NADPH Oxidase Subunits P47phox Research Articles

Sirtuin1 contributes to the overexpression of Giα proteins and hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats.

We earlier demonstrated that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit the overexpression of Giα proteins and hyperproliferation that is attributed to the enhanced levels of endogenous angiotensin II (Ang II). In addition, the implication of Sirtuin1 (Sirt1) a histone deacetylase class III family in Ang II-induced hypertension has also been shown. We recently demonstrated that Ang II increased the expression of Sirt1 in aortic VSMC that contributed to the overexpression of Giα proteins. However, whether Sirt1 is overexpressed in VSMC from SHR and is linked to the enhanced expression of Giα proteins and hyperproliferation remains unexplored. In the present study, we show that Sirt1 is upregulated in VSMC from SHR and this upregulation was attenuated by AT1 receptor antagonist losartan. In addition, the inhibition or knockdown of Sirt1 by specific inhibitors EX 527 and NAM and/or siRNA attenuated the enhanced expression of Giα proteins, cell cycle proteins and hyperproliferation of VSMC from SHR. Furthermore, the enhanced levels of reactive oxygen species (ROS), hydrogen peroxide and NADPH oxidase subunits NOX2 and p47phox, increased phosphorylation of EGFR, ERK1/2 and AKT displayed by VSMC from SHR were also attenuated by knocking down of Sirt1 by siRNA. In summary, our results demonstrate that Sirt1 is overexpressed in VSMC from SHR which through augmenting oxidative stress contributes to the enhanced expression of Giα proteins, cell cycle proteins and resultant hyperproliferation of VSMC.

Uncoupling of IL-6 signaling and LC3-associated phagocytosis drives immunoparalysis during sepsis

Immune deactivation of phagocytes is a central event in the pathogenesis of sepsis. Herein, we identify a master regulatory role of IL-6 signaling on LC3-associated phagocytosis (LAP) and reveal that uncoupling of these two processes during sepsis induces immunoparalysis in monocytes/macrophages. In particular, we demonstrate that activation of LAP by the human fungal pathogen Aspergillus fumigatus depends on ERK1/2-mediated phosphorylation of p47phox subunit of NADPH oxidase. Physiologically, autocrine IL-6/JAK2/Ninein axis orchestrates microtubule organization and dynamics regulating ERK recruitment to the phagosome and LC3+ phagosome (LAPosome) formation. In sepsis, loss of IL-6 signaling specifically abrogates microtubule-mediated trafficking of ERK, leading to defective activation of LAP and impaired killing of bacterial and fungal pathogens by monocytes/macrophages, which can be selectively restored by IL-6 supplementation. Our work uncovers a molecular pathway linking IL-6 signaling with LAP and provides insight into the mechanisms underlying immunoparalysis in sepsis.

The atypical small GTPase GEM/Kir is a negative regulator of the NADPH oxidase and NETs production through macroautophagy.

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22phox and p47phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils.

Mitochondrial respiration and redox protein expression in peripheral blood mononuclear cells from Non‐Hispanic Black and White Males

Background Non-Hispanic Black (NHB) adults exhibit a higher prevalence of cardiovascular disease (CVD) compared to Non-Hispanic White (NHW) adults. Vascular oxidative stress may be one contributing factor in these racial disparities. Peripheral blood mononuclear cells (PBMCs) are primary contributors to systemic oxidative stress and there are documented racial disparities in the PBMC-derived reactive oxygen species (ROS) production. However, it is unclear if the racial differences in PBMC-derived ROS is associated with alterations in mitochondrial function. Therefore, the purpose of this study was to investigate potential racial differences in cellular metabolism and redox protein expression in PBMCs from NHW and NHB adults. Methods Commercial PBMCs (n=10 cell lines; 5 NHW & 5 NHB males) were cultured 14-18 hours prior to cell respiration and protein harvest. Basal and maximal respiration were measured using a Clark electrode (Oxytherm, Hanstech) and then calculated coupling efficiency and leak respiration. Western blotting was used to measure protein expression for the antioxidant proteins: superoxide dismutase (SOD) isoforms 1 and 2, as well as the acetylated (i.e., inactivated) SOD2 (AcSOD2) and the NAD-dependent deacetylase sirtuin-3 (Sirt3). The pro-oxidant complex NADPH oxidase subunits p47phox and gp91phox were also assessed. Welch unpaired t-tests were used to assess the racial differences in respiration measures and independent t-tests were used for protein expression. Results For the respiration measures, NHW had significantly higher levels of basal respiration (Fig. 1A, p=0.048) and leak respiration (Fig. 1D, p=0.016), while having a lower coupling efficiency (Fig. 1C, p=0.003). There was no difference in maximal respiration (Fig. 1B, p=0.188). No baseline racial differences were found in PBMC expression for the redox regulating proteins: SOD1 (Fig. 2A, p=0.42), SOD2 (Fig. 2B, p=0.40), AcSOD2 (Fig. 2C, p=0.45), Sirt3 (Fig. 2D, p=0.45), p47phox (Fig 3A, p=0.23), or gp91phox (Fig. 3B, p=0.86). Conclusion The lower coupling efficiency and higher leak respiration in PBMCs from NHW may be beneficial in decreasing ROS production and overall oxidative damage. While we found no racial differences in the redox regulating proteins, additional data are needed, particularly in PBMCs from female participants.

Leishmania donovani Metacyclic Promastigotes Impair Phagosome Properties in Inflammatory Monocytes.

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

Involvement of the Microglial Aryl Hydrocarbon Receptor in Neuroinflammation and Vasogenic Edema after Ischemic Stroke.

Microglia are activated after ischemic stroke and induce neuroinflammation. The expression of the aryl hydrocarbon receptor (AhR) has recently been reported to elicit cytokine expression. We previously reported that microglial activation mediates ischemic edema progression. Thus, the purpose of this study was to examine the role of AhR in inflammation and edema after ischemia using a mouse middle cerebral artery occlusion (MCAO) model. MCAO upregulated AhR expression in microglia during ischemia. MCAO increased the expression of tumor necrosis factor α (TNFα) and then induced edema progression, and worsened the modified neurological severity scores, with these being suppressed by administration of an AhR antagonist, CH223191. In THP-1 macrophages, the NADPH oxidase (NOX) subunit p47phox was significantly increased by AhR ligands, especially under inflammatory conditions. Suppression of NOX activity by apocynin or elimination of superoxide by superoxide dismutase decreased TNFα expression, which was induced by the AhR ligand. AhR ligands also elicited p47phox expression in mouse primary microglia. Thus, p47phox may be important in oxidative stress and subsequent inflammation. In MCAO model mice, P47phox expression was upregulated in microglia by ischemia. Lipid peroxidation induced by MCAO was suppressed by CH223191. Taken together, these findings suggest that AhR in the microglia is involved in neuroinflammation and subsequent edema, after MCAO via p47phox expression upregulation and oxidative stress.

Imperatorin alleviates metabolic and vascular alterations in high-fat/high-fructose diet-fed rats by modulating adiponectin receptor 1, eNOS, and p47phox expression

In the present study, the therapeutic effects of imperatorin on metabolic and vascular alterations and possible underlying mechanisms were investigated in high-fat/high-fructose diet (HFFD)-fed rats. Male Sprague-Dawley rats were fed a high-fat diet plus 15% fructose in drinking water for 16 weeks. HFFD-fed rats were treated with imperatorin (15 or 30 mg/kg/day) for the last 4 weeks. In HFFD-fed rats, imperatorin significantly reduced obesity, hypertension, dyslipidemia, and insulin resistance. Imperatorin markedly improved vascular endothelial function and alleviated changes in vascular morphology. Furthermore, imperatorin treatment significantly increased the plasma levels of the nitric oxide metabolite and adiponectin, and upregulated adiponectin receptor 1 and endothelial nitric oxide synthase (eNOS) protein expression in the thoracic aorta. Imperatorin treatment decreased vascular superoxide anion production and downregulated aortic NADPH oxidase subunit p47phox protein expression. These findings indicated that imperatorin alleviates HFFD-induced metabolic and vascular alterations in rats. The possible underlying mechanism may involve the restoration of adiponectin receptor 1 and eNOS expression and suppression of p47phox expression.

Peroxynitrite activates NLRP3 inflammasome and contributes to hemorrhagic transformation and poor outcome in ischemic stroke with hyperglycemia

This study aims to test the hypothesis that peroxynitrite-mediated inflammasome activation could be a crucial player in the blood-brain barrier (BBB) disruption, hemorrhagic transformation (HT) and poor outcome in ischemic stroke with hyperglycemia. We used an experimental rat stroke model subjected to 90 min of middle cerebral artery occlusion plus 24 h or 7 days of reperfusion with or without acute hyperglycemia. We detected the production of peroxynitrite, the expression of NADPH oxidase, iNOS, MMPs and NLRP3 inflammasome in the ischemic brains, and evaluated infarct volume, brain edema, HT, neurological deficit score and survival rates. Our results show that: (1) Hyperglycemia increased the expression of NADPH oxidase subunits p47phox and p67phox, and iNOS, and the production of peroxynitrite. (2) Hyperglycemia increased infarct volume, aggravated the BBB hyperpermeability, induced brain edema and HT, and worsened neurological outcomes. These brain damages and poor outcome were reversed by the treatments of FeTmPyP (a representative peroxynitrite decomposition catalyst, PDC), peroxynitrite scavenger uric acid, and iNOS inhibitor 1400W. Furthermore, the activations of MMPs and NLRP3 inflammasome including pro/active-caspase-1 and IL-1β were inhibited both PDC and 1400W, indicating the roles of peroxynitrite in the inductions of MMPs and NLRP3 inflammasome in the ischemic brains under hyperglycemia. (3) NLRP3 inflammasome inhibitor MCC950, caspase-1 inhibitor VX-765 and IL-1β inhibitor diacerein attenuated brain edema, minimized hemorrhagic transformation and improved neurological outcome, demonstrating the roles of NLRP3 inflammasome in the hyperglycemia-mediated HT and poor outcome in the ischemic stroke rats with acute hyperglycemia. In conclusion, peroxynitrite could mediate activations of MMPs and NLRP3 inflammasome, aggravate the BBB damage and HT, and induce poor outcome in ischemic stroke with hyperglycemia. Therefore, targeting peroxynitrite–mediated NLRP3 inflammasome could be a promising strategy for ischemic stroke with hyperglycemia.

Neuroprotective Effects of Early Hypothermia Induced by Phenothiazines and DHC in Ischemic Stroke.

Methods Adult male Sprague Dawley rats were studied in 4 groups: (1) sham; (2) stroke; (3) stroke treated with pharmacological hypothermia before reperfusion (interischemia hypothermia); and (4) stroke treated with pharmacological hypothermia after reperfusion is initiated (inter-reperfusion hypothermia). The combination of chlorpromazine and promethazine with dihydrocapsaicin (DHC) was used to induce hypothermia. To compare the neuroprotective effects of drug-induced hypothermia between the interischemia and inter-reperfusion groups, brain damage was evaluated using infarct volume and neurological deficits at 24 h reperfusion. In addition, mRNA expressions of NADPH oxidase (NOX) subunits (gp91phox, p67phox, p47phox, and p22phox) and glucose transporter subtypes (GLUT1 and GLUT3) were determined by real-time PCR at 6 and 24 h reperfusion. ROS production was measured by flow cytometry assay at the same time points. Results In both hypothermia groups, the cerebral infarct volumes and neurological deficits were reduced in the ischemic rats. At 6 and 24 h reperfusion, ROS production and the expressions of NOX subunits and glucose transporter subtypes were also significantly reduced in both hypothermia groups as compared to the ischemic group. While there were no statistically significant differences between the two hypothermia groups at 6 h reperfusion, brain damage was significantly further decreased by interischemia hypothermia at 24 h. Conclusion Both interischemia and inter-reperfusion pharmacological hypothermia treatments play a role in neuroprotection after stroke. Interischemia hypothermia treatment may be better able to induce stronger neuroprotection after ischemic stroke. This study provides a new avenue and reference for stronger neuroprotective hypothermia before vascular recanalization in stroke patients.

Serum of limb remote ischemic postconditioning inhibits fMLP-triggered activation and reactive oxygen species releasing of rat neutrophils

ABSTRACT Objectives The study explores the protective role of the peripheral serum of limb remote ischemic postconditioning (LRIP) in reducing the reactive oxygen species (ROS) levels and neutrophil activation, which are responsible for the deleterious reperfusion injury. Methods LRIP was induced in Sprague–Dawley rats by three cycles of 5 min occlusion /5 min reperfusion on the left hind limb. The blood samples were collected before LRIP or 0 and 1 h after LRIP (named SerumSham, SerumLRIP0, SerumLRIP1, respectively). The effects of LRIP serum on ROS level and neutrophils activation were determined. The expression of MyD88-TRAF6-MAPKs and PI3K/AKT pathways in neutrophils were examined. Results When compared with SerumSham, SerumLRIP0 and SerumLRIP1 significantly reduced the ROS released from neutrophils activated by fMLP. Meanwhile, the mRNA expression levels of NADPH oxidase subunit p22phox and multiple ROS-producing related key proteins, such as NADPH oxidase subunit p47phox ser 304, ser 345. MyD88, p-ERK, p-JNK and p-P38 expression of neutrophils were downregulated by SerumLRIP0 and SerumLRIP1. SerumLRIP1 also downregulated p47phox mRNA expression and tumor necrosis factor receptor-associated factor 6 (TRAF6) protein expression. Conclusion LRIP serum protects against ROS level and neutrophils activation involving the MyD88-TRAF6-MAPKs. This finding provides new insight into the understanding of LRIP mechanisms.

Medical Image Analysis of Telmisartan-Induced Protection in Hippocampal CA1 After Chronic Intermittent Hypoxia

Objective: In this paper, we evaluate medical image analysis in hippocampus, immunohistochemistry analyzed and oxidative stress-mediated injury induced by chronic intermittent hypoxia on the mouse hippocampal CA1 area and the protective effects of telmisartan (TEL) on oxidative stress-mediated injury. Methodology: C57B6J mice have been randomly categorized as chronic intermittent hypoxia (CIH) group, CIH + telmisartan (CIH + TEL) group, the room air control group and blank control group, and examined during a period of 12 weeks. Brain images were obtained by Computed tomography scan, spatial learning and memory abilities were tested, and p47 NADPH oxidase subunit (p47PHOX) and 8-hydroxy-guanosine (8-OHG) were measured in the hippocampal CA1 area by immunohistochemistry and analyzed by Image-Pro Plus 6.0. Results: (1) After 12 weeks, there were no abnormalities in mice brain by CT scan. (2) Spatial learning ability measured by escape latency from the Morris water maze in the hypoxia group decreased, while intervention with telmisartan improved the hypoxiainduced spatial learning deficit (F = 3.04, P = 0.045). There exist no differences in spatial memory among any groups. (3) p47PHOX immunostaining significantly increased in hippocampal CA1 area after CIH, which was reduced by TEL intervention. (4) CIH significantly increased 8-OHG immunostaining in the hippocampal CA1 area, while intervention with TEL significantly reduced 8-OHG immunostaining. Conclusions: Telmisartan significantly reduced expression of NADPH oxidase p47PHOX and 8-OHG in CIH mice, and improved hippocampusdependent learning, suggesting that telmisartan can reduce CIH-induced neuronal oxidative stress-mediated injury.

Myeloperoxidase and CYBA genetic variants in polycystic ovary syndrome.

Oxidative stress plays a pivotal role in the pathogenesis of polycystic ovary syndrome (PCOS). Genetic variations in myeloperoxidase (MPO; G-463A) and NADPH oxidase p22phox subunit (CYBA; C242T) cause inter-individual variability in enzyme activities. Here, we investigated the associations between MPO activity and the MPO G-463A and CYBA C242T polymorphisms in Chinese women with PCOS. This case-control study included 1003 patients with PCOS and 810 controls. The G-463A and C242T polymorphisms were detected by polymerase chain reaction and restriction analysis, and clinical, hormonal, metabolic and oxidative stress parameters and MPO activity were analysed. The frequencies of the GA+AA genotype and A allele frequency of the MPO G-463A polymorphism were significantly higher in the PCOS group than in the control group. Logistic regression analysis showed that the MPO-463A allele is a risk factor for PCOS (OR=1.261, 95% CI: 1.042-1.526, P=.017). Patients with the AA genotype tended to have higher plasma MPO activity than those with the GG genotype. No statistical significance was found in the genotype and allele frequencies of the CYBA C242T polymorphism between the PCOS and control groups. However, we demonstrated that the coexistence of the MPO A allele (GA+AA genotypes) and the CYBA CC genotype was associated with an increased risk of PCOS when compared with the wild-type GG/CC genotypes (OR=1.302, 95% CI: 1.030-1.646, P=.027). The MPO G-463A variant, but not CYBA C242T variant, is associated with a risk of PCOS in Chinese women.

Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47phox

Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidant N-acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression in p53-/- and INK4a-/-/Arf-/- mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47phox, which results in NADPH oxidase activation. Expression of Akt3 in p47phox-/- MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47phox, but not by the phosphorylation site mutant of p47phox In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important, Akt3 alterations correlate with a higher frequency of mutation of p53, suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.

Effects of late aerobic exercise on cardiac remodeling of rats with small myocardial infarction

Abstract Background Physical exercise has been highlighted as an important non-pharmacological therapy for prevention and treatment of several cardiovascular diseases. However, its effects on hearts with minor cardiac remodeling are not clear. Purpose To evaluate the influence of aerobic physical exercise on functional capacity, cardiac structure, left ventricular (LV) function, and gene expression of NADPH oxidase subunits in rats with small-sized myocardial infarction (MI). Methods Three months after MI induction, Wistar rats were divided into three groups: Sham; sedentary MI (MI-SED); and aerobic exercised MI (MI-EA). Rats exercised three times a week for 12 weeks on a treadmill. Echocardiogram was performed before and after experimental period. Infarction size and cardiomyocyte diameters were evaluated by histology. Gene expression was assessed by RT-PCR. Results Only rats with MI size lower than 30% of LV total area were included in the study. Functional capacity was higher in MI-AE than the other groups. Infarction size did not differ between groups. Infarcted rats had increased LV diastolic and systolic diameter, left atrial diameter, and LV mass, with systolic dysfunction. LV diastolic posterior wall thickness was higher in MI-AE than Sham, and relative wall thickness was lower in MI-SED than MI-AE and Sham groups. Cardiomyocyte diameter was smaller in infarcted groups than Sham. Myocardial gene expression of the NADPH oxidase subunits NOX2, NOX4, p22phox, and p47phox did not differ between groups. Conclusion Small-sized myocardial infarction changes cardiac structures and left ventricular systolic function. Late aerobic physical exercise improves functional capacity and cardiac remodeling by preserving left ventricular geometry. NADPH oxidase subunits gene expression is not involved in cardiac remodeling or modulated by aerobic exercise in rats with small myocardial infarction. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): CNPq; CAPES

Lipid Droplets Contribute to Sepsis-Associated Organ Dysfunction by Disrupting Tissue Tolerance Through the Amplification of Inflammation and Lipid Peroxidation

Sepsis is a complex life-threatening syndrome caused by dysregulated inflammatory and metabolic host response to infection. Alterations in lipid metabolism and an increased number of lipid droplets (LDs) are observed during sepsis; however LD involvement in maladaptive tissue tolerance that culminates in organ dysfunction during sepsis is poorly understood. Here we show that increased accumulation of LDs is associated with peroxidized lipid production, liver dysfunction and sepsis severity. Through the use of antioxidants or CRISPR/Cas9 deletion of the NADPH oxidase subunit p22phox we demonstrate that ROS induced LD biogenesis and oxidatively altered LD content. Moreover, the LDs associated with mitochondria and peridroplet mitochondria in cells from the septic mice showed increased ultrastructural damage. Strikingly, dampening LD accumulation by DGAT1 inhibition decreased the production of inflammatory mediators, reduced lipid peroxidation while improving tissue function. Altogether, we demonstrate that LDs contribute to sepsis-associated organ dysfunction by disrupting tissue tolerance through the amplification of lipid peroxidation, suggesting that LD and changes in lipid metabolism in sepsis may be a target for therapy.

THE PROTECTIVE EFFECT AND MECHANISM OF MESENCHYMAL STEM CELLS ON DIABETIC PULMONARY FIBROSIS IN RATS

Background and Objective: Diabetes affects a variety of organs such as the kidneys, eyes, and liver, and there is increasing evidence that the lung is also one of the target organs of diabetes and imbalance of Sirt3-mediated stress responses such as inflammation, oxidative stress, apoptosis, autophagy, and ER stress may contribute to diabetic pulmonary fibrosis. Although previous studies have reported that mesenchymal stem cells (MSCs) have beneficial effects on various diabetic complications, the effect and mechanisms of MSCs on diabetes-induced lung injury is not clear. Therefore, the present study aims to investigate the protective effect and possible mechanisms of MSCs against diabetic pulmonary fibrosis in rats. Methods and Results: In this study, the STZ-induced diabetes model was constructed in rats, and the effect and potential mechanisms of bone marrow MSCs on diabetic pulmonary fibrosis were investigated. The results revealed that fibrotic changes in the lung were successfully induced in the diabetic rats, while MSCs significantly inhibited or even reversed the changes. Specifically, MSCs up-regulated the expression levels of Sirt3 and SOD2 and then activated the Nrf2/ARE signaling pathway, thereby controlling MDA, GSH content and iNOS, NADPH oxidase subunit p22phox expression levels in the lung tissue. Meanwhile, high levels of Sirt3 and SOD2 induced by MSCs reduced the expression levels of IL-1β, TNF-α, ICAM-1 and MMP9 by suppressing the NF-κB/HMGB1/NLRP3/Caspase1 signaling pathway, as well as regulated the expression levels of cleaved caspasese3, Bax and Bcl2 by up-regulating the expression level of P-Akt, thereby inhibiting the apoptosis of the lung tissue. In addition, MSCs also regulated the expression levels of LC3, P62, BiP, Chop, and PERK, thereby enhancing autophagy and attenuating endoplasmic reticulum stress. Conclusions: In conclusion, our study demonstrates that MSCs effectively ameliorate diabetic pulmonary fibrosis via regulating Sirt3-mediated stress responses, including enhancement of autophagy and inhibition of oxidative stress, inflammation, apoptosis, and endoplasmic reticulum stress. These results not only shed new light on the pathogenesis of diabetic lung injury but also provide a theoretical foundation for further exploration of diabetic pulmonary fibrosis therapeutics. Keywords: Mesenchymal stem cells, diabetic pulmonary fibrosis, Sirt3, stress response This work was supported by the National Natural Science Foundation of China (grant numbers 81870609, 31571474, 81802210).

Cytoskeleton-disrupting agent cytochalasin B reduces oxidative stress caused by high glucose in the human arterial smooth muscle

The role of cytoskeleton dynamics in the oxidative stress toward human vasculature has been unclear. The current study examined whether the cytoskeleton-disrupting agent cytochalasin B reduces oxidative stress caused by high glucose in the human arterial smooth muscle. All experiments in the human omental arteries without endothelium or the cultured human coronary artery smooth muscle cells were performed in d-glucose (5.5 mmol/L). The exposure toward d-glucose (20 mmol/L) for 60 min reduced the relaxation or hyperpolarization to an ATP sensitive K+ channel (KATP) opener levcromakalim (10−8 to 3 × 10−6 mol/L and 3 × 10−6 mol/L, respectively). Cytochalasin B and a superoxide inhibitor Tiron, restored them similarly. Cytochalasin B reduced the NADPH oxidase activity, leading to a decrease in superoxide levels of the arteries treated with high d-glucose. Also, cytochalasin B impaired the F-actin constitution and the membrane translocation of an NADPH oxidase subunit p47phox in artery smooth muscle cells treated with high d-glucose. A clinical concentration of cytochalasin B prevented human vascular smooth muscle malfunction via the oxidative stress caused by high glucose. Regulation of the cytoskeleton may be essential to keep the normal vascular function in patients with hyperglycemia.

Matrine ameliorates cognitive deficits via inhibition of microglia mediated neuroinflammation in an Alzheimer's disease mouse model.

Amyloid β (Aβ) induced microglial activation and attendant neuroinflammation play pivotal roles in Alzheimer's disease (AD) pathogenesis. Matrine is a natural anti-inflammation compound from the Chinese herbal medicine Sophora flavescens Ait. (Kushen). This study aimed to investigate the effects of matrine on memory deficit and neuroinflammation in an oligomeric Aβ (oAβ)-induced AD mice model. Whether microglial activation and NADPH oxidase were involved in these effects were further studied. Different doses of matrine (10, 20, or 40 mg/kg) were intragastrically administered once a day after intracerebroventricular oAβ injection (2.5 μg/μl, 4 μl). 15 days after the oAβ injection, behavioral experiments including novel object recognition (NOR) test and Morris water maze (MWM) test were performed. 21 days after the oAβ injection, concentration of ROS, TNF-α, IL-1β and IL-6 as well as expression of NADPH oxidase subunits gp91phox and p47phox in mice hippocampal tissues were assessed, and microglial activation were evaluated by Iba-1 immunohistochemical staining. Results of NOR test and MWM test revealed that oAβ injection could remarkably impair learning and memory function in AD mice, and matrine administration could significantly ameliorate the impairment. ROS, TNF-α, IL-1β and IL-6 levels increased after oAβ injection, while matrine could significantly reduce the concentrations of these inflammatory factors. OAβ induced protein expression of NADPH oxidase subunits gp91phox and p47phox were also significantly reduced by matrine. Iba-1 immunohistochemistry results showed less activated microglia in matrine-treated mice brain. These results indicate that matrine could ameliorate learning and memory impairment and neuroinflammation induced by oAβ injection. These effects were found to be mediated through inhibition of microglial activation and NADPH oxidase expression in hippocampal tissue. The results suggest that matrine may be a valuable natural compound with therapeutic potential against AD.

P0352P-SELECTIN BLOCKADE AMELIORATES RENAL HYPOXIA OF LUPUS NEPHRITIS IN MRI/LPR MICE

Abstract Background and Aims Lupus nephritis is one of the most common and severe complications of systemic lupus erythematosus, of which poor prognosis is indicated by aggravated renal hypoxia and tubulointerstitial fibrosis. Cell adhesion molecules play a key role in the progression of lupus nephritis tubulointerstitial lesion, including P-selectin, which mediates the rolling of leukocytes and subsequent adhesion and infiltration and then initiates the inflammatory immune response and ischemia and hypoxia injury. However, the effects and mechanisms of P-selectin in lupus nephritis remain to be investigated, and a noninvasive measurement of lupus nephritis tubulointerstitial hypoxia and fibrosis remains to be explored. Method Thirty-four MRL/lpr mice were randomly divided into the following three groups: MRL/lpr, saline, and anti–P-selectin, which consisted of no treatment, treatment with normal saline, and treatment with anti–P-selectin monoclonal antibody (mAb) from 12 to 16 weeks of age, respectively. Ten male C57BL/6 mice of the same age served as normal controls. Twenty-four-hour urinary protein, urinary albumin–creatinine ratio, and periodic acid–Schiff were used to assess kidney damage; Western blot or immunohistochemical staining of the hypoxia probe HypoxyprobeTM-1, hypoxia-inducible factor 1α (HIF-1α), and CD31 were used to evaluate hypoxia in renal tissue; and NADPH oxidase subunit gp91phox and p22phox were used to examine renal oxidative stress. The correlation between kidney injury and blood oxygen level–dependent magnetic resonance imaging (BOLD-MRI) was calculated to assess the clinical value of BOLD-MRI. Results P-selectin is upregulated in lupus nephritis. Blocking P-selectin with mAb alleviated renal tubulointerstitial fibrosis, renal hypoxia, and peritubular capillary loss, without alteration of the levels of lupus activity indicators, anti-dsDNA antibody, or complement C3. BOLD-MRI showed that the reduced R2* values in the renal cortex and medulla of lupus mice were increased when treated with anti–P-selectin mAb as compared with those treated with normal saline, which were negatively correlated with HypoxyprobeTM-1 hypoxia probe and the expression of HIF-1α. Conclusion Early intervention of lupus nephritis with anti–P-selectin mAb can significantly improve the hypoxic state of the kidney and reduce the severity of tubulointerstitial lesions. BOLD-MRI techniques are noninvasive and can dynamically evaluate the changes in renal lesions and intrarenal oxygenation levels before and after treatment in lupus nephritis.

Effects of walnut oil on metabolic profile and transcription factors in rats fed high-carbohydrate-/-fat diets.

The aim of this study was to examine the effects of walnut oil (WO) on metabolic profile and transcription factors in rats fed high carbohydrate (HCD) and high-fat diet (HFD). Forty-two male rats were divided in to six groups: (a) Control, (b) WO (20mg/kg BW), (c) HCD (20% of sucrose), (d) HCD+WO (e) HFD (42% of calories as fat), and (f) HFD+WO. HFD and HCD intake increased final body weights by 19% and 23% and visceral fat weights by 3- and 5-fold, respectively (p<.05 for all). In addition, serum glucose, total cholesterol, triglyceride, and free fatty acids (FFA) insulin, leptin, and MDA levels increased in rats fed with HFD and HCD. WO supplementation improved these metabolic parameters (p<.05 for all). HFD+WO and HCD+WO treated groups had a significant reduction in serum and liver malondialdehyde (MDA) levels by 12% or 15% (p<.05 for both). In addition, WO supplementation lowered the levels of hepatic nuclear factor kappa B (NF-κB) and NADPH oxidase subunit p22phox , whereas increased the endothelial-NO synthase (e-NOS), nuclear factor erythroid 2-related factor-2, and sirtuin-1 levels. In conclusion, WO supplementation could alleviate the adverse impacts of both HCD and HFD in the rats. PRACTICAL APPLICATIONS: This study suggests that WO intake can modulate carbohydrate metabolism and increase antioxidant capacity. These properties might be partially mediated through the regulation of the transcription factors and some proteins involved in energy metabolism, as well as a balance of oxidative stress, and insulin sensitivity.