Purine-rich Element-binding Protein B Mediates Ferroptosis in Lipopolysaccharide-induced Raw264.7 Macrophage Inflammation.

ABSTRACT: Lipopolysaccharide (LPS) plays an important role in Raw264.7 macrophage ferroptosis and inflammation. Purine-rich element-binding protein B (Purb) influences cellular processes by regulating gene expression as a transcription factor. However, the effect and molecular mechanism of Purb in regulating Raw264.7 macrophage ferroptosis is still unknown. In this study, we used malondialdehyde, glutathione (GSH) assays, Fe 2+ fluorescence, reactive oxygen species staining, and western blotting to determine the effect of Purb on LPS-induced Raw264.7 macrophage ferroptosis. Pharmacological inhibitor of ferroptosis was utilized to explore its potential effects for inflammation by reverse transcription-quantitative polymerase chain reaction analysis. Meanwhile, chromatin immunoprecipitation was performed to verify the binding of Purb and the GSH-dependent peroxidase 4 (Gpx4). The results showed that LPS-induced inflammation in Raw264.7 macrophages was inhibited by ferroptosis inhibitor Fer-1 treatment. LPS inhibited the expression of Purb in Raw264.7 macrophages. In addition, Purb overexpression relieved the ferroptosis, and inflammatory response of Raw264.7 macrophages induced by LPS. Mechanistically, the binding of Purb to the Gpx4 promoter was decreased after LPS stimulation. Therefore, we concluded for the first time that Purb played a critical role in LPS-induced ferroptosis and inflammatory response by regulating the transcription of Gpx4. These results provide a theoretical basis for further research on the macrophage ferroptosis and inflammation.

Farnesoid X Receptor Protects Murine Lung against IL-6-promoted Ferroptosis Induced by Polyriboinosinic-Polyribocytidylic Acid.

Various infections trigger a storm of proinflammatory cytokines in which IL-6 acts as a major contributor and leads to diffuse alveolar damage in patients. However, the metabolic regulatory mechanisms of IL-6 in lung injury remain unclear. Polyriboinosinic-polyribocytidylic acid [poly(I:C)] activates pattern recognition receptors involved in viral sensing and is widely used in alternative animal models of RNA virus-infected lung injury. In this study, intratracheal instillation of poly(I:C) with or without an IL-6-neutralizing antibody model was combined with metabonomics, transcriptomics, and so forth to explore the underlying molecular mechanisms of IL-6-exacerbated lung injury. We found that poly(I:C) increased the IL-6 concentration, and the upregulated IL-6 further induced lung ferroptosis, especially in alveolar epithelial type II cells. Meanwhile, lung regeneration was impaired. Mechanistically, metabolomic analysis showed that poly(I:C) significantly decreased glycolytic metabolites and increased bile acid intermediate metabolites that inhibited the bile acid nuclear receptor farnesoid X receptor (FXR), which could be reversed by IL-6-neutralizing antibody. In the ferroptosis microenvironment, IL-6 receptor monoclonal antibody tocilizumab increased FXR expression and subsequently increased the Yes-associated protein (YAP) concentration by enhancing PKM2 in A549 cells. FXR agonist GW4064 and liquiritin, a potential natural herbal ingredient as an FXR regulator, significantly attenuated lung tissue inflammation and ferroptosis while promoting pulmonary regeneration. Together, the findings of the present study provide the evidence that IL-6 promotes ferroptosis and impairs regeneration of alveolar epithelial type II cells during poly(I:C)-induced murine lung injury by regulating the FXR-PKM2-YAP axis. Targeting FXR represents a promising therapeutic strategy for IL-6-associated inflammatory lung injury.

Paeonol represses A549 cell glycolytic reprogramming and proliferation by decreasing m6A modification of Acyl-CoA dehydrogenase.

Aberrant glycolytic reprogramming is involved in lung cancer progression by promoting the proliferation of non-small cell lung cancer cells. Paeonol, as a traditional Chinese medicine, plays a critical role in multiple cancer cell proliferation and inflammation. Acyl-CoA dehydrogenase (ACADM) is involved in the development of metabolic diseases. N6-methyladenosine (m6A) modification is important for the regulation of messenger RNA stability, splicing, and translation. Here, we investigated whether paeonol regulates the proliferation and glycolytic reprogramming via ACADM with m6A modification in A549 cells (human non-small cell lung cancer cells). Cell counting kit 8, 5-Bromo-2-deoxyuridine, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, flow cytometry analysis, western blotting and seahorse XFe24 extracellular flux analyzer assays showed that paeonol had a significant inhibitory effect against A549 cell proliferation and glycolysis. Mechanistically, ACADM was a functional target of paeonol. We also showed that the m6A reader YTH domain containing 1 plays an important role in m6A-modified ACADM expression, which is negatively regulated by paeonol, and is involved in A549 cell proliferation and glycolytic reprogramming. These results indicated the central function of paeonol in regulating A549 cell glycolytic reprogramming and proliferation via m6A modification of ACADM.

Ubiquitinated AIF is a major mediator of hypoxia-induced mitochondrial dysfunction and pulmonary artery smooth muscle cell proliferation.

BACKGROUND: Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is the main cause of hypoxic pulmonary hypertension (PH), and mitochondrial homeostasis plays a crucial role. However, the specific molecular regulatory mechanism of mitochondrial function in PASMCs remains unclear. METHODS: In this study, using the CCK8 assay, EdU incorporation, flow cytometry, Western blotting, co-IP, mass spectrometry, electron microscopy, immunofluorescence, Seahorse extracellular flux analysis and echocardiography, we investigated the specific involvement of apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase in regulating mitochondrial energy metabolism and mitophagy in PASMCs. RESULTS: In vitro, AIF deficiency in hypoxia leads to impaired oxidative phosphorylation and increased glycolysis and ROS release because of the loss of mitochondrial complex I activity. AIF was also downregulated and ubiquitinated under hypoxia leading to the abnormal occurrence of mitophagy and autophagy through its interaction with ubiquitin protein UBA52. In vivo, treatment with the adeno-associated virus vector to overexpress AIF protected pulmonary vascular remodeling from dysfunctional and abnormal proliferation. CONCLUSIONS: Taken together, our results identify AIF as a potential therapeutic target for PH and reveal a novel posttranscriptional regulatory mechanism in hypoxia-induced mitochondrial dysfunction.