Baicalein alleviates cisplatin-induced acute kidney injury by inhibiting ALOX12-dependent ferroptosis.

BACKGROUND: In acute kidney injury (AKI), ferroptosis is the main mechanism of cell death in the renal tubular epithelium. Baicalein, a traditional Chinese medicine monomer, plays a protective role in various kidney diseases; however, the effect of baicalein on ferroptosis in AKI still needs further exploration. PURPOSE: In this study, we explored the role of baicalein and its specific mechanism in mediating ferroptosis in cisplatin-induced AKI. METHODS: We used a cisplatin-induced AKI model to study the effects of baicalein on renal tissue and tubular epithelial cell injury. The effects of baicalein on tubular epithelial cell ferroptosis were detected in cisplatin-induced AKI and further verified by folic acid-induced AKI. The Swiss Target Prediction online database was used to predict the possible mechanism by which baicalein regulates ferroptosis, and the specific target proteins were further verified. Molecular docking and SPR were used to further determine the binding potential of baicalein to the target protein. Finally, RNA interference (RNAi) technology and enzymatic inhibition were used to determine whether baicalein regulates ferroptosis through target proteins. RESULTS: Baicalein alleviated cisplatin- and folic acid-induced renal dysfunction and pathological damage and improved cisplatin-induced HK2 cell injury. Mechanistically, baicalein reduced the expression of 12-lipoxygenase (ALOX12), which inhibits phospholipid peroxidation and ferroptosis in AKI. Molecular docking and SPR demonstrated direct binding between baicalein and ALOX12. Finally, we found that silencing ALOX12 had a regulatory effect similar to that of baicalein. Comparable results were also obtained with the ALOX12 inhibitor ML355. CONCLUSION: This was the first study to confirm that baicalein regulates ferroptosis both in vitro and in vivo in cisplatin-induced AKI and to verify the regulatory effect of baicalein in folic acid-induced AKI. Our results reveal the critical role of ALOX12 in kidney damage and ferroptosis caused by cisplatin and emphasize the regulatory effect of baicalein on renal tubular epithelial cell ferroptosis mediated by ALOX12. Baicalein is an effective drug for treating AKI, and ALOX12 is a potential drug target.

Enhanced ALOX12 Gene Expression Predicts Therapeutic Susceptibility to 5-Azacytidine in Patients with Myelodysplastic Syndromes.

5-azacytidine (AZA), a representative DNA-demethylating drug, has been widely used to treat myelodysplastic syndromes (MDS). However, it remains unclear whether AZA's DNA demethylation of any specific gene is correlated with clinical responses to AZA. In this study, we investigated genes that could contribute to the development of evidence-based epigenetic therapeutics with AZA. A DNA microarray identified that AZA specifically upregulated the expression of 438 genes in AZA-sensitive MDS-L cells but not in AZA-resistant counterpart MDS-L/CDA cells. Of these 438 genes, the ALOX12 gene was hypermethylated in MDS-L cells but not in MDS-L/CDA cells. In addition, we further found that (1) the ALOX12 gene was hypermethylated in patients with MDS compared to healthy controls; (2) MDS classes with excess blasts showed a relatively lower expression of ALOX12 than other classes; (3) a lower expression of ALOX12 correlated with higher bone marrow blasts and a shorter survival in patients with MDS; and (4) an increased ALOX12 expression after AZA treatment was associated with a favorable response to AZA treatment. Taking these factors together, an enhanced expression of the ALOX12 gene may predict favorable therapeutic responses to AZA therapy in MDS.

Identifying ALOX15-initiated lipid peroxidation increases susceptibility to ferroptosis in asthma epithelial cells.

Ferroptosis is a programmed form of cell death regulated by iron and has been linked to the development of asthma. However, the precise mechanisms driving ferroptosis in asthma remain elusive. To gain deeper insights, we conducted an analysis of nasal epithelial and sputum samples from the GEO database using three machine learning methods. Our investigation identified a pivotal gene, Arachidonate 15-lipoxygenase (ALOX15), associated with ferroptosis in asthma. Through both in vitro and in vivo experiments, we further confirmed the significant role of ALOX15 in ferroptosis in asthma. Our results demonstrate that ferroptosis manifests in an HDM/LPS-induced allergic airway inflammation (AAI) mouse model, mimicking human asthma, and in HDM/LPS-stimulated 16HBE cells. Moreover, we observed an up-regulation of ALOX15 expression in HDM/LPS-induced mice and cells. Notably, silencing ALOX15 markedly decreased HDM/LPS-induced ferroptosis in 16HBE cells. These findings indicate that ferroptosis may be implicated in the onset and progression of asthma, with ALOX15-induced lipid peroxidation raising the susceptibility to ferroptosis in asthmatic epithelial cells.

Rhythm gene PER1 mediates ferroptosis and lipid metabolism through SREBF2/ALOX15 axis in polycystic ovary syndrome.

OBJECTIVE: This work aimed to investigate the role of rhythm gene PER1 in mediating granulosa cell ferroptosis and lipid metabolism of polycystic ovary syndrome (PCOS). METHODS: We injected dehydroepiandrosterone and Ferrostatin-1 (Fer-1) into mice to explore the mechanism of ferroptosis in PCOS. The effect of PER1 on ferroptosis-like changes in granulosa cells was explored by overexpression of PER1 plasmid transfection and Fer-1 treatment. RESULTS: We found that Fer-1 ameliorated the characteristic polycystic ovary morphology, suppressed ferroptosis in the PCOS mice. PER1 and ALOX15 were highly expressed in PCOS, whereas SREBF2 was lowly expressed. Overexpression of PER1 decreased granulosa cell viability and inhibited proliferation. Meanwhile, overexpression of PER1 increased lipid reactive oxygen species, 4-Hydroxynonenal (4-HNE), Malondialdehyde (MDA), total Fe, and Fe2+ levels in granulosa cells and decreased Glutathione (GSH) content. Fer-1, SREBF2 overexpression, or ALOX15 silencing treatment reversed the effects of PER1 overexpression on granulosa cells. PER1 binds to the SREBF2 promoter and represses SREBF2 transcription. SREBF2 binds to the ALOX15 promoter and represses ALOX15 transcription. Correlation analysis of clinical trials showed that PER1 was positively correlated with total cholesterol, low-density lipoprotein cholesterol, luteinizing hormone, testosterone, 4-HNE, MDA, total Fe, Fe2+, and ALOX15. In contrast, PER1 was negatively correlated with SREBF2, high-density lipoprotein cholesterol, follicle-stimulating hormone, progesterone, and GSH. CONCLUSION: This study demonstrates that the rhythm gene PER1 promotes ferroptosis and dysfunctional lipid metabolism in granulosa cells in PCOS by inhibiting SREBF2/ALOX15 signaling.

Caffeic acid phenethyl ester analogues as selective inhibitors of 12-lipoxygenase product biosynthesis in human platelets.

The inflammatory response is an essential process for the host defence against pathogens. Lipid mediators are important in coordinating the pro-inflammatory and pro-resolution phases of the inflammatory process. However, unregulated production of these mediators has been associated with chronic inflammatory diseases such as arthritis, asthma, cardiovascular diseases, and several types of cancer. Therefore, it is not surprising that enzymes implicated in the production of these lipid mediators have been targeted for potential therapeutic approaches. Amongst these inflammatory molecules, the 12-hydroxyeicosatetraenoic acid (12(S)-HETE) is abundantly produced in several diseases and is primarily biosynthesized via the platelet's 12-lipoxygenase (12-LO) pathway. To this day, very few compounds selectively inhibit the 12-LO pathway, and most importantly, none are currently used in the clinical settings. In this study, we investigated a series of polyphenol analogues of natural polyphenols that inhibit the 12-LO pathway in human platelets without affecting other normal functions of the cell. Using an ex vivo approach, we found one compound that selectively inhibited the 12-LO pathway, with IC50 values as low as 0.11 µM, with minimal inhibition of other lipoxygenase or cyclooxygenase pathways. More importantly, our data show that none of the compounds tested induced significant off-target effects on either the platelet's activation or its viability. In the continuous search for specific and better inhibitors targeting the regulation of inflammation, we characterized two novel inhibitors of the 12-LO pathway that could be promising for subsequent in vivo studies.

Functional Characterization of Transgenic Mice Overexpressing Human 15-Lipoxygenase-1 (ALOX15) under the Control of the aP2 Promoter.

Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory, hyperproliferative, neurodegenerative, and metabolic diseases, but the physiological function of ALOX15 still remains a matter of discussion. To contribute to this discussion, we created transgenic mice (aP2-ALOX15 mice) expressing human ALOX15 under the control of the aP2 (adipocyte fatty acid binding protein 2) promoter, which directs expression of the transgene to mesenchymal cells. Fluorescence in situ hybridization and whole-genome sequencing indicated transgene insertion into the E1-2 region of chromosome 2. The transgene was highly expressed in adipocytes, bone marrow cells, and peritoneal macrophages, and ex vivo activity assays proved the catalytic activity of the transgenic enzyme. LC-MS/MS-based plasma oxylipidome analyses of the aP2-ALOX15 mice suggested in vivo activity of the transgenic enzyme. The aP2-ALOX15 mice were viable, could reproduce normally, and did not show major phenotypic alterations when compared with wildtype control animals. However, they exhibited gender-specific differences with wildtype controls when their body-weight kinetics were evaluated during adolescence and early adulthood. The aP2-ALOX15 mice characterized here can now be used for gain-of-function studies evaluating the biological role of ALOX15 in adipose tissue and hematopoietic cells.

Angiotensin II-induced vascular endothelial cells ferroptosis via P53-ALOX12 signal axis.

OBJECTIVES: The present study aimed to investigate the effect and mechanism of angiotensin II-induced ferroptosis in vascular endothelial cells. METHODS: In vitro, HUVECs were treated with AngII, AT1/2 R antagonist, P53 inhibitor, or their combinations. MDA and intracellular iron content were evaluated using an ELISA assay. The expression of ALOX12, P53, P21, and SLC7A11 were determined by western blotting in HUVECs and then confirmed through RT-PCR. RESULTS: As the concentration of Ang II (0, 0.1,1,10,100, and 1000uM for 48 h) increased, the level of MDA and intracellular iron content increased in HUVECs. Compared with the single AngII group, ALOX12, p53, MDA, and intracellular iron content in AT1/2R antagonist group decreased significantly. In pifithrin-α hydrobromide-treated, ALOX12, P21,MDA, and intracellular iron content decreased significantly as compared to the single AngII group. Similarly, the effect of combined use of blockers is stronger than that of blockers alone. CONCLUSIONS: AngII can induce ferroptosis of vascular endothelial cells. The mechanism of AngII-induced ferroptosis may be regulated through the signal axis of p53-ALOX12.

S1PR1/S1PR3-YAP signaling and S1P-ALOX15 signaling contribute to an aggressive behavior in obesity-lymphoma.

BACKGROUND: Excess body weight has been found to associate with an increased risk of lymphomas and some metabolic pathways are currently recognized in lymphomagenesis. Bioactive lipid metabolites such as sphingosine-1-phosphate (S1P) have been proposed to play an important role linking obesity and lymphomas. However, the underlying mechanism(s) of S1P signaling in obesity-lymphomagenesis have not been well addressed. METHODS: The gene expression of sphingosine kinase (SPHK), lymphoma prognosis, and S1P production were analyzed using Gene Expression Omnibus (GEO) and human lymphoma tissue array. Obesity-lymphoma mouse models and lymphoma cell lines were used to investigate the S1P/SPHK-YAP axis contributing to obesity-lymphomagenesis. By using the mouse models and a monocyte cell line, S1P-mediated polarization of macrophages in the tumor microenvironment were investigated. RESULTS: In human study, up-regulated S1P/SPHK1 was found in human lymphomas, while obesity negatively impacted progression-free survival and overall survival in lymphoma patients. In animal study, obesity-lymphoma mice showed an aggressive tumor growth pattern. Both in vivo and in vitro data suggested the existence of S1P-YAP axis in lymphoma cells, while the S1P-ALOX15 signaling mediated macrophage polarization towards TAMs exacerbated the lymphomagenesis. In addition, treatment with resveratrol in obesity-lymphoma mice showed profound effects of anti-lymphomagenesis, via down-regulating S1P-YAP axis and modulating polarization of macrophages. CONCLUSION: S1P/S1PR initiated the feedback loops, whereby S1P-S1PR1/S1PR3-YAP signaling mediated lymphomagenesis contributing to tumor aggressive growth, while S1P-ALOX15 signaling mediated TAMs contributing to immunosuppressive microenvironment in obesity-lymphoma. S1P-targeted therapy could be potentially effective and immune-enhancive against obesity-lymphomagenesis.

Daidzein phosphorylates and activates 5-lipoxygenase via the MEK/ERK pathway: a mechanism for inducing the production of 5-lipoxygenase metabolite that inhibit influenza virus intracellular replication.

We previously reported that the soy isoflavone daidzein (Dz) suppresses the intracellular replication of influenza virus and that arachidonic acid-derived oxidation product via lipid oxidase 5-lipoxygenase (5-LOX) is involved in its antiviral effect. The activation of 5-LOX by Dz triggers anti-influenza activity; however, the mechanism of activation of 5-LOX remains unclear. Therefore, in this study, we aimed to clarify the activation mechanism using human monocyte-derived THP-1 cells differentiated using phorbol 12-myristate 13-acetate. THP-1 cells expressed 5-LOX endogenously and Dz did not induce 5-LOX expression. However, 8 h after treatment with Dz, the amount of 5-hydroxyeicosatetraenoic acid (5-HETE), an arachidonic acid oxidation product via 5-LOX, increased significantly suggesting that the enzyme is activated regardless of changes in 5-LOX protein levels. Intracellular Ca2+ content, ATP concentration, 5-LOX protein phosphorylation, and 5-LOX intracellular localization are known 5-LOX activation factors. The intracellular Ca2+ and ATP concentrations were not affected by Dz treatment. The enzymatic activity of 5-LOX is regulated by the phosphorylation of three serine residues and four tyrosine residues. Pretreatment with inhibitors of each kinase revealed that Dz-induced 5-HETE production was suppressed by the MEK/ERK inhibitor. 5-LOX in which the Ser663 residue was phosphorylated was found to be increased in the nuclear fraction of Dz-treated THP-1 cells. Furthermore, immunocytochemistry showed that 5-LOX translocates to the nuclear envelope following Dz treatment. These results indicate that Dz activates 5-LOX by phosphorylating Ser663 via the MEK/ERK pathway. Thus, these results demonstrate that Dz exerts anti-influenza virus activity via the MEK/ERK signal transduction pathway.

Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder.

Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology.

Biology and pharmacology of platelet-type 12-lipoxygenase in platelets, cancer cells, and their crosstalk.

Platelet-type lipoxygenase (pl12-LOX), encoded by ALOX12, catalyzes the production of the lipid mediator 12S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12S-HpETE), which is quickly reduced by cellular peroxidases to form 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12S-HETE). Platelets express high levels of pl12-LOX and generate considerable amounts of 12S-HETE from arachidonic acid (AA; C20:4, n-6). The development of sensitive chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods has allowed the accurate quantification of 12S-HETE in biological samples. Moreover, advances in the knowledge of the mechanism of action of 12S-HETE have been achieved. The orphan G-protein-coupled receptor 31 (GPR31) has been identified as the high-affinity 12S-HETE receptor. Moreover, upon platelet activation, 12S-HETE is produced, and significant amounts are found esterified to membrane phospholipids (PLs), such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC), promoting thrombin generation. Platelets play many roles in cancer metastasis. Among them, the platelets' ability to interact with cancer cells and transfer platelet molecules by the release of extracellular vesicles (EVs) is noteworthy. Recently, it was found that platelets induce epithelial-mesenchymal transition(EMT) in cancer cells, a phenomenon known to confer high-grade malignancy, through the transfer of pl12-LOX contained in platelet-derived EVs. These cancer cells now generate 12-HETE, considered a key modulator of cancer metastasis. Interestingly, 12-HETE was mainly found esterified in plasmalogen phospholipids of cancer cells. This review summarizes the current knowledge on the regulation and functions of pl12-LOX in platelets and cancer cells and their crosstalk.Novel approaches to preventing cancer and metastasis by the pharmacological inhibition of pl12-LOX and the internalization of mEVs are discussed.

MiR-702-5p ameliorates diabetic encephalopathy in db/db mice by regulating 12/15-LOX.

The purpose of this study was to investigate the effect of miR-702-5p on diabetic encephalopathy (DE) and the interaction of miR-702-5p/12/15-LOX in the central nervous system (CNS). In this study, db/db mice were used as DE animal model and HT22 cells were treated with high-glucose (HG). Based on the bioinformatics prediction of possible binding sites between miR-702-5p and 12/15-LOX, we found that the expression of miR-702-5p was significantly down-regulated while 12/15-LOX up-regulated in vivo and in vitro, and the expression changes were inversely correlated. In vivo, diabetic mice with cognitive dysfunction and hippocampal neuronal damage had a concomitant increase in amyloid precursor protein (APP), amyloid beta(Aß), tau, BAX protein expressions; by contrast, Bcl-2 protein expression was significantly decreased. Overexpression of miR-702-5p significantly reduced the histopathological damage of the hippocampus, improved the learning and memory function of db/db mice, down-regulated 12/15-LOX, APP, Aß, tau, BAX protein expressions significantly and up-regulated the expression of Bcl-2. In vitro, miR-702-5p mimic reversed the decline in cell viability and the increase in cell apoptosis induced by HG. Simultaneously, reduced 12/15-LOX, APP, Aß, BAX protein expressions, and increased Bcl-2 protein expression were detected in the miR-702-5p mimic group. Moreover, combined administration of miR-702-5p mimic and 12/15-LOX overexpression lentivirus significantly reversed the protective effect of up-regulation of miR-702-5p. In conclusion, miR-702-5p has a neuroprotective effect on DE, and this effect was achieved by inhibiting 12/15-LOX. However, miR-702-5p had an endogenous regulatory effect on 12/15-LOX rather than a direct targeting relationship.

ALOX12: A Novel Insight in Bevacizumab Response, Immunotherapy Effect, and Prognosis of Colorectal Cancer.

Colorectal cancer is a highly malignant cancer with poor prognosis and mortality rates. As the first biological agent approved for metastatic colorectal cancer (mCRC), bevacizumab was confirmed to exhibit good performance when combined with chemotherapy and immunotherapy. However, the efficacy of both bevacizumab and immunotherapy is highly heterogeneous across CRC patients with different stages. Thus, exploring a novel biomarker to comprehensively assess the prognosis and bevacizumab and immunotherapy response of CRC is of great significance. In our study, weighted gene co-expression network analysis (WGCNA) and the receiver operating characteristic (ROC) curves were employed to identify bevacizumab-related genes. After verification in four public cohorts and our internal cohort, ALOX12 was identified as a key gene related to bevacizumab response. Prognostic analysis and in vitro experiments further demonstrated that ALOX12 was closely associated with the prognosis, tumor proliferation, invasion, and metastasis. Multi-omics data analysis based on mutation and copy number variation (CNV) revealed that RYR3 drove the expression of ALOX12 and the deletion of 17p12 inhibited ALOX12 expression, respectively. Moreover, we interrogated the relationship between ALOX12 and immune cells and checkpoints. The results exhibited that high ALOX12 expression predicted a higher immune infiltration and better immunotherapy response, which was further validated in Tumor Immune Dysfunction and Exclusion (TIDE) and Subclass Mapping (SubMap) methods. Above all, our study provides a stable biomarker for clinical protocol optimization, prognostic assessment, precise treatment, and individualized treatment of CRC.

Survival-related indicators ALOX12B and SPRR1A are associated with DNA damage repair and tumor microenvironment status in HPV 16-negative head and neck squamous cell carcinoma patients.

OBJECTIVES: To investigate prognostic-related gene signature based on DNA damage repair and tumor microenvironment statue in human papillomavirus 16 negative (HPV16-) head and neck squamous cell carcinoma (HNSCC). METHODS: For the RNA-sequence matrix in HPV16- HNSCC in the Cancer Genome Atlas (TCGA) cohort, the DNA damage response (DDR) and tumor microenvironment (TM) status of each patient sample was estimated by using the ssGSEA algorithm. Through bioinformatics analysis in DDR_high/TM_high (n = 311) and DDR_high/TM_low (n = 53) groups, a survival-related gene signature was selected in the TCGA cohort. Two independent external validation cohorts (GSE65858 (n = 210) and GSE41613 (n = 97)) with HPV16- HNSCC patients validated the gene signature. Correlations among the clinical-related hub differentially expressed genes (DEGs) and infiltrated immunocytes were explored with the TIMER2.0 server. Drug screening based on hub DEGs was performed using the CellMiner and GSCALite databases. The loss-of-function studies were used to evaluate the effect of screened survival-related gene on the motility of HPV- HNSCC cells in vitro. RESULTS: A high DDR level (P = 0.025) and low TM score (P = 0.012) were independent risk factors for HPV16- HNSCC. Downregulated expression of ALOX12B or SPRR1A was associated with poor survival rate and advanced cancer stages. The pathway enrichment analysis showed the DDR_high/TM_low samples were enriched in glycosphingolipid biosynthesis-lacto and neolacto series, glutathione metabolism, platinum drug resistance, and ferroptosis pathways, while the DDR_high/TM_low samples were enriched in Th17 cell differentiation, Neutrophil extracellular trap formation, PD - L1 expression and PD - 1 checkpoint pathway in cancer. Notably, the expression of ALOX12B and SPRR1A were negatively correlated with cancer-associated fibroblasts (CAFs) infiltration and CAFs downstream effectors. Sensitivity to specific chemotherapy regimens can be derived from gene expressions. In addition, ALOX12B and SPRR1A expression was associated with the mRNA expression of insulin like growth factor 1 receptor (IGF1R), AKT serine/threonine kinase 1 (AKT1), mammalian target of rapamycin (MTOR), and eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) in HPV negative HNSCC. Down-regulation of ALOX12B promoted HPV- HNSCC cells migration and invasion in vitro. CONCLUSIONS: ALOX12B and SPRR1A served as a gene signature for overall survival in HPV16- HNSCC patients, and correlated with the amount of infiltrated CAFs. The specific drug pattern was determined by the gene signature.

Vagus nerve stimulation promotes resolution of inflammation by a mechanism that involves Alox15 and requires the α7nAChR subunit.

Nonresolving inflammation underlies a range of chronic inflammatory diseases, and therapeutic acceleration of resolution of inflammation may improve outcomes. Neural reflexes regulate the intensity of inflammation (for example, through signals in the vagus nerve), but whether activation of the vagus nerve promotes the resolution of inflammation in vivo has been unknown. To investigate this, mice were subjected to electrical vagus nerve stimulation (VNS) or sham surgery at the cervical level followed by zymosan-induced peritonitis. The duration of inflammation resolution was significantly reduced and efferocytosis was significantly increased in mice treated with VNS as compared with sham. Lipid mediator (LM) metabololipidomics revealed that mice treated with VNS had higher levels of specialized proresolving mediators (SPMs), particularly from the omega-3 docosahexaenoic (DHA) and docosapentaenoic (n-3 DPA) metabolomes, in peritoneal exudates. VNS also shifted the ratio between proinflammatory and proresolving LMs toward a proresolving profile, but this effect by VNS was inverted in mice deficient in 12/15-lipoxgenase (Alox15), a key enzyme in this SPM biosynthesis. The significant VNS-mediated reduction of neutrophil numbers in peritoneal exudates was absent in mice deficient in the cholinergic α7-nicotinic acetylcholine receptor subunit (α7nAChR), an essential component of the inflammatory reflex. Thus, VNS increased local levels of SPM and accelerated resolution of inflammation in zymosan-induced peritonitis by a mechanism that involves Alox15 and requires the α7nAChR.

[Clinical and genetic analysis of a patient with autosomal recessive congenital ichthyosis due to compound heterozygous variants of ALOX12B gene].

OBJECTIVE: To explore the clinical and genetic characteristics of a pediatric patient suspected for Autosomal Recessive Congenital Ichthyosis (ARCI). METHODS: Clinical data of the patient was analyzed. Peripheral blood samples were collected from the patient and his parents for the extraction of genomic DNA. Next-generation sequencing (NGS) was then carried out. Candidate variants were confirmed by Sanger sequencing. A variety of bioinformatic tools including Mutation Taster, PROVEAN, and PolyPhen2 were used to predict the pathogenicity of the variants based on guidelines from the American College of Medical Genetics and Genomics (ACMG). RESULTS: The patient, a 1-month-and-7-day-old male, had presented with cutaneous erythema and fine scaling of the whole body. NGS revealed that he has harbored compound heterozygous variants c.1579G>A (p.Val527Met) (paternal) and c.923T>C (p.Leu308Pro) (maternal) of the ALOX12B gene. The former was known to be likely pathogenic, while the latter was unreported previously and categorized as "likely pathogenic" based on the ACMG guidelines. Based on the clinical and genetic findings, the patient was diagnosed with ARCI. CONCLUSION: The c.1579G>A and c.923T>C variants of the ALOX12B genes probably underlay the ARCI in this patient. Above finding has enriched the spectrum of ALOX12B mutations and enabled molecular diagnosis of the patient, based on which genetic counseling and prenatal diagnosis may be provided.

Senescence-associated tumor growth is promoted by 12-Lipoxygenase.

Radiation therapy is a commonly used treatment modality for cancer. Although effective in providing local tumor control, radiation causes oxidative stress, inflammation, immunomodulatory and mitogenic cytokine production, extracellular matrix production, and premature senescence in lung parenchyma. The senescence associated secretory phenotype (SASP) can promote inflammation and stimulate alterations in the surrounding tissue. Therefore, we hypothesized that radiation-induced senescent parenchymal cells in irradiated lung would enhance tumor growth. Using a murine syngeneic tumor model of melanoma and non-small cell lung cancer lung metastasis, we demonstrate that radiation causes a significant increase in markers of premature senescence in lung parenchyma within 4 to 8 weeks. Further, injection of B16F0 (melanoma) or Lewis Lung carcinoma (epidermoid lung cancer) cells at these time points after radiation results in an increase in the number and size of pulmonary tumor nodules relative to unirradiated mice. Treatment of irradiated mice with a senolytic agent (ABT-737) or agents that prevent senescence (rapamycin, INK-128) was sufficient to reduce radiation-induced lung parenchymal senescence and to mitigate radiation-enhanced tumor growth. These agents abrogated radiation-induced expression of 12-Lipoxygenase (12-LOX), a molecule implicated in several deleterious effects of senescence. Deficiency of 12-LOX prevented radiation-enhanced tumor growth. Together, these data demonstrate the pro-tumorigenic role of radiation-induced senescence, introduces the dual TORC inhibitor INK-128 as an effective agent for prevention of radiation-induced normal tissue senescence, and identifies senescence-associated 12-LOX activity as an important component of the pro-tumorigenic irradiated tissue microenvironment. These studies suggest that combining senotherapeutic agents with radiotherapy may decrease post-therapy tumor growth.

Ferroptosis plays an important role in promoting ionizing radiation-induced intestinal injuries.

The intestinal tract is an essential component of the body's immune system, and is extremely sensitive to exposure of ionizing radiation. While ionizing radiation can effectively induce multiple forms of cell death, whether it can also promote ferroptosis in intestinal cells and the possible interrelationship between ferroptosis and intestinal immune function has not been reported so far. Here, we found that radiation-induced major ultrastructural changes in mitochondria of small intestinal epithelial cells and the changes induced in iron content and MDA levels in the small intestine were consistent with that observed during cellular ferroptosis, thus suggesting occurrence of ferroptosis in radiation-induced intestinal damage. Moreover, radiation caused a substantial increase in the expression of ferroptosis-related factors such as LPCAT3 and ALOX15 mRNA, augmented the levels of immune-related factors INF-γ and TGF-ß mRNA, and decreased the levels of IL-17 mRNA thereby indicating that ionizing radiation induced ferroptosis and impairment of intestinal immune function. Liproxstatin-1 is a ferroptosis inhibitor that was found to ameliorate radiation-induced ferroptosis and promote the recovery from immune imbalances. These findings supported the role of ferroptosis in radiation-induced intestinal immune injury and provide novel strategies for protection against radiation injury through regulation of the ferroptosis pathway.

Natural chalcones elicit formation of specialized pro-resolving mediators and related 15-lipoxygenase products in human macrophages.

Specialized pro-resolving mediators (SPMs) comprise lipid mediators (LMs) produced from polyunsaturated fatty acids (PUFAs) via stereoselective oxygenation particularly involving 12/15-lipoxygenases (LOXs). In contrast to pro-inflammatory LMs such as leukotrienes formed by 5-LOX and prostaglandins formed by cyclooxygenases, the SPMs have anti-inflammatory and inflammation-resolving properties. Although glucocorticoids and non-steroidal anti-inflammatory drugs (NSAIDs) that block prostaglandin production are still prime therapeutics for inflammation-related diseases despite severe side effects, novel concepts focus on SPMs as immunoresolvents for anti-inflammatory pharmacotherapy. Here, we studied the natural chalcone MF-14 and the corresponding dihydrochalcone MF-15 from Melodorum fruticosum, for modulating the biosynthesis of LM including leukotrienes, prostaglandins, SPM and their 12/15-LOX-derived precursors in human monocyte-derived macrophage (MDM) M1- and M2-like phenotypes. In MDM challenged with Staphylococcus aureus-derived exotoxins both compounds (10 µM) significantly suppressed 5-LOX product formation but increased the biosynthesis of 12/15-LOX products, especially in M2-MDM. Intriguingly, in resting M2-MDM, MF-14 and MF-15 strikingly evoked generation of 12/15-LOX products and of SPMs from liberated PUFAs, along with translocation of 15-LOX-1 to membranous compartments. Enhanced 12/15-LOX product formation by the chalcones was evident also when exogenous PUFAs were supplied, excluding increased substrate supply as sole underlying mechanism. Rather, MF-14 and MF-15 stimulate the activity of 15-LOX-1, supported by experiments with HEK293 cells transfected with either 5-LOX, 15-LOX-1 or 15-LOX-2. Together, the natural chalcone MF-14 and the dihydrochalcone MF-15 favorably modulate LM biosynthesis in human macrophages by suppressing pro-inflammatory leukotrienes but stimulating formation of SPMs by differential interference with 5-LOX and 15-LOX-1.

Effect of oridonin on oxylipins in the livers of mice with acute liver injury induced by D-galactosamine and lipopolysaccharide.

BACKGROUND AND PURPOSE: Oridonin (Ori) has been shown to protect against acute liver injury (ALI) induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) and are key proinflammatory mediators. This study aimed to investigate the changes in oxylipins in the livers of mice with D-GalN/LPS-induced ALI and the effects of Ori on these changes. RESULTS: 54 oxylipins in liver tissues were identified and qualitatively and quantitatively analyzed by ultra-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UPLC-QTRAP/MS/MS). The levels of 12-HETE, 12-HEPE, 14(S)-HDHA, PGE2, dihomo-γ-linolenic acid and 13-HOTrE in the liver were significantly increased in the D-GalN/LPS-induced ALI group compared with the control group, and the levels of EPA and 7-HDHA were significantly decreased. However, pretreatment with Ori dramatically decreased the levels of 12-HETE, 12-HEPE, 14(S)-HDHA, PGE2 and 13-HOTrE compared with those of the ALI group and induced 7-HDHA and 15-oxoETE. Moreover, Ori reduced the protein levels of COX-1, COX-2, ALOX5, ALOX12 and ALOX15 induced by D-GalN/LPS, indicating that Ori altered oxylipins through the COX and LOX pathways. CONCLUSIONS: These results suggest that the protective effect of Ori on ALI is partly mediated by affecting the oxylipin pathway.