevated level of endothelial NOX4, which aggravated eNOS uncoupling after SAH, leading to decreased cortical perfusion and worse neurological outcome.

[This corrects the article DOI: 10.3389/fcvm.2021.756098.].

UNASSIGNED: Acute lung injury (ALI) is a common complication following severe burns. The underlying mechanisms of ALI are incompletely understood; thus, available treatments are not sufficient to repair the lung tissue after ALI.
UNASSIGNED: To investigate the relationship between the Notch pathway and burn-induced lung injury, we established a rat burn injury model by scalding and verified lung injury via lung injury evaluations, including hematoxylin and eosin (H&E) staining, lung injury scoring, bronchoalveolar lavage fluid and wet/dry ratio analyses, myeloperoxidase immunohistochemical staining and reactive oxygen species (ROS) accumulation analysis. To explore whether burn injury affects Notch1 expression, we detected the expression of Notch1 and Hes1 after burn injury. Then, we extracted pulmonary microvascular endothelial cells (PMVECs) and conducted Notch pathway inhibition and activation experiments, via a γ-secretase inhibitor (GSI) and OP9-DLL1 coculture, respectively, to verify the regulatory effect of the Notch pathway on ROS accumulation and apoptosis in burn-serum-stimulated PMVECs. To investigate the regulatory effect of the Notch pathway on ROS accumulation, we detected the expression of oxidative-stress-related molecules such as superoxide dismutase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 2, NOX4 and cleaved caspase-3. NOX4-specific small interfering RNA (siRNA) and the inhibitor GKT137831 were used to verify the regulatory effect of the Notch pathway on ROS via NOX4.
UNASSIGNED: We successfully established a burn model and revealed that lung injury, excessive ROS accumulation and an inflammatory response occurred. Notch1 detection showed that the expression of Notch1 was significantly increased after burn injury. In PMVECs challenged with burn serum, ROS and cell death were elevated. Moreover, when the Notch pathway was suppressed by GSI, ROS and cell apoptosis levels were significantly increased. Conversely, these parameters were reduced when the Notch pathway was activated by OP9-DLL1. Mechanistically, the inhibition of NOX4 by siRNA and GKT137831 showed that the Notch pathway reduced ROS production and cell apoptosis by downregulating the expression of NOX4 in PMVECs.
UNASSIGNED: The Notch pathway reduced ROS production and apoptosis by downregulating the expression of NOX4 in burn-stimulated PMVECs. The Notch-NOX4 pathway may be a novel therapeutic target to treat burn-induced ALI.

Neointima formation is a serious complication caused by mechanical trauma to the vessel. (R)-4,6-dimethoxy-3-(4-methoxy phenyl)-2,3-dihydro-1H-indanone [(R)-TML 104] is a synthesized analog of the natural product resveratrol sesquiterpenes (±)-isopaucifloral F. The present study aimed to investigate the effects and underlying mechanisms of (R)-TML104 on neointima formation. Our results showed that (R)-TML104 prevented neointima formation based on a carotid artery injury model in mice. Furthermore, (R)-TML104 inhibited platelet-derived growth factor-BB (PDGF-BB)-induced vascular smooth muscle cells (VSMC) phenotypic transformation, evidenced by increased α-smooth muscle actin, reduced VSMC proliferation, and migration. Simultaneously, (R)-TML104 upregulated sirtuin-1 (SIRT1) expression in VSMC. We further uncovered that SIRT1 expression is critical for the inhibitory effects of (R)-TML104 on PDGF-BB-induced VSMC phenotypic transformation in vitro and injury-induced neointima formation in vivo. Finally, (R)-TML104-upregulated SIRT1 inhibited PDGF-BB-induced VSMC phenotypic transformation by downregulating nicotinamide adenine dinucleotide phosphate oxidase 4 expression via decreasing nuclear factor-κB acetylation. Taken together, these results revealed that (R)-TML104 upregulates SIRT1 expression and ameliorates neointima formation. Therefore, the application of (R)-TML104 may constitute an effective strategy to ameliorate neointima formation.

Objective: To study the effect of benazepril on the expression of nuclear factor E2 related factor 2 (Nrf2), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and reactive oxygen species (ROS) concentration in rats with hepatic fibrosis and to explore the possible antifibrotic mechanism of benazepril. Methods: Twenty-two healthy male Sprague-Dawley rats were randomly divided into 3 groups: control group (6 rats), model group (8 rats) and benazepril treatment group (8 rats). Two rats died during modeling and treatment in the model group and the benazepril treatment group, and a model of hepatic fibrosis induced by carbon tetrachloride (CCL(4)) was established. The rats in benazepril group were given benazepril for 8 weeks by gastric gavage. The assessment of liver tissue damage in each group was measured using conventional hematoxylin-eosin and Masson staining. The mRNA level of Nrf2, NOX4 in liver tissue was detected by RT-PCR, and serum ROS concentration was determined by colorimetry. All data were expressed in mean ± standard deviations, and were analyzed using SPSS21.0 statistical software. The data were compared using one-way analysis of variance, and the LSD-t method was used for pairwise comparison between the two groups. The correlation analysis was performed by Spearman\'s correlation analysis. Results: In the liver of the model group, with the aggravation of liver fibrosis the expression of Nrf2mRNA, NOX4 mRNA and ROS concentration were higher than control group [(4.01 ± 3.40), (31.78 ± 3.96), (1.82 ± 0.46) μg/ ml vs. (0.12 ± 0.11), (2.03 ± 0.31), (1.56±0.84) μg/ml, P < 0.05]. After benazepril treatment, NOX4 mRNA expression and ROS concentration were decreased than the model group [(15.93 ± 5.01), (0.78 ± 0.44) μg/ml vs. (31.78 ± 3.96), (1.82 ± 0.46) μg /ml, P < 0.05], while Nrf2 mRNA expression was higher than the model group [(6.69 ± 4.86) vs. (4.01 ± 3.40), P < 0.05]. There was a positive correlation between Nrf2 and NOX4, Nrf2 and ROS, and NOX4 and ROS (r = 0.616, 0.411, 0.802, P < 0.05). Conclusion: Benazepril may exert an anti-hepatic fibrosis effect by activating Nrf2 expression, or may inhibit the ROS-mediated oxidative stress in response to NOX4.
目的： 研究贝那普利对肝纤维化大鼠肝脏核因子E2相关因子2（Nrf2）、还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（NOX4）表达及血清活性氧簇（ROS）浓度的影响，探讨贝那普利可能的抗纤维化机制。 方法： 将22只清洁级健康雄性SD大鼠随机分3组，其中对照组6只，模型组和贝那普利治疗组每组8只，在造模及治疗过程中模型组和贝拉普利治疗组各有2只大鼠死亡，建立四氯化碳诱导的肝纤维化模型，贝那普利治疗组同时应用贝那普利灌胃，共8周，各组肝组织损伤的评估采用常规苏木精-伊红和Masson染色，RT-PCR检测肝组织中Nrf2 mRNA及NOX4 mRNA的表达量、比色法测定血清ROS浓度。所有数据表示为均数±标准差，采用SPSS21.0统计软件分析。计量资料组间比较采用单因素方差分析，两两比较采用LSD-t法；相关性分析采用Spearman相关分析。 结果： 随着肝纤维化的加重，模型组肝脏Nrf2 mRNA、NOX4 mRNA表达及血清ROS浓度均较对照组升高[（4.01±3.40）、（31.78±3.96）、（1.82±0.46）μg/ml对比（0.12±0.11）、（2.03±0.31）、（1.56±0.84）μg/ml，P值均<0.05]。贝那普利治疗后NOX4 mRNA表达和ROS浓度较模型组下降[（15.93±5.01）、（0.78±0.44）μg/ml对比（31.78±3.96）、（1.82±0.46）μg/ml，P值均<0.05]，而Nrf2 mRNA表达较模型组升高（相对表达量6.69±4.86对比4.01±3.40，P < 0.05）。Nrf2与NOX4、Nrf2与ROS、NOX4与ROS三组之间均呈正相关（r值分别为0.616、0.411、0.802，P值均< 0.05）。 结论： 贝那普利可能通过激活Nrf2表达发挥抗肝纤维化作用，亦可能抑制NOX4产生的ROS介导的氧化应激反应，发挥抗肝纤维化作用。.

Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-derived reactive oxygen species (ROS) serve an important role in cerebral ischemia/reperfusion (I/R) injury. However, the mechanism by which ROS generation is regulated has not yet been fully elucidated. The present study aimed to explore the role of transforming growth factor-β signaling in ROS generation. Sprague Dawley rats were subjected to I/R injury and PC-12 cells were transfected with small interfering RNA against activin receptor-like kinase (ALK)5 or hypoxia/reoxygenation (H/R). The results indicated that I/R or H/R significantly increased ALK5 expression, SMAD2/3 phosphorylation and NOX2/4 expression and activity, concomitant with ROS generation and apoptosis. In addition, ALK5 knockdown significantly reversed changes induced by H/R treatment in PC-12 cells. These results suggest that ALK5/SMAD2/3 signaling serves a key role in oxidative stress. To the best of our knowledge, this is the first study to demonstrate that ALK5/SMAD2/3 activation is associated with the regulation of NOX2/4 expression and exacerbates I/R injury.