4-HNE Levels Research Articles

Naringenin attenuates cerebral ischemia/reperfusion injury by inhibiting oxidative stress and inflammatory response via the activation of SIRT1/FOXO1 signaling pathway in vitro.

To explore the protection of naringenin against oxygen-glucose deprivation/reperfusion (OGD/R)-induced HT22 cell injury, a cell model of cerebral ischemia/reperfusion (I/R) injury in vitro, focusing on SIRT1/FOXO1 signaling pathway. Cytotoxicity, apoptosis, reactive oxygen species (ROS) generation, malondialdehyde (MDA) content, 4-hydroxynonenoic acid (4-HNE) level, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activities were measured by commercial kits. Inflammatory cytokines levels were determined by enzyme-linked immunosorbent assay (ELISA). The protein expressions were monitored by Western blot analysis. Naringenin significantly ameliorated OGD/R-induced cytotoxicity and apoptosis in HT22 cells. Meanwhile, naringenin promoted SIRT1 and FOXO1 protein expressions in OGD/R-subjected HT22 cells. In addition, naringenin attenuated OGD/R-induced cytotoxicity, apoptosis, oxidative stress (the increased ROS, MDA and 4-HNE levels, and the decreased SOD, GSH-Px and CAT activities) and inflammatory response (the increased tumor necrosis factor-α, interleukin [IL]-1β, and IL-6 levels and the decreased IL-10 level), which were blocked by the inhibition of the SIRT1/FOXO1 signaling pathway induced by SIRT1-siRNA transfection. Naringenin protected HT22 cells against OGD/R injury depending on its antioxidant and anti-inflammatory activities via promoting the SIRT1/FOXO1 signaling pathway.

Expression profile of interleukin-6, 4-hydroxy-2-nonenal, and hypoxia-inducible factor 1-α in women with breast cancer and their association with clinicopathological parameters.

Breast cancer (BC) is the most common malignancy in women worldwide, representing a major public health burden. Therefore, there is a need to identify circulating biomarkers for the early detection of BC and to facilitate the diagnosis. Interleukin-6 (IL-6), 4-hydroxy-2-nonenal (4-HNE), and hypoxia-inducible factor 1-α (HIF-1α) are biomarkers involved in the initiation and progression of this disease. The aim of this study was to evaluate the concentrations of these molecules and to find a possible correlation with the clinicopathological parameters of patients with BC from western Algeria. We evaluated the serum levels of IL-6, 4-HNE, and HIF-1α by ELISA technique and compare them in different age groups and BC molecular subtypes, and then correlated them with clinicopathological parameters. Our study revealed a significant increase in 4-HNE (p < 0.05), and a negative correlation (p < 0.05) was found between IL-6 serum levels and lymph node count, but not (p > 0.05) between 4-HNE, HIF-1α and lymph node count. No significant correlation (p > 0.05) was found between IL-6, 4-HNE, HIF-1α , and Scarff-Bloom-Richardson grade. Furthermore, there was no significant difference (p > 0.05) in serum levels of IL-6, 4-HNE, and HIF-1α in the different age groups and molecular subtypes of BC. The data obtained show that the presence of lipid peroxidation (4-HNE) is a marker of oxidative stress, and that IL-6 is a good prognostic factor due to its negative effect on the number of lymph nodes. Furthermore, age and BC molecular subtypes do not influence the serum concentrations of IL-6,4-HNE, and HIF-1α.

δ-Opioid Receptor Activation Inhibits Ferroptosis by Activating the Nrf2 Pathway in MPTP-Induced Parkinson Disease Models.

Recent studies suggest the involvement of ferroptosis in the pathogenesis of Parkinson disease (PD). δ-Opioid receptors (DORs) have neuroprotective effects in PD. It is not known whether the neuroprotective effects of DORs in PD are attributable to the inhibition of ferroptosis. Therefore, we aimed to investigate the role of DORs in ferroptosis in MPTP-induced PD models. To identify the influence of DORs on ferroptosis in MPTP-induced PD models, we measured the malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels, analyzed the levels of ferroptosis-related proteins (GXP4 and SLC7a11) and Nrf2 expression by using western blotting, and assessed mitochondrial dysfunction by using JC-1 staining and transmission electron microscopy. DOR activation reduced the 4-HNE and MDA levels, increased the GXP4 and SLC7a11 levels, and ameliorated mitochondrial dysfunction in MPTP-induced PD models. These neuroprotective effects of DORs could be blocked by Nrf2-siRNA. Thus, the effects of DORs on ferroptosis in PD models were partially controlled by Nrf2, which regulated GXP4 and SLC7a11 synthesis. DORs exert neuroprotective effects in PD models by inhibiting ferroptosis partially via activating the Nrf2 pathway.

Stroke by inducing HDAC9-dependent deacetylation of HIF-1 and Sp1, promotes TfR1 transcription and GPX4 reduction, thus determining ferroptotic neuronal death.

Background: The inhibition of histone deacetylase 9 (HDAC9) represents a promising druggable target for stroke intervention. Indeed, HDAC9 is overexpressed in neurons after brain ischemia where exerts a neurodetrimental role. However, mechanisms of HDAC9-dependent neuronal cell death are not yet well established. Methods: Brain ischemia was obtained in vitro by primary cortical neurons exposed to glucose deprivation plus reoxygenation (OGD/Rx) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcript and protein levels. Chromatin immunoprecipitation was used to evaluate the binding of transcription factors to the promoter of target genes. Cell viability was measured by MTT and LDH assays. Ferroptosis was evaluated by iron overload and 4-hydroxynonenal (4-HNE) release. Results: Our results showed that HDAC9 binds to hypoxia-inducible factor 1 (HIF-1) and specificity protein 1 (Sp1), two transcription activators of transferrin 1 receptor (TfR1) and glutathione peroxidase 4 (GPX4) genes, respectively, in neuronal cells exposed to OGD/Rx. Consequently, HDAC9 induced: (1) an increase in protein level of HIF-1 by deacetylation and deubiquitination, thus promoting the transcription of the pro-ferroptotic TfR1 gene; and (2) a reduction in Sp1 protein levels by deacetylation and ubiquitination, thus resulting in a down-regulation of the anti-ferroptotic GPX4 gene. Supporting these results, the silencing of HDAC9 partially prevented either HIF-1 increase and Sp1 reduction after OGD/Rx. Interestingly, silencing of the neurodetrimental factors, HDAC9, HIF-1, or TfR1 or the overexpression of the prosurvival factors Sp1 or GPX4 significantly reduced a well-known marker of ferroptosis 4-HNE after OGD/Rx. More important, in vivo, intracerebroventricular injection of siHDAC9 reduced 4-HNE levels after stroke by preventing: (1) HIF-1 and TfR1 increase and thus the augmented intracellular iron overload; and (2) a reduction of Sp1 and its target gene GPX4. Conclusions: Collectively, results obtained suggest that HDAC9 mediates post-traslational modifications of HIF-1 and Sp1 that, in turn, increases TfR1 and decreases GPX4 expression, thus promoting neuronal ferroptosis in in vitro and in vivo models of stroke.

Formononetin ameliorates ferroptosis-associated fibrosis in renal tubular epithelial cells and in mice with chronic kidney disease by suppressing the Smad3/ATF3/SLC7A11 signaling

AimsChronic kidney disease (CKD) is characterized by interstitial fibrosis, while limited treatment drugs are available. Ferroptosis is a newly identified process that can trigger tubular atrophy and fibrosis. The aim of this study is to investigate the effects of formononetin (FN), a bioflavonoid, on ferroptosis and renal fibrosis. Main methodsIn vivo experiments, unilateral ureteral obstruction (UUO)- and folic acid (FA, 250 mg/kg)-induced CKD models were constructed in C57BL/6 mice of 6–8 weeks old, followed by the administration with 40 mg/kg/day FN by gavage. For in vitro experiments, ferroptosis was induced with RSL3 or erastin in primary mouse renal tubular epithelial cells (TECs), followed by the addition of indicated concentrations of FN. Then, the levels of ferroptosis and fibrosis were analyzed. The translocation of Smad3, ATF3, and Nrf2 from the cytoplasm to the nucleus was checked by western blotting. The interaction of Smad3 and ATF3 was detected by Co-immunoprecipitation. Key findingsFN dramatically ameliorated tubular injury along with reduced expression of the profibrotic genes including α-SMA, Col1a1, and fibronectin in both two CKD mouse models and RSL3/erastin-treated TECs. Furthermore, FN administration also significantly suppressed ferroptosis, as evidenced by increased expression of SLC7A11 and GPX4, and decreased levels of 4-HNE. In mechanism, FN disrupted the interaction between Smad3 and ATF3, resulting in the blocking of their translocation from the cytoplasm to the nucleus. In addition, FN also promoted the separation of the Nrf2/Keap1 complex and enhanced Nrf2 nuclear accumulation. SignificanceFN alleviates CKD by impeding ferroptosis-associated fibrosis by suppressing the Smad3/ATF3/SLC7A11 signaling and could serve as a candidate therapeutic drug for CKD.

The receptor for advanced glycation end products (RAGE) is involved in mitochondrial function and cigarette smoke-induced oxidative stress

The mechanisms underlying muscle dysfunction with Chronic Obstructive Pulmonary Disease (COPD) are poorly understood. Indirect evidence has recently suggested a role of Advanced Glycation End Products (AGEs) and their receptor (RAGE) in the pathophysiology of COPD. Accordingly, this study aimed to examine the redox balance and mitochondrial alterations in the skeletal muscle of a mouse model deficient in the receptor for AGE (RAGE-KO) and wild-type C57BL/6 exposed to cigarette smoke for 8-months using immunoblotting, spectrophotometry, and high-resolution respirometry. Cigarette smoke exposure increased by two-fold 4-HNE levels (P < 0.001), a marker of oxidative stress, and markedly downregulated contractile proteins, mitochondrial respiratory complexes, and uncoupling proteins levels (P < 0.001). Functional alterations with cigarette smoke exposure included a greater reliance on complex-I supported respiration (P < 0.01) and lower relative respiratory capacity for fatty acid (P < 0.05). RAGE knockout resulted in 47% lower 4-HNE protein levels than the corresponding WT control mice exposed to cigarette smoke (P < 0.05), which was partly attributed to increased Complex III protein levels. Independent of cigarette smoke exposure, RAGE KO decreased mitochondrial specific maximal respiration (P < 0.05), resulting in a compensatory increase in mitochondrial content measured by citrate synthase activity (P < 0.001) such that muscle respiratory capacity remained unaltered. Together, these findings suggest that knockout of RAGE protected the skeletal muscle against oxidative damage induced by 8 months of cigarette smoke exposure. In addition, this study supports a role for RAGE in regulating mitochondrial content and function and can thus serve as a potential therapeutic target.

Integration of metabolomics and transcriptomics to reveal ferroptosis is involved in Tripterygium wilfordii polyglycoside tablet-induced testicular injury

Ethnopharmacological relevanceTripterygium wilfordii polyglycoside tablet (TWP), a traditional Chinese medicine preparation, has multiple pharmacological properties, including anti-inflammatory, immune-modulatory and anti-proliferative activities. However, the reproductive toxicity of TWP greatly limits its clinical application and the mechanism of TWP-induced reproductive toxicity is not fully understood yet. Aim of the studyThis study was designed to explore the mechanism of TWP-induced testis injury in male rats. Materials and methodsThe mechanism underlying TWP-induced rat testicular injury was firstly investigated by integration of metabolomics and transcriptomics. Meanwhile, histopathological analysis, Western blot and RT-qPCR were performed to confirm the damaging effects and mechanisms of TWP on rat testis. ResultsHistopathological analysis revealed that TWP had significant testicular damage, which severely reduced the testis's tubular diameter and epithelium height. Further, TWP caused the protein level of ZO-1, CLDN11, PLZF, and OCT4 significantly downregulate, suggesting the blood-testis barrier function and spermatogenesis were damaged. Differentially expressed genes (DEGs), including 4952 upregulated and 2626 downregulated, were found in TWP-exposed testis compared to the normal group. Moreover, 77 changed metabolites were identified from testis samples. With integrated analysis of DEGs and changed metabolites, we found that glutathione metabolism and ferroptosis played an essential role in testicular injury. Additionally, the levels of ferroptosis-related protein GPX4, SLC7A11, and NRF2 were significantly downregulated, and the protein level of 4-HNE, a leading product of lipid peroxidation and oxidative stress, was upregulated. The changes in ferroptosis-related genes indicated that TWP might promote ferroptosis in rat testis. ConclusionThese results suggested that ferroptosis was involved in the testicular damage caused by TWP, which might provide a new strategy to alleviate TWP- induced testicular injury.

A Role of Immunothrombosis in Acquired Antibody-Mediated Thrombophilia's: Classical Complement and Neutrophil Activation in APS and HIT

Introduction: Heparin induced thrombocytopenia (HIT) and APS (antiphospholipid syndrome) are acquired thrombophilia's characterized by both arterial and venous thromboembolic events (TE). As a common feature APS and HIT share the presence of pathological antibodies, which play a crucial role in disease pathogenesis. In HIT, antibodies are directed to platelet factor (PF) 4 and heparin, respectively, and in APS, antibodies target anti-phospholipids (anti-cardiolipin antibody, anti-Beta2- glycoprotein I and lupus anticoagulant). There is growing evidence that complement- and neutrophil activation in the form of neutrophil extracellular traps (NETs) play an important role in the pathogenesis of thrombosis, the so-called "immunothrombosis". Insufficient control with subsequent unrestrained complement activation results in thrombosis as seen in patients with paroxysmal nocturnal hemoglobinuria (PNH). Therapeutic inhibition of complement activation abrogates thrombosis in these patients, emphasizing the important role of complement activation in the pathogenesis of thrombosis. It has been demonstrated, that complement activation products efficiently induce neutrophil activation, and therapeutic complement inhibition in sepsis and PNH prevents neutrophil activation. Here, we investigate the role of complement activation and neutrophil activation in patients with APS and HIT, respectively. Methods To assess the association of complement activation and NET Formation, we measured complement activation products (C4bc, C3bc) and marks for neutrophil activation, such as nucleosomes and human neutrophil elastase-α1- antitrypsin complexes (HNE) by ELISA in plasma of 34 primary APS- and 26 HIT- patients and 31 healthy controls. Groups have been compared using the Mann- Whitney-Rank Test. Values are indicated as median and range, p<0.05 is considered statistically significant. Results Patients with APS and HIT showed sign. higher levels of complement activation products as compared to healthy controls. Complement activation mainly of the classical (C4b/c) and alternate pathway (C3b/c) in APS (11 nM and 263 nM) and in HIT (33 nM and 324 nM) were sign. higher (p<0.0001) as compared to the healthy controls (7 nM and 19 nM). C4bc levels were sign. higher in HIT as compared to APS patients (p<0001), but there was no difference in C3b/c levels between these two groups (see figure 1). Patients with HIT had a sig. (p<0.0001) higher nucleosomes (440 U/ml) and HNE (121 ng/ml) as compared to healthy controls (12 U/ml and 26 ng/ml). HNE levels were sign. (p<0.001) higher in APS patients (37 ng/ml) as compared to healthy controls (26 ng/ml), however no difference was observed in nucleosome levels (12 U/ml vs 14 U/ml) (see figure 2). Based on the number of positive antibodies, eleven APS patients have been stratified as high-risk and 24 APS patients as low- to intermediate risk. C4bc levels were sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in C3b/c levels could be observed between the 2 groups. Similarly, HNE complexes sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in nucleosome levels could be observed between the 2 groups. Six HIT patients (22%) had a diagnosed TE event whereas 17 patients (63%) had no diagnosed TE. In four patients (15%) no clinical data were available, and they have been excluded from subgroup analysis. Interestingly, the measured markers for complement - and neutrophil activation in HIT patient with and without thrombosis were not different. Conclusions We demonstrate complement activation in patients with HIT and APS. Based on the simultaneous raise in nucleosomes and HNE, there is evidence of NET formation in HIT, whereas only weak neutrophil activation can be found in APS. In APS classical complement activation and neutrophil activation correlates with APS risk stratification. Our results demonstrate classical complement pathway- and neutrophil activation in HIT and APS patients. These findings may form the base for new therapeutic strategies targeting complement and neutrophil activation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Serum 4-hydroxynonenal associates with the recurrence of patients with primary cerebral infarction.

4-Hydroxynonenal (4-HNE), an α, β-unsaturated hydroxyalkenal, has been found to be associated with aspirin resistance, which is a risk factor for recurrent cerebral infarction. However, its effect on recurrent cerebral infarction is less defined. We designed this study to investigate the association between 4-HNE and increased risk of recurrent cerebral infarction. We recruited 189 patients with primary cerebral infarction from 2017 to 2019. According to the recurrence of cerebral infarction during the 3-year follow-up period, they were divided into two groups, namely, the non-recurrence group (n = 93) and the recurrence group (n = 96). All patients were analyzed to explore the risk factors for the recurrence of primary cerebral infarction and the predictive value of serum 4-HNE for the recurrence of cerebral infarction. The levels of serum 4-HNE in patients of the recurrence group were significantly higher than that in patients of the non-recurrence group. There was a positive correlation between serum 4-HNE levels and the serum levels of triglyceride (r = 0.448, p = 0.008) and low-density lipoprotein cholesterol (LDL-C; r = 0.442, p = 0.002) in primary cerebral infarction patients. Cox proportional hazards modeling showed that demographic and certain clinical parameters, such as age, serum triglyceride levels, the National Institutes of Health Stroke Scale (NIHSS) scores, and serum 4-HNE levels, were independent factors for the recurrence in patients. The results of the receiver operating characteristic (ROC) curve showed that the area under the curve (AUC) value of serum 4-HNE in patients with cerebral infarction recurrence was 0.703, and when the cutoff value of serum 4-HNE was set at 42.34 ng/ml, the sensitivity and specificity values of serum 4-HNE in predicting recurrent cerebral infarction were 79.20 and 52.70%, respectively. Serum 4-HNE is an independent risk factor for the recurrence of patients with primary cerebral infarction, and it may become a new intervention way to prevent the recurrence of patients with cerebral infarction.

A noncanonical function of EIF4E limits ALDH1B1 activity and increases susceptibility to ferroptosis

Ferroptosis is a type of lipid peroxidation-dependent cell death that is emerging as a therapeutic target for cancer. However, the mechanisms of ferroptosis during the generation and detoxification of lipid peroxidation products remain rather poorly defined. Here, we report an unexpected role for the eukaryotic translation initiation factor EIF4E as a determinant of ferroptotic sensitivity by controlling lipid peroxidation. A drug screening identified 4EGI-1 and 4E1RCat (previously known as EIF4E-EIF4G1 interaction inhibitors) as powerful inhibitors of ferroptosis. Genetic and functional studies showed that EIF4E (but not EIF4G1) promotes ferroptosis in a translation-independent manner. Using mass spectrometry and subsequent protein-protein interaction analysis, we identified EIF4E as an endogenous repressor of ALDH1B1 in mitochondria. ALDH1B1 belongs to the family of aldehyde dehydrogenases and may metabolize the aldehyde substrate 4-hydroxynonenal (4HNE) at high concentrations. Supraphysiological levels of 4HNE triggered ferroptosis, while low concentrations of 4HNE increased the cell susceptibility to classical ferroptosis inducers by activating the NOX1 pathway. Accordingly, EIF4E-dependent ALDH1B1 inhibition enhanced the anticancer activity of ferroptosis inducers in vitro and in vivo. Our results support a key function of EIF4E in orchestrating lipid peroxidation to ignite ferroptosis.

Leuconostoc mesenteroides utilizes glucose fermentation to produce electricity and ameliorates high-fat diet-induced abdominal fat mass.

Bacteria capable of producing electricity in intestinal microbiota have been discovered. However, no studies have explored butyric acid which generated by electrogenic bacteria on the host organism have significant physiological impacts on certain organs. We found that the capacity for electrical current generation by the commensal gut Leuconostoc mesenteroides EH-1 (L. mesenteroides EH-1) during glucose fermentation. The electricity production was essential for the gut colonization of L. mesenteroides EH-1 since the inhibition of electricity production by cyclophilin A inhibitor (TMN355) significantly diminished the number of bacteria attached to the human gut epithelial cell surface. The adipocyte differentiation contributes to the increased 4-hydroxy-2-nonenal (4-HNE), considered as a biomarker of reactive oxygen species (ROS). The effect of intestinal electrogenic microbiota in the high-fat diet (HFD)-induced 4-HNE and abdominal fat accumulation in mice was investigated in this study. The oral administration of glucose with a butyric acid-producing L. mesenteroides EH-1 bacterium attenuated the expression of 4-HNE and abdominal fat. The level of 4-HNE and abdominal fat depot were markedly increased in mice administered with cyclophilin A inhibitor-pretreated bacteria or GLPG-0974, an antagonist of free fatty acid receptor 2 (Ffar2). Our studies suggest a novel means by which the probiotic bacteria can modulate fat mass deposition and oxidative stress via the cyclophilin A-mediated electron production and the butyric acid-activated Ffar2 pathway.

Aqueous humor and serum levels of 4-hydroxynonenal and 8-hydroxy-2'deoxyguanosine in pseudoexfoliation syndrome and glaucoma.

To compare the aqueous humor (AH) and serum levels of 4-hydroxynenal (4-HNE) and 8-hydroxy-2'-deoxyguanosine (8-OhdG) in patients with pseudoexfoliation syndrome (PES) and pseudoexfoliation glaucoma (PEG) with each other and with age- and sex-matched control group. Thisprospective study included 66patientsdivided into three groups:PES (n = 24), PEG (n = 21), and acontrol group (n = 21). 4-HNEand 8-OhdG levelswere analyzed using theenzyme-linked immunosorbent assay. Aqueous and serum 4-HNE levels were significantly higher inthe PEG(466.52 ± 62.12pg/mL and313.47 ± 47.41pg/mL) and PES (290.69 ± 63.63pg/mL and201.53 ± 39.57pg/mL) groups than the control group (144.02 ± 39.58pg/mL and99.10 ± 16.96pg/mL;p < 0.001, for all). Both aqueous and serum levels of 4-HNE in the PEG group were significantly higher than in the PES group (p < 0.001, for both). Similar to 4-HNE, the AH 8-OhdG levelswere higher in the PEG group (21.18 ± 2.23ng/mL) compared to the PES (14.90 ± 3.37ng/mL) and control(4.86 ± 1.94ng/mL) groups(p < 0.001, for all).Serum 8-OhdG levels were significantly higher in the PEG and PES groups than the control (p < 0.001, for both); however,there was no significant difference between the PES andPEG groups (p = 0.097). There were strong significant correlations between the aqueous and serum levels of 4-HNE (p < 0.001, r = 0.857) and 8-OhdG (p < 0.001, r = 0.807) amongall the patients. Aqueous humor and serum levels of 4-HNE and 8-OhdG increased in the PES and PEG patients. These findings are potentially significant and add to the growing body of evidence concerning oxidative stress in PES and PEG.

Overexpression of miR-210 ameliorates endothelial dysfunction in type 2 diabetic mice

Abstract Background MicroRNA (miR)-210 plays a protective role in many cardiometabolic diseases, and its levels are reduced in whole blood, erythrocytes, and plasma in type 2 diabetes mellitus (T2DM). Our recent study demonstrated that miR-210 is downregulated in carotid artery plaques from patients with T2DM compared to non-diabetic patients. However, the functional role of miR-210 in endothelial dysfunction is not fully understood. Purpose This study aimed to investigate the potential therapeutic value of miR-210 overexpression against T2DM-associated endothelial dysfunction. Methods miR-210 transgenic mice (TG) at age of 8 weeks were subjected to Western Diet (WD) for 12 weeks. During the last 10 days of the diet regime, doxycycline or vehicle dissolved in drinking water was given to switch on miR-210 (miR-210/on) expression or keep it off (miR-210/off), respectively. Age-matched wild-type (WT) control mice received normal chow through the same period. Additionally, WT and T2DM db/db mice at age of 15–20 weeks received tail vein injections of miR-210 mimic or miR-210 scramble oligonucleotides. All animals were euthanized at the end of the treatment or 72h after the i.v. injections for determination of endothelial function by acetylcholine-induced endothelium-dependent relaxation (EDR) of isolated aortic segments from all groups of mice using wire myographs. The expression of protein tyrosine phosphatase 1B (PTP1B; a miR-210 target protein) and the levels of 4-hydroxynonenal (4-HNE; an oxidative stress marker) were measured in aortic segments by immunohistochemistry. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). Results EDR in aortic segments from miR-210/off TG mice fed WD and T2DM db/db mice injected with miR-210 scramble was significantly impaired compared to vessels from WT controls fed with regular chow (Fig. 1A, B). Of note, EDR was markedly improved or even restored in aortae from miR-210/on TG mice treated with WD and T2DM db/db mice injected with miR-210 mimic (Fig. 1A, B). Furthermore, the expression of PTP1B and the levels of 4-HNE, which is formed by lipid peroxidation, were significantly elevated in the aortae from miR-210/off TG mice treated with WD when compared to WT controls (Fig. 2A–D). The expression was attenuated in aortae of miR-210/on TG mice treated with WD compared to miR-210/off TG mice (Fig. 2A–D). There was a significant increase in the expression of PTP1B and a trend to increased 4-HNE levels in aortae of db/db mice injected with miR-210 scramble vs. control mice (Fig. 2E–H). A significant reduction in PTP1B but not 4-HNE was observed in db/db mice injected with miR-210 mimic (Fig. 2E–H). Conclusion Genetic and pharmacological overexpression of miR-210 ameliorates endothelial dysfunction in mice fed with WD and db/db mice. Increasing miR-210 levels may become a potential treatment strategy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation

The relation among myocardial iron load and the severity of heart failure

Abstract Heart failure (HF) symptoms are related to reduced circulatory perfusion, peripheral congestion, also impaired peripheral muscle strength &amp; oxygen utilization due to iron deficiency. Despite many premises, the correlation between myocardial iron load and failing heart function was not proved. The purpose of this study was to assess iron itself and iron-related proteins in human failing (FH) vs non-failing (NFH) myocardium. The study group consisted of 58 explanted FHs vs the control consisted of 31 NFHs unsuitable for transplantation. HF study group presented with LV dilatation (LVDD 67±8 mm; LVSD 59±13 mm), reduced contraction (LVEF 20±7.5%), RV enlargement (RVD 42±6.5 mm), pulmonary hypertension (2.21±0.93 Wu) and elevated NTproBNP (3955.5±2250.5pg/ml). We assessed iron serum markers: ferritin (FR), transferrin/saturation (TSAT), transferrin receptor (soluble sTfR), sTFR/log FR; myocardial iron homeostasis assessment: M-Iron (Instrumental Neutron Activation Analysis, μg/g) and iron gathering proteins (by ELISA): divalent metal transporter (DMT-1); L-type calcium channel (L-CH), TfR-1/TfR-2; ferritins: heavy (FT-H), light (FT-L) chain, mitochondrial (FT-MT); ferroportin (FPN) and oxidative stress marker: 4-hydroxynonenal (4-HNE). In FH vs NFH, M-Iron was reduced (164.6±25.2 vs 182.3±23.9; p=0.0358) however, did not apply to the entire group. Therefore we divvied the FH group into two subgroups (based on M-Iron concentration in NFH controls). Average iron concertation in non-iron-deficient (NIDM) subgroup was 172.5±19.1 (n=35) and in iron-deficient (IDM) subgroup wad 127.0±16.5; (n=23, p=0.0001). Regardless M-Iron load in FH vs NFH myocardial proteins expressions were reduced: L-CH (NIDM vs IDM vs NFH: 49.2±9.2; 42.5±4.2; 67.3±23.0, p=0.0022); FT-L (16.4±7.3; 11.6±4.8; 26.3±7.5, p= &amp;lt;0.0001, respectively); FT-MT (41.2±7.6; 38.7±4.1; 59.3±10.4 p≤0,0001, respectively). With regard to M-Iron load, only in DMT-1 expression was changed (NIDM vs IDM vs NFH 4.8±0.7; 4.0±0.5; 5.8±0.8; p≤0.0001). 4-HNE levels in FH vs NFH were increased, regardless M-Iron load (NIDM-Iron vs IDM-Iron vs NFH; 55.7±29.2; 57.2±47.8; 9.9±8.1 p≤0.0001). Interestingly, the iron load (NIDM-Iron vs IDM-Iron) did not modify the assessed clinical parameters. With regard to commonly used systemic iron markers only sTFR/log FR proved M-Iron load difference (NIDM-Iron vs IDM-Iron 0.6±0.2; 0.7±0.3; p=0.047910) In FH, we observed, the subgroup with the reduced M-Iron load. The observed reduction of iron gathering proteins were independent on M-Iron load, except DMT-1 the main iron transportation protein into the cell. The results showed that the HF degree and concertation of iron proteins are unlikely to be affected by the M-Iron load, except DMT-1 protein. The oxidative stress was also regardless from M-Iron load, and increased in the whole FH group. Commonly used systemic iron markers except sTFR/log FR did not define M-Iron load. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): The Cardinal Stefan Wyszyński National Institute of Cardiology

AD-9308 ameliorates the impacts of 4-HNE on the progress of pulmonary arterial hypertension in aldehyde dehydrogenase 2*1*2 knock-in mice

Abstract Endothelial dysfunctions play a critical role on the development of pulmonary arterial hypertension (PAH). It has been reported that the one-year mortality rate is still up to 15% even with PAH-targeted therapy, implying that there may be untargeted pathways. 4-hydroxynenonal (4-HNE), an unsaturated aldehyde, is highly induced in the lungs of PAH animals and its serum levels were also reported to be higher in PAH patients. 4-HNE is metabolized by mitochondrial aldehyde dehydrogenase (ALDH2), which is dysfunctional in near 40% of East Asian people. Currently, the impacts of 4-HNE on endothelial dysfunctions in the development of PAH are unclear. In terms of translational medicine, we proposed that modulation of 4-HNE level may alleviate the progress of PAH patients with ALDH2 deficiency. We found that 4-HNE alone was not sufficient to induce pulmonary artery endothelial cell (PAEC) functional changes, including proliferation, migration and tube formation, whereas their effects emerge from the depletion of ALDH2. We further mimicked human ALDH2 functional deficiency by using daidzin (DZN), an inhibitor which is able to block the substrate binding site of ALDH2. ALDH2 functional inhibition alone did not induce any PAEC functional change, while an add-on of 4-HNE impaired PAEC functions. In addition, 4-HNE significantly reduced eNOS activity with combined DZN treatment. Consistent with the mechanism of ALDH2 activity-mediated angiogensis, ALDH2 enhancers Alda-1 and AD-5591 completely reverse the anti-angiogenic effects of 4-HNE in the presence of DZN. To further confirm whether ALDH2 functional deficiency impact on PAH development in mammals, heterozygous ALDH2*1/*2 transgenic and wild-type mice were subjected to chronic hypoxia to induce PAH. ALDH2*1/*2 transgenic mice had similar right ventricular systolic pressure (RVSP) as wild-type mice. However, after exposure to chronic hypoxia, ALDH2*1/*2 transgenic mice indeed developed a significantly higher RVSP than that in wild-type mice. Furthermore, we demonstrated that 4-HNE expression was profoundly enhanced in ALDH2*1/*2 transgenic mice by chronic hypoxia-induced PAH with pulmonary artery smooth muscle cell hyperplasia. More importantly, we found that AD-9308, an enhancer of ALDH2 significantly decreased hypoxia-induced RVSP elevation in heterozygous ALDH2*1/*2 transgenic mice. Taken together, our data demonstrate that 4-HNE and ALDH2 functional deficiency potentially contribute to PAH development and worsening, and that ALDH2 enhancers may be promising as a PAH adjunct therapy, particularly for patients with ALDH2 nonfunctional alleles. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science and Technology, Taiwan

Quercetin and lycopene co-administration prevents oxidative damage induced by d-galactose in mice

Quercetin and lycopene are strong dietary antioxidants that often co-exist in foods. Here, the cardiac and neuroprotective effects of quercetin and lycopene combination were analyzed in d-galactose-induced oxidative stress in mice. ICR mice were divided into control group, d-galactose group (D-GAL), quercetin treatment (Q45: 45 mg kg−1 d−1quercetin, Q60: 60 mg kg−1 d−1 quercetin), lycopene treatment (15 mg kg−1 d−1 lycopene), and M groups (45 mg kg−1 d−1quercetin+15 mg kg−1 d−1 lycopene). Mice were given quercetin, lycopene, and their combination through oral gavage for 6 weeks. They were injected with d-galactose (150 mg kg−1 d−1) simultaneously except for the control group. Results showed that the quercetin-lycopene combination could ameliorate histopathological injuries in the heart and hippocampus. They significantly decreased the serum and heart malonaldehyde (MDA) levels, heart 4-Hydroxynonenal (4-HNE) levels, and increased the activity of serum and heart superoxide dismutase (SOD), catalase (CAT), and the hippocampal SOD and CAT mRNA level. Quercetin-lycopene combination exhibited anti-inflammation effects by reducing inflammatory genes such as cyclooxygenase-2 (COX-2) and interleukin-1β (IL-1β). The heart and hippocampal mRNA level of nuclear factor erythroid 2-related factor 2 (Nrf2) expression was up-regulated, and the antioxidant genes related to Nrf2 including heme oxygenase-1 (HO-1), NAD(P)H Quinone Dehydrogenase 1 (NQO1) were elevated. Quercetin-lycopene combination significantly promoted the mRNA level of deacetylase sirtuin-1 (SIRT1), increased 3.3-fold and 3-fold SIRT1 expression in the heart and hippocampus, respectively. They could bind SIRT1 at the active site predicted by molecular docking. These results suggested that they may interact with SIRT1 to activate Nrf2, inhibit pro-inflammatory factors, and prevent oxidative stress in D-GAL-induced mice.

Determine the effect of the lipid peroxidation end product (4-Hydroxynonenal) in the context of atherogenic among COVID-19 cases

Background: Coronavirus disease 2019 (COVID-19) manifests as multiple clinical and pathological organ dysfunctions. It also disrupts metabolic profile due to the release of pro-inflammatory cytokines causing a systemic inflammation reaction. As the number of COVID-19 complications continues to rise worldwide, the identification of risk factors for the disease is an urgent issue, and it remains controversial whether atherogenic lipid-related traits including lipid peroxidation product 4-Hydroxynonenal and lipid profile panel levels, are risk factors. The aim of this study was to estimate causal effects of lipid-related factors on COVID-19 risk Methods: This case control study includes (90) subjects, were divided into: (30) critical, (30) sever COVID-19 groups, and (30) control group. Samples of COVID-19 patients were collected from Al-Hayat unit at Imam Hussein Medical City/ Iraq. This study was included all patients that confirmed with positive RT-PCR in the hospital, and only smoked patients were excluded….Results: The results of this study found significant increase in HNE levels in sever and critical groups compared to control group.

SIRT1-mediated p53 deacetylation inhibits ferroptosis and alleviates heat stress-induced lung epithelial cells injury

Objective Acute lung injury (ALI) is a common complication of heat stroke (HS) and a direct cause of death. However, the mechanism underlying ALI following HS remains unclear. Method To investigate whether ferroptosis is involved in HS-ALI. We established a HS model of mice and mouse lung epithelial-2 cells (MLE-2). The severity of lung injury was measured by H&E staining, the wet-to-dry lung weight ratio, and Transmission electron microscopy. Potential markers of ferroptosis Fe2+, malondialdehyde (MDA), hydroxynonenal (4-HNE) and lipid peroxidation were detected. The percentages of cell death and viability induced by HS were assessed by LDH and CCK8 assays. SLC7A11, ACSL4, GPX4, SIRT1, p53, and p53 K382 acetylation levels were measured by Western blot. Results The administration of ferroptosis inhibitor ferrostatin-1(Fer-1) could significantly ameliorate lung injury, inhibiting levels of MDA and 4-HNE, and ameliorating HS-induced increased ACSL4, decreased SLC7A11 and GPX4, suggesting ferroptosis was involved in HS-induced ALI in vivo and in vitro. Moreover, SIRT1 expression decreased, and p53 K382 acetylation levels increased in MLE-2 cells. Activation of SIRT1 could improve lung epithelial ferroptosis caused by HS in vivo ang in vitro. Besides, the activation of SIRT1 could significantly reduce the p53 K382 acetylation levels, suggesting that activation of SIRT1 could prevent ferroptosis via inhibiting p53 acetylation. Conclusion These findings substantiate the vital role of the SIRT1/p53 axis in mediating ferroptosis in HS-ALI, suggesting that targeting SIRT1 may represent a novel therapeutic strategy to ameliorate ALI during HS.

Puerarin Attenuates Oxidative Stress and Ferroptosis via AMPK/PGC1α/Nrf2 Pathway after Subarachnoid Hemorrhage in Rats.

Puerarin was shown to exert anti-oxidative and anti-ferroptosis effects in multiple diseases. The goal of this study was to explore the neuroprotective effect of puerarin on early brain injury (EBI) after subarachnoid hemorrhage (SAH) in rats. A total of 177 adult male Sprague Dawley rats were used. SAH was included via endovascular perforation. Intranasal puerarin or intracerebroventricular dorsomorphin (AMPK inhibitor) and SR18292 (PGC1α inhibitor) were administered. The protein levels of pAMPK, PGC1α, Nrf2, 4HNE, HO1, MDA, ACSL4, GSSG, and iron concentration in the ipsilateral hemisphere were significantly increased, whereas SOD, GPX4, and GSH were decreased at 24 h after SAH. Moreover, puerarin treatment significantly increased the protein levels of pAMPK, PGC1α, Nrf2, HO1, SOD, GPX4, and GSH, but decreased the levels of 4HNE, MDA, ACSL4, GSSG, and iron concentration in the ipsilateral hemisphere at 24 h after SAH. Dorsomorphin or SR18292 partially abolished the beneficial effects of puerarin exerted on neurological dysfunction, oxidative stress injury, and ferroptosis. In conclusion, puerarin improved neurobehavioral impairments and attenuated oxidative-stress-induced brain ferroptosis after SAH in rats. The neuroprotection acted through the activation of the AMPK/PGC1α/Nrf2-signaling pathway. Thus, puerarin may serve as new therapeutics against EBI in SAH patients.

Endoplasmic Reticulum Stress Contributes to Ventilator-Induced Diaphragm Atrophy and Weakness in Rats

Background: Accumulating evidence indicates that endoplasmic reticulum (ER) stress plays a critical role in the regulation of skeletal muscle mass. In recent years, much attention has been given to ventilator-induced diaphragm dysfunction (VIDD) because it strongly impacts the outcomes of critically ill patients. Current evidence suggests that the enhancement of oxidative stress is essential for the development of VIDD, but there are no data on the effects of ER stress on this pathological process. Methods: VIDD was induced by volume-controlled mechanical ventilation (MV) for 12 h; Spontaneous breathing (SB, for 12 h) rats were used as controls. The ER stress inhibitor 4-phenylbutyrate (4-PBA), the antioxidant N-acetylcysteine (NAC), and the ER stress inducer tunicamycin (TUN) were given before the onset of MV or SB. Diaphragm function, oxidative stress, and ER stress in the diaphragms were measured at the end of the experiments. Results: ER stress was markedly increased in diaphragms relative to that in SB after 12 h of MV (all p < 0.001). Inhibition of ER stress by 4-PBA downregulated the expression levels of proteolysis-related genes in skeletal muscle, including Atrogin-1 and MuRF-1, reduced myofiber atrophy, and improved diaphragm force-generating capacity in rats subjected to MV (all p < 0.01). In addition, mitochondrial reactive oxygen species (ROS) production and protein level of 4-HNE (4-hydroxynonenal) were decreased upon 4-PBA treatment in rats during MV (all p < 0.01). Interestingly, the 4-PBA treatment also markedly increased the expression of peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α) (p < 0.01), a master regulator for mitochondrial function and a strong antioxidant. However, the antioxidant NAC failed to reduce ER stress in the diaphragm during MV (p > 0.05). Finally, ER stress inducer TUN largely compromised diaphragm dysfunction in the absence of oxidative stress (all p < 0.01). Conclusion: ER stress is induced by MV and the inhibition of ER stress alleviates oxidative stress in the diaphragm during MV. In addition, ER stress is responsible for diaphragm dysfunction in the absence of oxidative stress. Therefore, the inhibition of ER stress may be another promising therapeutic approach for the treatment of VIDD.