Melatonin suppresses atherosclerosis by ferroptosis inhibition via activating NRF2 pathway.

Melatonin (MLT), a conserved small indole compound, exhibits anti-inflammatory and antioxidant properties, contributing to its cardioprotective effects. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with atherosclerosis disease risk, and is known as an atherosclerosis risk biomarker. This study aimed to investigate the impact of MLT on Lp-PLA2 expression in the atherosclerotic process and explore the underlying mechanisms involved. In vivo, ApoE-/- mice were fed a high-fat diet, with or without MLT administration, after which the plaque area and collagen content were assessed. Macrophages were pretreated with MLT combined with ox-LDL, and the levels of ferroptosis-related proteins, NRF2 activation, mitochondrial function, and oxidative stress were measured. MLT administration significantly attenuated atherosclerotic plaque progression, as evidenced by decreased plaque area and increased collagen. Compared with those in the high-fat diet (HD) group, the levels of glutathione peroxidase 4 (GPX4) and SLC7A11 (xCT, a cystine/glutamate transporter) in atherosclerotic root macrophages were significantly increased in the MLT group. In vitro, MLT activated the nuclear factor-E2-related Factor 2 (NRF2)/SLC7A11/GPX4 signaling pathway, enhancing antioxidant capacity while reducing lipid peroxidation and suppressing Lp-PLA2 expression in macrophages. Moreover, MLT reversed ox-LDL-induced ferroptosis, through the use of ferrostatin-1 (a ferroptosis inhibitor) and/or erastin (a ferroptosis activator). Furthermore, the protective effects of MLT on Lp-PLA2 expression, antioxidant capacity, lipid peroxidation, and ferroptosis were decreased in ML385 (a specific NRF2 inhibitor)-treated macrophages and in AAV-sh-NRF2 treated ApoE-/- mice. MLT suppresses Lp-PLA2 expression and atherosclerosis processes by inhibiting macrophage ferroptosis and partially activating the NRF2 pathway.

Ultrasound and microbubble-mediated delivery of miR-424-5p has a therapeutic effect in preeclampsia.

OBJECTIVE: To determine the influence of ultrasound/microbubble-mediated miR-424-5p delivery on trophoblast cells and the underlying mechanism. METHODS: Blood pressure and 24-h proteinuria of patients with preeclampsia (PE) were measured as well as the levels of miR-424-5p and amine oxidase copper containing 1 (AOC1) in placental tissues. HTR-8/Svneo and TEV-1 cells were subjected to cell transfection or ultrasonic microbubble transfection for determination of the expression of miR-424-5p, AOC1, ß-catenin and c-Myc as well as cell proliferation, apoptosis, migration and invasiveness. The concentrations of placental growth factor (PLGF), human chorionic gonadotropin (ß-hCG) and tumor necrosis factor-α (TNF-α) were measured in HTR-8/Svneo and TEV-1 cells. RNA immunoprecipitation (RIP) and dual luciferase reporter assay detected the binding of miR-424-5p to AOC1. A PE mouse model was induced by subcutaneous injection of L-NAME, where the influence of ultrasound/microbubble-mediated miR-424-5p delivery was evaluated. RESULTS: miR-424-5p was downregulated while AOC1 was upregulated in the placental tissues from PE patients. Overexpression of miR-424-5p activated Wnt/ß-catenin signaling pathway and promoted the proliferation of HTR-8/Svneo and TEV-1 cells as well as enhanced the migratory and invasive behaviors. AOC1 overexpression partly eliminated the effects of miR-424-5p on HTR-8/Svneo and TEV-1 cells. Ultrasound and microbubble mediated gene delivery enhanced the transfection efficiency of miR-424-5p and further promoted the effects of miR-424-5p in trophoblast cells. Ultrasound/microbubble-mediated miR-424-5p delivery alleviated experimental PE in mice. CONCLUSION: Ultrasound and microbubble-mediated miR-424-5p delivery targets AOC1 and activates Wnt/ß-catenin signaling pathway, thus promoting the aggressive phenotype of trophoblast cells, which indicating that miR-424-5p/AOC1 axis might be involved with PE pathogenesis.

Nomograms based on preoperative multimodal ultrasound of papillary thyroid carcinoma for predicting central lymph node metastasis.

OBJECTIVES: To establish a nomogram for predicting central lymph node metastasis (CLNM) based on the preoperative clinical and multimodal ultrasound (US) features of papillary thyroid carcinoma (PTC) and cervical LNs. METHODS: Overall, 822 patients with PTC were included in this retrospective study. A thyroid tumor ultrasound model (TTUM) and thyroid tumor and cervical LN ultrasound model (TTCLNUM) were constructed as nomograms to predict the CLNM risk. Areas under the curve (AUCs) evaluated model performance. Calibration and decision curves were applied to assess the accuracy and clinical utility. RESULTS: For the TTUM training and test sets, the AUCs were 0.786 and 0.789 and bias-corrected AUCs were 0.786 and 0.831, respectively. For the TTCLNUM training and test sets, the AUCs were 0.806 and 0.804 and bias-corrected AUCs were 0.807 and 0.827, respectively. Calibration and decision curves for the TTCLNUM nomogram exhibited higher accuracy and clinical practicability. The AUCs were 0.746 and 0.719 and specificities were 0.942 and 0.905 for the training and test sets, respectively, when the US tumor size was ≤ 8.45 mm, while the AUCs were 0.737 and 0.824 and sensitivity were 0.905 and 0.880, respectively, when the US tumor size was > 8.45 mm. CONCLUSION: The TTCLNUM nomogram exhibited better predictive performance, especially for the CLNM risk of different PTC tumor sizes. Thus, it serves as a useful clinical tool to supply valuable information for active surveillance and treatment decisions. KEY POINTS: • Our preoperative noninvasive and intuitive prediction method can improve the accuracy of central lymph node metastasis (CLNM) risk assessment and guide clinical treatment in line with current trends toward personalized treatments. • Preoperative clinical and multimodal ultrasound features of primary papillary thyroid carcinoma (PTC) tumors and cervical LNs were directly used to build an accurate and easy-to-use nomogram for predicting CLNM. • The thyroid tumor and cervical lymph node ultrasound model exhibited better performance for predicting the CLNM of different PTC tumor sizes. It may serve as a useful clinical tool to provide valuable information for active surveillance and treatment decisions.

MCL attenuates atherosclerosis by suppressing macrophage ferroptosis via targeting KEAP1/NRF2 interaction.

BACKGROUND: Micheliolide (MCL), which is the active metabolite of parthenolide, has demonstrated promising clinical application potential. However, the effects and underlying mechanisms of MCL on atherosclerosis are still unclear. METHOD: ApoE-/- mice were fed with high fat diet, with or without MCL oral administration, then the plaque area, lipid deposition and collagen content were determined. In vitro, MCL was used to pretreat macrophages combined by ox-LDL, the levels of ferroptosis related proteins, NRF2 activation, mitochondrial function and oxidative stress were detected. RESULTS: MCL administration significantly attenuated atherosclerotic plaque progress, which characteristics with decreased plaque area, less lipid deposition and increased collagen. Compared with HD group, the level of GPX4 and xCT in atherosclerotic root macrophages were increased in MCL group obviously. In vitro experiment demonstrated that MCL increased GPX4 and xCT level, improved mitochondrial function, attenuated oxidative stress and inhibited lipid peroxidation to suppress macrophage ferroptosis induced with ox-LDL. Moreover, MCL inhibited KEAP1/NRF2 complex formation and enhanced NRF2 nucleus translocation, while the protective effect of MCL on macrophage ferroptosis was abolished by NRF2 inhibition. Additionally, molecular docking suggests that MCL may bind to the Arg483 site of KEAP1, which also contributes to KEAP1/NRF2 binding. Furthermore, Transfection Arg483 (KEAP1-R483S) mutant plasmid can abrogate the anti-ferroptosis and anti-oxidative effects of MC in macrophages. KEAP1-R483S mutation also limited the protective effect of MCL on atherosclerosis progress and macrophage ferroptosis in ApoE-/- mice. CONCLUSION: MCL suppressed atherosclerosis by inhibiting macrophage ferroptosis via activating NRF2 pathway, the related mechanism is through binding to the Arg483 site of KEAP1 competitively.

Analysis of active components and molecular mechanism of action of Rubia cordifolia L. in the treatment of nasopharyngeal carcinoma based on network pharmacology and experimental verification.

The aim of this study is to explore the active components and potential molecular mechanism of action of Rubia cordifolia L. against nasopharyngeal carcinoma (NPC). We used network pharmacology, molecular docking, and bioinformatics analysis to identify the active components and their role against NPC. The experimental verification was detected by MTT, AnnexinV-FITC/PI double fluorescence staining and Western blotting method. Network pharmacology identified that mollugin is one of the most effective components inRubia cordifolia L. Important NPC targets included HSP90AA1, CDK1, EGFR, PIK3CA, MAPK14, and CDK2. Molecular docking revealed considerable binding activity of mollugin with either of the 6 important NPC targets. Bioinformatics analysis showed that these 6 important targets were mutated in NPC, and the expression of HSP90AA1, PIK3CA, and CDK2 in cancer tissues was significantly different from that in normal tissues. MTT detection and AnnexinV-FITC/PI double fluorescence staining showed that mollugin inhibited the proliferation and induced apoptosis of NPC cells. Western blotting indicated that the molecular mechanism of mollugin against NPC was related to the regulation of the expression of Survivin and XIAP. This study predicted and partially verified the pharmacological and molecular mechanism of action of Rubia cordifolia L. against NPC. Mollugin was identified as a potential active ingredient against NPC. These results prove the reliability of network pharmacology approaches and provide a basis for further research and application of Rubia cordifolia L. against NPC.

Analysis of the Molecular Mechanism of Evodia rutaecarpa Fruit in the Treatment of Nasopharyngeal Carcinoma Using Network Pharmacology and Molecular Docking.

Background: Nasopharyngeal carcinoma (NPC), a neoplasm of the head and neck, has high incidence and mortality rates in East and Southeast Asia. Evodia rutaecarpa is a tree native to Korea and China, and its fruit (hereafter referred to as Evodia) exhibits remarkable antitumour properties. However, little is known about its mechanism of action in NPC. In this study, we employed network pharmacology to identify targets of active Evodia compounds in nasopharyngeal carcinoma and generate an interaction network. Methods: The active ingredients of Evodia and targets in NPC were obtained from multiple databases, and an interaction network was constructed via the Cytoscape and STRING databases. The key biological processes and signalling pathways were predicted using Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analyses. Molecular docking technology was used to identify the affinity and activity of target genes, and The Cancer Genome Atlas and Human Protein Atlas databases were used to analyse differential expression. Cell Counting Kit-8 (CCK-8) and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual-fluorescence staining were used for experimental verification. Results: Active Evodia compounds included quercetin, isorhamnetin, and evodiamine, and important NPC targets included MAPK14, AKT1, RELA, MAPK1, JUN, and p53, which were enriched in lipid and atherosclerosis signalling pathways. Additionally, we verified the high affinity and activity of the active compounds through molecular docking, and the target proteins were verified using immunohistochemistry and differential expression analyses. Furthermore, CCK-8 assays and Annexin V-FITC/PI dual-fluorescence staining showed that isorhamnetin inhibited the proliferation of NPC cells and induced apoptosis. Conclusion: Our results identified the molecular mechanisms of Evodia and demonstrated its ability to alter the proliferation and apoptosis of NPC cells through multiple targets and pathways, thereby providing evidence for the clinical application of Evodia.

Hyperthermia combined with immune checkpoint inhibitor therapy in the treatment of primary and metastatic tumors.

According to the difference in temperature, thermotherapy can be divided into thermal ablation and mild hyperthermia. The main advantage of thermal ablation is that it can efficiently target tumors in situ, while mild hyperthermia has a good inhibitory effect on distant metastasis. There are some similarities and differences between the two therapies with respect to inducing anti-tumor immune responses, but neither of them results in sustained systemic immunity. Malignant tumors (such as breast cancer, pancreatic cancer, nasopharyngeal carcinoma, and brain cancer) are recurrent, highly metastatic, and highly invasive even after treatment, hence a single therapy rarely resolves the clinical issues. A more effective and comprehensive treatment strategy using a combination of hyperthermia and immune checkpoint inhibitor (ICI) therapies has gained attention. This paper summarizes the relevant preclinical and clinical studies on hyperthermia combined with ICI therapies and compares the efficacy of two types of hyperthermia combined with ICIs, in order to provide a better treatment for the recurrence and metastasis of clinically malignant tumors.

Surface-Eroding Poly(ortho ester amides) for Highly Efficient Oral Chemotherapy.

Two new poly(ortho ester amide) copolymers (POEA-4 and POEA-5) were synthesized via polycondensation of a new ortho ester diamine monomer with active esters of different aliphatic diacids. The kinetics of POEA mass loss and release of 5-FU were both nearly zero-order, suggesting predominantly surface-restricted polymer erosion and drug release. In vitro cytotoxicity tests demonstrated that both copolymers have excellent biocompatibility. In vivo acute toxicity tests suggested that oral administration of POEA-4 and POEA-5 did not cause any adverse effects on mice even at a very high dose (2000 mg/kg). In vivo antitumor efficacy against H22 transplanted tumors of 5-FU-loaded POEA tablets were fully examined. We envision that, with further optimization, POEA-based materials could have great potential as drug carriers for oral chemotherapy.

Amphiphilic block copolymers bearing six-membered ortho ester ring in side chains as potential drug carriers: synthesis, characterization, and in vivo toxicity evaluation.

A new type of amphiphilic block copolymers, poly(ethylene glycol)-block-poly(2-methyl-acrylicacid 2-methoxy-5-methyl-[1,3]dioxin-5-ylmethyl ester) (PEG-b-PMME), bearing acid-labile six-membered ortho ester rings in side chains was synthesized by reversible addition-fragmentation chain-transfer polymerization, and the influence of chain length of the hydrophobic PMME block on micelle properties was investigated. The PEG-b-PMME micelles were stable in aqueous buffer at physiological pH with a low critical micelle concentration. Nile Red as a model drug was encapsulated into the micelles to explore the release profiles. The Nile Red-loaded polymeric micelles showed rapid release of Nile Red in weakly acidic environments (pH 5) but slow release under physiological condition (pH 7.4), due to different hydrolysis rate of ortho ester side chains of PEG-b-PMME. The Paclitaxel (PTX)-loaded micelles retained potency in killing lung cancer cells (A549), compared with the free PTX. No obvious toxicity was found in vitro and in vivo after intraperitoneal injection of the micelles, which confirms that the PEG-b-PMME micelles with unique acid-labile characteristic have great potential as nano-scaled carriers for drug delivery.