LPS-induced Lipid Peroxidation Research Articles

Activation of lipophagy is required for RAB7 to regulate ferroptosis in sepsis-induced acute kidney injury

Sepsis-induced acute kidney injury (S-AKI) is the most common type of acute kidney injury (AKI), accompanied by elevated morbidity and mortality rates. This study investigated the mechanism by which lipid droplets (LDs) degraded via autophagy (lipophagy)required for RAB7 regulated ferroptosis in the pathogenesis of S-AKI. Here, we constructed the S-AKI model in vitro and in vivo to elucidate the potential relationship of lipophagy and ferroptosis, and we first confirmed that the activation of lipophagy promoted renal tubular epithelial cell ferroptosis and renal damage in S-AKI. The results showed that lipopolysaccharide (LPS) induced a marked increase in lipid peroxidation and ferroptosis, which were rescued by ferrstain-1 (Fer-1), an inhibitor of ferroptosis. In addition, LPS induced the remarkable activation of RAB7-mediated lipophagy. Importantly, silencing RAB7 alleviated LPS-induced lipid peroxidation and ferroptosis. Thus, the present study demonstrated the potential significant role of ferroptosis and lipophagy in sepsis-induced AKI, and contributed to better understanding of the pathogenesis and treatment targets of AKI.

Exploring the ferroptosis-related gene lipocalin 2 as a potential biomarker for sepsis-induced acute respiratory distress syndrome based on machine learning

BackgroundSepsis is a major cause of mortality in patients, and ARDS is one of the most common outcomes. The pathophysiology of acute respiratory distress syndrome (ARDS) caused by sepsis is significantly impacted by genes related to ferroptosis. MethodsIn this study, Weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) networks, functional enrichment analysis, and machine learning were employed to identify characterized genes and to construct receiver operating characteristic (ROC) curves. Additionally, DNA methylation levels were quantified and single-cell analysis was conducted. To validate the alterations in the expression of Lipocalin-2 (LCN2) and ferroptosis-related proteins in the in vitro model, Western blotting was carried out, and the changes in intracellular ROS and Fe2+ levels were detected. ResultsA combination of eight machine learning algorithms, including RFE, LASSO, RandomForest, SVM-RFE, GBDT, Bagging, XGBoost, and Boruta, were used with a machine learning model to highlight the significance of LCN2 as a key gene in sepsis-induced ARDS. Analysis of immune cell infiltration showed a positive correlation between neutrophils and LCN2. In a cell model induced by LPS, it was found that Fer-1, a ferroptosis inhibitor, was able to reverse the expression of LCN2. Knocking down LCN2 in BEAS-2B cells reversed the LPS-induced lipid peroxidation, Fe2+ levels, ACSL4, and GPX4 levels, indicating that LCN2, a FRG, plays a crucial role in mediating ferroptosis. ConclusionUpon establishing an FRG model for individuals with sepsis-induced ARDS, we determined that LCN2 could be a dependable marker for predicting survival in these patients. This finding provides a basis for more accurate ARDS diagnosis and the exploration of innovative treatment options.

Endotoxin lipopolysaccharide (LPS)-induced peripheral inflammation and its impact on brain and liver function

Endotoxin Lipopolysaccharide (LPS) induces an innate immune response and activates sickness behavior. During neuroinflammation, the activated microglial cells change its morphology and release various chemicals that could be harmful such as nitric oxide (NO), reactive oxygen species (ROS) and enzymes for proteolysis. Through the buildup of free radicals and ROS, these neuroinflammations can also result in oxidative stress. This ROS reacts with cellular macromolecules, triggering a cascade that results in membrane porosity caused by membrane lipid peroxidation (LPO), which is measured by the quantity of malondialdehyde (MDA). On LPS challenge, the complex behaviour that decreased common activities and explorations, is noticed and with rising anxiety, cognitive impairment and decreased social activities. It also decreased closed arm time, although closed arm entries were higher which may be related to the increased locomotion. It raised open arm time and open arm entries, suggesting of reduced anxiety and also increased exploratory behaviour. AChE and nNOS were increased in the forebrain part. Reduction in brainstem and MDA activity decreased on both forebrain and brain stem. MDA in the liver increased by LPS challenge indicating upregulated hepatic tissue damage and oxidative stress. As a result of LPS-induced lipid peroxidation and oxidative stress, brain Glutathione (GSH), a substrate for cytosolic Glutathione peroxidase, increased in the forebrain and decreased in the brainstem. Decreased scavenging of ROS was indicated by the LPS-induced decrease in liver GSH levels.

TMEM43 Protects against Sepsis-Induced Cardiac Injury via Inhibiting Ferroptosis in Mice

A previous study found that transmembrane protein 43 (TMEM43) was highly associated with arrhythmogenic right ventricular dysplasia/cardiomyopathy. However, as a transmembrane protein, TMEM43 may be involved in ferroptosis in cardiovascular disease. In this study, we aimed to explore the role of TMEM43 in lipopolysaccharide (LPS)-induced cardiac injury and the underlying mechanism. Mice were injected with LPS (10 mg/kg) for 12 h to generate experimental sepsis. Mice were also subjected to AAV9-shTMEM43 to knock down TMEM43 or AAV9-TMEM43 to overexpress TMEM43 in hearts. H9c2 rat cardiomyocytes were also transfected with Ad-TMEM43 or TMEM43 siRNA to overexpress/knock down TMEM43. As a result, TMEM43 knockdown in hearts deteriorated LPS-induced mouse cardiac injury and dysfunction. LPS increased cardiac ferroptosis as assessed by malonaldehyde (MDA) and cardiac iron density, which were aggravated by TMEM43 knockdown. Moreover, TMEM43 overexpression alleviated LPS-induced cardiac injury, dysfunction, and ferroptosis. In vitro experiments showed that TMEM43 overexpression inhibited LPS-induced lipid peroxidation and cardiomyocyte injury while TMEM43 knockdown aggravated LPS-induced ferroptosis and injury in cardiomyocytes. Mechanistically, LPS increased the expression of P53 and ferritin but decreased the level of Gpx4 and SLC7A11. TMEM43 could inhibit the level of P53 and ferritin enhanced the level of Gpx4 and SLC7A11. Furthermore, ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, could protect against LPS-induced cardiac injury and also counteracted the deteriorating effects of TMEM43 silencing in the heart. Based on these findings, we concluded that TMEM43 protects against sepsis-induced cardiac injury via inhibiting ferroptosis in mice. By targeting ferroptosis in cardiomyocytes, TMEM43 may be a therapeutic strategy for preventing sepsis in the future.

Effects of a Short-Term Lipopolysaccharides Challenge on Mouse Brain and Liver Peroxisomal Antioxidant and β-oxidative Functions: Protective Action of Argan Oil.

During sepsis, the imbalance between oxidative insult and body antioxidant response causes the dysfunction of organs, including the brain and liver. Exposing mice to bacterial lipopolysaccharides (LPS) results in a similar pathophysiological outcome. The protection offered by argan oil was studied against LPS-induced oxidative stress, dysregulation of peroxisomal antioxidants, and β-oxidation activities in the brain and liver. In a short-term LPS treatment, lipid peroxidation (malonaldehyde assay) increased in the brain and liver with upregulations of proinflammatory tumor necrosis factor (Tnf)-α and anti-inflammatory interleukin (Il)-10 genes, especially in the liver. Although exposure to olive oil (OO), colza oil (CO), and argan oil (AO) prevented LPS-induced lipid peroxidation in the brain and liver, only AO exposure protected against liver inflammation. Remarkably, only exposure to AO prevented LPS-dependent glutathione (GSH) dysregulation in the brain and liver. Furthermore, exposure to AO increased more efficiently than OO and CO in both organs, peroxisomal antioxidant capacity via induction of catalase (Cat) gene, protein and activity expression levels, and superoxide dismutase (Sod1) mRNA and activity levels. Interestingly, LPS decreased protein levels of the peroxisomal fatty acid-ATP binding cassette (ABC) transporters, ABCD1 and ABCD2, and increased acyl-CoA oxidase 1 (ACOX1) protein expression. Moreover, these LPS effects were attenuated for ABCD1 and ACOX1 in the brain of mice pretreated with AO. Our data collectively highlight the protective effects of AO against early oxidative stress caused by LPS in the brain and liver and their reliance on the preservation of peroxisomal functions, including antioxidant and β-oxidation activities, making AO a promising candidate for the prevention and management of sepsis.

Itaconate inhibits ferroptosis of macrophage via Nrf2 pathways against sepsis-induced acute lung injury

Itaconate, a metabolite produced during inflammatory macrophage activation, has been extensively described to be involved in immunoregulation, oxidative stress, and lipid peroxidation. As a form of iron and lipid hydroperoxide-dependent regulated cell death, ferroptosis plays a critical role in sepsis-induced acute lung injury (ALI). However, the relationship between itaconate and ferroptosis remains unclear. This study aims to explore the regulatory role of itaconate on ferroptosis in sepsis-induced ALI. In in vivo experiments, mice were injected with LPS (10 mg/kg) for 12 h to generate experimental sepsis models. Differential gene expression analysis indicated that genes associated with ferroptosis existed significant differences after itaconate pretreatment. 4-octyl itaconate (4-OI), a cell-permeable derivative of endogenous itaconate, can significantly alleviate lung injury, increase LPS-induced levels of glutathione peroxidase 4 (GPX4) and reduce prostaglandin-endoperoxide synthase 2 (PTGS2), malonaldehyde (MDA), and lipid ROS. In vitro experiments showed that both 4-OI and ferrostatin-1 inhibited LPS-induced lipid peroxidation and injury of THP-1 macrophage. Mechanistically, we identified that 4-OI inhibited the GPX4-dependent lipid peroxidation through increased accumulation and activation of Nrf2. The silence of Nrf2 abolished the inhibition of ferroptosis from 4-OI in THP-1 cells. Additionally, the protection of 4-OI for ALI was abolished in Nrf2-knockout mice. We concluded that ferroptosis was one of the critical mechanisms contributing to sepsis-induced ALI. Itaconate is promising as a therapeutic candidate against ALI through inhibiting ferroptosis.

Ferritinophagy-mediated ferroptosis is involved in sepsis-induced cardiac injury

Ferroptosis is a reactive oxygen species (ROS)- and iron-dependent form of regulated cell death (RCD), playing critical roles in organ injury and targeting therapy of cancers. Previous studies have demonstrated that ferroptosis participates in the development of cardiomyopathy including cardiac hypertrophy, diabetic cardiomyopathy and doxorubicin-induced cardiotoxicity. However, the role of ferroptosis in sepsis-induced cardiac injury remains unclear. This study aimed to explore the role and underlying mechanism of ferroptosis on lipopolysaccharide (LPS)-induced cardiac injury. Mice were injected with LPS (10 mg/kg) for 12 h to generate experimental sepsis. Ferrostatin-1 (Fer-1) and Dexrazoxane (DXZ) were used to suppress ferroptosis of mice with sepsis-induced cardiac injury. LPS increased the levels of ferroptotic markers involving prostaglandin endoperoxide synthase 2 (PTGS2), malonaldehyde (MDA) and lipid ROS, apart from resulting in obvious mitochondria damage, which were alleviated by Fer-1 and DXZ. In vitro experiments showed that Fer-1 inhibited LPS-induced lipid peroxidation and injury of H9c2 myofibroblasts while erastin and sorafenib aggravated LPS-induced ferroptosis. Additionally, Fer-1 and DXZ improved survival rate and cardiac function of mice with sepsis. Mechanistically, LPS increased the expression of nuclear receptor coactivator 4 (NCOA4) and the level of intracellular Fe2+ but decreased the level of ferritin. NCOA4 could directly interact with ferritin and degrade it in a ferritinophagy-dependent manner, which subsequently released a great amount of iron. Cytoplasmic Fe2+ further activated the expression of siderofexin (SFXN1) on mitochondrial membrane, which in turn transported cytoplasmic Fe2+ into mitochondria, giving rise to the production of mitochondrial ROS and ferroptosis. Based on these findings, we concluded that ferritinophagy-mediated ferroptosis is one of the critical mechanisms contributing to sepsis-induced cardiac injury. Targeting ferroptosis in cardiomyocytes may be a therapeutic strategy for preventing sepsis in the future.

Targeted High Resolution LC/MS3 Adductomics Method for the Characterization of Endogenous DNA Damage.

DNA can be damaged through covalent modifications of the nucleobases by endogenous processes. These modifications, commonly referred to as DNA adducts, can persist and may lead to mutations, and ultimately to the initiation of cancer. A screening methodology for the majority of known endogenous DNA adducts would be a powerful tool for investigating the etiology of cancer and for the identification of individuals at high-risk to the detrimental effects of DNA damage. This idea led to the development of a DNA adductomic approach using high resolution data-dependent scanning, an extensive MS2 fragmentation inclusion list of known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, the detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and their relative quantitation using the corresponding full scan extracted ion chromatograms. The method's inclusion list consists of the majority of known endogenous DNA adducts, compiled, and reported here, as well as adducts specific to tobacco exposure included to compare the performance of the method with previously developed targeted approaches. The sensitivity of the method was maximized by reduction of extraneous background signal through the purification and minimization of the amount of commercially obtained enzymes used for the DNA hydrolysis. In addition, post-hydrolysis sample purification was performed using off-line HPLC fraction collection to eliminate the highly abundant unmodified bases, and to avoid introduction of plasticizers found in solid-phase extraction cartridges. Also, several instrument parameters were evaluated to optimize the ion signal intensities and fragmentation spectra quality. The method was tested on an animal model of lung carcinogenesis where A/J mice were exposed to the tobacco specific lung carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) with its effects enhanced by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS). Lung DNA were screened for endogenous DNA adducts known to result from oxidative stress and LPS-induced lipid peroxidation, as well as for adducts due to NNK exposure. The relative quantitation of the detected DNA adducts was performed using parallel reaction monitoring MS2 analysis, demonstrating a general workflow for analysis of endogenous DNA adducts.

In vivo modulation of LPS induced leukotrienes generation and oxidative stress by sesame lignans

The role of inflammation and oxidative stress is critical during onset of metabolic disorders and this has been sufficiently established in literature. In the present study, we evaluated the effects of sesamol and sesamin, two important bioactive molecules present in sesame oil, on the generation of inflammatory and oxidative stress factors in LPS injected rats. Sesamol and sesamin lowered LPS induced expression of cPLA2 (61 and 56%), 5-LOX (44 and 51%), BLT-1(32 and 35%) and LTC4 synthase (49 and 50%), respectively, in liver homogenate. The diminished serum LTB4 (53 and 64%) and LTC4 (67 and 44%) levels in sesamol and sesamin administered groups, respectively, were found to be concurrent with the observed decrease in the expression of cPLA2 and 5-LOX. The serum levels of TNF-α (29 and 19%), MCP-1 (44 and 57%) and IL-1β (43 and 42%) were found to be reduced in sesamol and sesamin group, respectively, as given in parentheses, compared to LPS group. Sesamol and sesamin offered protection against LPS induced lipid peroxidation in both serum and liver. Sesamol, but not sesamin, significantly restored the loss of catalase and glutathione reductase activity due to LPS (P<.05). However, both sesamol and sesamin reverted SOD activities by 92 and 98%, respectively. Thus, oral supplementation of sesamol and sesamin beneficially modulated the inflammatory and oxidative stress markers, as observed in the present study, in LPS injected rats. Our report further advocates the potential use of sesamol and sesamin as an adjunct therapy wherein, inflammatory and oxidative stress is of major concern.

Endotoxin-induced acute lung injury in mice is protected by 5,7-dihydroxy-8-methoxyflavone via inhibition of oxidative stress and HIF-1α.

Up to date, the morbidity and mortality rates of acute lung injury (ALI) still rank high among clinical illnesses. Endotoxin, also called lipopolysaccharide (LPS), induced sepsis is the major cause for ALI. Beneficial biological effects, such as antioxidation, anti-inflammation, and neuroprotection was found to express by 5,7-dihydroxy-8-methoxyflavone (DHMF). The purpose of present study was to investigate the potential protective effects of DHMF and the possibile mechanisms involved in LPS-induced ALI. In our experimental model, ALI was induced in mice by intratracheal injection of LPS, and DHMF at various concentrations was injected intraperitoneally for 30 min prior to LPS administration. Pretreatment with DHMF inhibited not only the histolopatholgical changes occurred in lungs but also leukocytes infiltration in LPS-induced ALI. Decreased activity of antioxidative enzymes (AOE) such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) caused by LPS was reversed by DHMF. LPS-induced lipid peroxidation HIF-1α accumulation, NF-κB phosphorylation, and IκBα degradation were all inhibited by DHMF. In addition, LPS-induced expression of proinflammatory mediators such as TNF-α and IL-1β were also inhibited by 5,7-dihydroxy-8-methoxyflavone. These results suggested that the protective mechanisms of DHMF on endotoxin-induced ALI might be via up-regulation of antioxidative enzymes, inhibition of NFκB phosphorylation, and HIF-1α accumulation. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1700-1709, 2016.

Silver-zeolite combined to polyphenol-rich extracts of Ascophyllum nodosum: potential active role in prevention of periodontal diseases.

The purpose of this study was to evaluate various biological effects of silver-zeolite and a polyphenol-rich extract of A. nodosum (ASCOP) to prevent and/or treat biofilm-related oral diseases. Porphyromonas gingivalis and Streptococcus gordonii contribute to the biofilm formation associated with chronic periodontitis. In this study, we evaluated in vitro antibacterial and anti-biofilm effects of silver-zeolite (Ag-zeolite) combined to ASCOP on P. gingivalis and S. gordonii growth and biofilm formation capacity. We also studied the anti-inflammatory and antioxidant capacities of ASCOP in cell culture models. While Ag-zeolite combined with ASCOP was ineffective against the growth of S. gordonii, it showed a strong bactericidal effect on P. gingivalis growth. Ag-zeolite combined with ASCOP was able to completely inhibit S. gordonii monospecies biofilm formation as well as to reduce the formation of a bi-species S. gordonii/P. gingivalis biofilm. ASCOP alone was ineffective towards the growth and/or biofilm formation of S. gordonii and P. gingivalis while it significantly reduced the secretion of inflammatory cytokines (TNFα and IL-6) by LPS-stimulated human like-macrophages. It also exhibited antioxidant properties and decreased LPS induced lipid peroxidation in gingival epithelial cells. These findings support promising use of these products in future preventive or therapeutic strategies against periodontal diseases.

Systemic taurine prevents brain from lipopolysaccharide-induced lipid peroxidation in rats

Lipopolysaccharide (LPS), the major component of the outer membran of Gram-negative bacteria, can reproduce neuroinflammation and leads to oxidative stress in brain. Becausetaurine exhibits antioxidative properties, in this study, we aimed to evaluate the protective effects of taurine at the subtoxic dose of lipopolysaccharide-induced lipid peroxidation and oxidative stress. We measured malondialdehyde (MDA), gluthathione (GSH) and nitrite + nitrate (NOx) levels of different brain regions in rats after the i.p administration of LPS and taurine. Wistar male rats (220 to 300 g) were divided into five groups: 1. Control; 2. Saline + LPS (500 μg/kg, i.p.); 3. LPS (500 μg/kg, i.p.) + LPS (500 μg/kg, i.p.); 4. LPS (500 μg/kg, i.p.) + taurine (500 mg/kg, i.p.); 5. Taurine (500 mg/kg, i.p.). i.p. LPS was administred 6 h before sacrification. Taurine was administered after 3 h of LPS injection and the animals sacrified after 3 h of taurine administration. Hippocampus, temporal cortex, frontal cortex, parietal cortex, basal forebrain, cerebellum and brain stem were dissected. MDA, GSH and NOx levels were assayed spectrophotometrically. MDA levels were found increased significantly in all mentioned brain regions after i.p. LPS (p<0.05) except basal forebrain. In LPS + taurine group, we found significantly decreased MDA levels in all mentioned brain regions except an insignificant decrease in cerebellum after i.p. LPS administaration. These results demonstrate that taurine administration with LPS protects brain against LPS-induced lipid peroxidation and oxidative stress. Key words: Lipopolysaccaride, taurine, lipid peroxidation, nitric oxide, brain.

Reactive Oxygen Species Contribute to Lipopolysaccharide-Induced Teratogenesis in Mice

Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including embryonic resorption, fetal death and growth retardation, and preterm delivery. In the present study, we showed that an ip injection with LPS daily from gestational day (gd) 8 to gd 12 resulted in the incidence of external malformations. The highest incidence of malformed fetuses was observed in fetuses from dams exposed to 20 microg/kg LPS, in which 34.9% of fetuses per litter were externally malformed. In addition, 17.4% of fetuses per litter in 30 microg/kg group and 12.5% of fetuses per litter in 10 microg/kg group were externally malformed. Importantly, external malformations were also observed in fetuses from dams exposed to only two doses of LPS (20 microg/kg, ip) on gd 8, in which 76.5% (13/17) of litters and 39.1% of fetuses per litter were affected. LPS-induced teratogenicity seemed to be associated with oxidative stress in fetal environment, measured by lipid peroxidation, nitrotyrosine residues, and glutathione (GSH) depletion in maternal liver, embryo, and placenta. alpha-Phenyl-N-t-butylnitrone (PBN, 100 mg/kg, ip), a free radical spin-trapping agent, abolished LPS-induced lipid peroxidation, nitrotyrosine residues, and GSH depletion. Consistent with its antioxidant effects, PBN decreased the incidence of external malformations. Taken together, these results suggest that reactive oxygen species might be, at least partially, involved in LPS-induced teratogenesis.

Maternally administered lipopolysaccharide (LPS) upregulates the expression of heme oxygenase-1 in fetal liver: The role of reactive oxygen species

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the rate-limiting step in the degradation of heme to biliverdin, carbon monoxide and iron. Previous studies have demonstrated that lipopolysaccharide (LPS) upregulates the expression of HO-1 in adult mouse liver. The present study aimed to investigate the effects of maternal LPS exposure on the expression of HO-1 in fetal liver. The pregnant mice were intraperitoneally injected with different doses of LPS (1, 10, 75 μg/kg) on gestational day 17. Results showed that the expression of HO-1 in fetal liver was increased, beginning 2 h after LPS, being at the highest level 24 h after LPS, and remaining elevated up to 48 h after LPS, whereas HO-2, the constitutive form, did not change at the various time points observed. LPS-induced upregulation of HO-1 was blocked by alpha-phenyl- N- t-butylnitrone (PBN), a free radical spin trapping agent. Correspondingly, PBN pretreatment significantly attenuated LPS-induced lipid peroxidation and glutathione (GSH) depletion in fetal liver. However, aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase (iNOS), and pentoxifylline (PTX), an inhibitor of tumor necrosis factor alpha (TNF-α) synthesis, had no effect on LPS-induced upregulation of HO-1 in fetal liver. In conclusion, reactive oxygen species (ROS), rather than TNF-α or nitric oxide (NO), are involved in LPS-induced upregulation of HO-1 in fetal liver. These results provide new evidence that maternal LPS exposure results in oxidative stress in fetuses, which may contribute to LPS-induced developmental toxicity.

Lipopolysaccharide (LPS) Up-Regulates the Expression of Haem Oxygenase-1 in Mouse Placenta

Haem oxygenase-1 (HO-1) is an inducible enzyme that catalyses the rate-limiting step in the degradation of haem to biliverdin, carbon monoxide and iron. There is increasing evidence that HO plays important roles in the cellular defence against oxidative stress and the deleterious effects of pro-inflammatory cytokines. In the present study, we investigated the effects of lipopolysaccharide (LPS) on the expression of HO-1 in mouse placenta. When a single dose of LPS (75 μg/kg, i.p.) was administered to the pregnant mice, the expression of HO-1 in mouse placenta was markedly increased at 12 h after LPS treatment and remained elevated up to 48 h after LPS administration. The expression of HO-2, the constitutive form, did not change at the various time points observed. LPS-induced up-regulation of placental HO-1 was blocked after the pregnant mice were pre-treated with alpha-phenyl- N- t-butylnitrone (PBN), a free radical spin trapping agent. Correspondingly, PBN pre-treatment significantly inhibited LPS-induced lipid peroxidation and glutathione (GSH) depletion in mouse placenta. Furthermore, pentoxifylline (PTX), an inhibitor of tumour necrosis factor alpha (TNF-α) synthesis, also significantly attenuated LPS-induced up-regulation of placental HO-1. However, aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase (iNOS), had little effect on LPS-induced up-regulation of HO-1 in mouse placenta. Taken together, these results indicate that LPS up-regulates the expression of HO-1 in mouse placenta. LPS-induced up-regulation of placental HO-1 is probably mediated, at least in part, by reactive oxygen species (ROS) and TNF-α, rather than nitric oxide.

Ascorbic acid protects against lipopolysaccharide-induced intra-uterine fetal death and intra-uterine growth retardation in mice

Lipopolysaccharide (LPS) has been associated with adverse developmental outcomes including embryonic resorption, intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR) and preterm labor. Reactive oxygen species (ROS) mediate LPS-induced developmental toxicity. Ascorbic acid is an antioxidant. In the present study, we investigated the effect of ascorbic acid on LPS-induced IUFD and IUGR in mice. All ICR pregnant mice except controls received an intraperitoneal (75 μg/kg, i.p.) injection of LPS daily on gd 15–17. The experiment was carried out in three different modes. In mode A, the pregnant mice were pretreated with a single dose (500 mg/kg, i.p.) of ascorbic acid before LPS. In mode B, the pregnant mice were administered with a single dose (500 mg/kg, i.p.) of ascorbic acid at 3 h after LPS. In mode C, the pregnant mice were administered with 500 mg/kg (i.p.) of ascorbic acid at 30 min before LPS, followed by additional dose (500 mg/kg, i.p.) of ascorbic acid at 3 h after LPS. The number of live fetuses, dead fetuses and resorption sites was counted on gd 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. Results showed that maternally administered LPS significantly increased fetal mortality, decreased fetal weight and crown-rump and tail lengths of live fetuses, and retarded skeletal ossification in caudal vertebrae, anterior and posterior phalanges, and supraoccipital bone. LPS-induced IUFD and IUGR were associated with lipid peroxidation and GSH depletion in maternal liver, placenta and fetal liver. Pre-treatment with ascorbic acid significantly attenuated LPS-induced lipid peroxidation, decreased fetal mortality, and reversed LPS-induced fetal growth and skeletal development retardation. By contrast to pre-treatment, post-treatment with ascorbic acid had less effect on LPS-induced IUFD, although post-treatment significantly attenuated LPS-induced lipid peroxidation and reversed LPS-induced fetal growth and skeletal development retardation. Furthermore, post-treatment with ascorbic acid reduced the protective effects of pre-treatment on LPS-induced IUFD. All these results suggest that pre-treatment with ascorbic acid protected against LPS-induced fetal death and reversed LPS-induced growth and skeletal development retardation via counteracting LPS-induced oxidative stress, whereas post-treatment had less effect on LPS-induced IUFD.

Melatonin protects against lipopolysaccharide‐induced intra‐uterine fetal death and growth retardation in mice

Lipopolysaccharide (LPS) has been associated with adverse developmental outcomes, including intra-uterine fetal death (IUFD) and intra-uterine growth retardation (IUGR). However, the exact mechanism for LPS-induced IUFD and IURD remains unclear. LPS stimulates macrophages to generate reactive oxygen species (ROS). Therefore, we hypothesize that ROS may be involved in LPS-induced IUFD and IURD. Melatonin is a powerful endogenous antioxidant. In this study, we investigated the protective effects of melatonin on LPS-induced IUFD and IURD in ICR mice. All pregnant mice except controls received an intraperitoneal (75 microg/kg, i.p.) injection of LPS on gestational day (gd) 15-17. The experiment was carried out in two different modes. In mode A, the pregnant mice received two doses of melatonin within 24 hr, one (5 or 10 mg/kg) injected immediately after LPS and the other (5 or 10 mg/kg) injected at 3 hr after LPS. In mode B, the pregnant mice were pretreated with 10 mg/kg of melatonin 18 hr before LPS and then received two doses of melatonin in 24 hr, one (10 mg/kg) injected immediately after LPS and the other (10 mg/kg) injected 3 hr after LPS. The number of live fetuses, dead fetuses and resorption sites were counted on gd 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. Results showed that post-treatments with melatonin significantly attenuated LPS-induced IUFD in a dose-dependent manner. Surprisingly, pre- plus post-treatments with melatonin almost blocked LPS-induced IUFD. In addition, both post-treatments and pre- plus post-treatments with melatonin significantly alleviated LPS-induced decreases in crown-rump and tail lengths and reversed LPS-induced skeletal developmental retardation. However, melatonin had little effect on LPS-induced decrease in fetal weight. Furthermore, pre- plus post-treatments with melatonin significantly attenuated LPS-induced lipid peroxidation in maternal liver. These results indicate that melatonin protects against LPS-induced IURD and IUGR via counteracting LPS-induced oxidative stress.

α-Tocopherol reduces lipopolysaccharide-induced peroxide radical formation and interleukin-6 secretion in primary murine microglia and in brain

The purpose of this study was to determine if α-tocopherol—a reactive oxygen species (ROS) scavenging agent—inhibits LPS-induced oxidative stress and interleukin-6 (IL-6) production in murine microglia and brain. LPS increased intracellular peroxides and IL-6 in a dose-dependent fashion in primary microglia. The LPS-induced increase in ROS and IL-6 was reduced by pretreatment of α-tocopherol. Intraperitoneal injection (i.p.) of LPS induced lipid peroxidation and IL-6 in brain. Importantly, three daily injections (20 mg) of α-tocopherol increased brain α-tocopherol and decreased LPS-induced lipid peroxidation and IL-6 in brain. Taken together, these data indicate that α-tocopherol can mitigate inflammatory cytokine production in the brain.

Evidence that glutathione depletion is a mechanism responsible for the anti-inflammatory effects of ethyl pyruvate in cultured lipopolysaccharide-stimulated RAW 264.7 cells.

Ethyl pyruvate (EP), an effective scavenger of reactive oxygen species, is also an anti-inflammatory agent in a variety of in vivo and in vitro model systems. To gain a better understanding of the molecular basis for the anti-inflammatory effects of EP, we compared the pharmacological properties of EP andN-acetyl-l-cysteine (NAC), a well studied scavenger of reactive oxygen species and a precursor for the endogenous antioxidant glutathione (GSH). The studies were performed using RAW 264.7 murine macrophage-like cells that were stimulated with lipopolysaccharide (LPS). Although EP and NAC both inhibited LPS-induced nitric oxide and interleukin (IL)-6 secretion, the former compound was considerably more potent than the latter. EP markedly inhibited inducible nitric-oxide synthase, IL-6, and IL-10 mRNA induction, whereas the effects of NAC were minimal. EP inhibited LPS-induced nuclear factor-kappaB DNA binding to a much greater extent than did NAC. Both compounds inhibited LPS-induced lipid peroxidation, but the two compounds had qualitatively different effects on cellular levels of GSH. Although NAC increased GSH levels, EP had the opposite effect. The anti-inflammatory effects of EP were partially reversed when RAW 264.7 cells were treated with a cell-permeable GSH analog, glutathione ethyl ester. These data support the view that the anti-inflammatory effects of EP are mediated, at least in part, by the ability of EP to deplete cellular GSH stores. Moreover, the findings presented here suggest that an unusual combination of biochemical effects (inhibition of lipid peroxidation and GSH depletion) might account for the anti-inflammatory effects of EP.

The pineal hormone melatonin inhibits LPS-induced lipid peroxidation in vitro

The pineal hormone melatonin inhibits LPS-induced lipid peroxidation in vitro