Down-regulation of NF-κB signalling by polyphenolic compounds prevents endotoxin-induced liver injury in a rat model

Activation of NF-κB has been reported to play a key role in causing endotoxin-induced hepatic damage through enhanced production of reactive oxygen species and pro-inflammatory mediators. In this context, the potential of polyphenolic phytochemicals in preventing endotoxin-induced liver damage remains unclear. Here, we demonstrate that catechin and quercetin have the potential to down-regulate the initial signalling molecule NF-κB which may further inhibit the downstream cascade including TNF-α and NO. These results were confirmed using N-nitro-L-arginine methyl ester (L-NAME), the inhibitor of inducible nitric oxide synthase (iNOS) along with the biochemical and histological alterations occurring in the presence and absence of supplementation with both the polyphenols. However, catechin was found to be more effective than quercetin against endotoxin-induced liver injury. These findings suggest that these polyphenols may form a pharmacological basis for designing a therapeutic agent against endotoxin-mediated oxidative damage.     

Small interfering RNA targeting NF-κB attenuates lipopolysaccharide-induced acute lung injury in rats

Background

Veterinary cardiology, especially electrocardiography, has shown major advancements for all animal species. Consequently, the number of ovine species used as experimental animals has increased to date. Few studies have been published on ovine systematic electrocardiography, particularly with respect to lamb physiology and neonatology. This study aimed to standardize the values of normal waves, complexes, and intervals of the electrocardiogram (ECG) in clinically Bergamasca healthy neonatal lambs, used as experimental animals. Serial computerized electrocardiography was performed in 10 male and 12 female neonates on the 1st, 7th, 14th, 21st, 28th, and 35th days of age. The following parameters were analyzed: heart rate and rhythm, duration and amplitude of waves, duration of intervals, and heart electrical axis.

Results

During the first 35 days of life, (1) the sinusal heart rhythm was predominant, (2) there was a progressive decrease in the heart rate and R and T wave amplitude, and (3) a progressive increase in the PR, QT, and RR intervals. Finally, we confirmed that various components of neonatal evolution were more discernible in the augmented unipolar leads (aVF), which we recommend should be preferentially used in future studies. No significant statistical alterations were observed between males and females in relation to the analyzed parameters.

Conclusions

The information assimilated in this study is anticipated to enhance the diagnosis of multiple congenital heart defects in Bergamasca lambs and could be implemented in studies that use ovine species as experimental models.

     

NF-κB activation in myeloid cells mediates ventilator-induced lung injury

Background

Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated.

Methods

To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKβ^△mye), IL-6^-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI.

Results

Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβ^△mye mice as well as in IL6^-/- to WT chimeric mice.

Conclusion

Given that IKKβ^△mye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB–IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.     

Gprc5a-deficiency confers susceptibility to endotoxin-induced acute lung injury via NF-κB pathway

Susceptibility to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) varies greatly among patients in sepsis/septic shock. The genetic and biochemical reasons for the difference are not fully understood. G protein coupled receptor family C group 5 member A (GPRC5A), a retinoic acid target gene, is predominately expressed in the bronchioalveolar epithelium of lung. We hypothesized that Gprc5a is important in controlling the susceptibility to ALI or ARDS. In this study, we examined the susceptibility of wild-type and Gprc5a-knockout (ko) mice to induced ALI. Administration of endotoxin LPS induced an increased pulmonary edema and injury in Gprc5a-ko mice, compared to wild-type counterparts. Consistently, LPS administration induced higher levels of inflammatory cytokines (IL-1β and TNFα) and chemokine (KC) in Gprc5a-ko mouse lungs than in wild-type. The enhanced pulmonary inflammatory responses were associated with dysregulated NF-κB signaling in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs. Importantly, selective inhibition of NF-κB through expression of the super-repressor IκBα in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs alleviated the LPS-induced pulmonary injury, and inflammatory response. Thus, Gprc5a is critical for lung homeostasis, and Gprc5a deficiency confers the susceptibility to endotoxin-induced pulmonary edema and injury, mainly through NF-κB signaling in bronchioalveolar epithelium of lung.     

Curcumin protects against acute liver damage in the rat by inhibiting NF-κB,proinflammatory cytokines production and oxidative stress

Curcumin, an anti-inflammatory and antioxidant compound, was evaluated for its ability to suppress acute carbon tetrachloride-induced liver damage. Acute hepatotoxicity was induced by oral administration of CCl₄ (4 g/kg, p.o.). Curcumin treatment (200 mg/kg, p.o.) was given before and 2 h after CCl₄ administration. Indicators of necrosis (alanine aminotransferase) and cholestasis (γ-glutamyl transpeptidase and bilirubins) resulted in significant increases after CCl₄ intoxication, but these effects were prevented by curcumin treatment. As an indicator of oxidative stress, GSH was oxidized and the GSH/GSSG ratio decreased significantly by CCl₄, but was preserved within normal values by curcumin. In addition to its antioxidants properties, curcumin is capable of preventing NF-κB activation and therefore to prevent the secretion of proinflammatory cytokines. Therefore, in this study we determined the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) mRNA, and NF-κB activation. CCl₄-administered rats depicted significant increases in TNF-α, IL-1β, and IL-6 production, while curcumin remarkably suppressed these mediators of inflammation in liver damage. These results were confirmed by measuring TNF-α, and IL-1β protein production using Western Blot analysis. Accordingly, these proteins were increased by CCl₄ and this effect was abolished by curcumin. Administration of CCl₄ induced the translocation of NF-κB to the nucleus; CCl₄ induced NF-κB DNA binding activity was blocked by curcumin treatment. These findings suggest that curcumin prevents acute liver damage by at least two mechanisms: acting as an antioxidant and by inhibiting NF-κB activation and thus production of proinflammatory cytokines.     

Rutin decreases lipopolysaccharide-induced acute lung injury via inhibition of oxidative stress and the MAPK–NF-κB pathway

Acute lung injury (ALI) is a serious disease with unacceptably high mortality and morbidity rates. Up to now, no effective therapeutic strategy for ALI has been established. Rutin, quercetin-3-rhamnosyl glucoside, expresses a wide range of biological activities and pharmacological effects, such as anti-inflammatory, antihypertensive, anticarcinogenic, vasoprotective, and cardioprotective activities. Pretreatment with rutin inhibited not only histopathological changes in lung tissues but also infiltration of polymorphonuclear granulocytes into bronchoalveolar lavage fluid in lipopolysaccharide (LPS)-induced ALI. In addition, LPS-induced inflammatory responses, including increased secretion of proinflammatory cytokines and lipid peroxidation, were inhibited by rutin in a concentration-dependent manner. Furthermore, rutin suppressed phosphorylation of NF-κB and MAPK and degradation of IκB, an NF-κB inhibitor. Decreased activities of antioxidative enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and heme oxygenase-1 caused by LPS were reversed by rutin. At the same time, we found that ALI amelioration by chelation of extracellular metal ions with rutin is more efficacious than with deferoxamine. These results indicate that the protective mechanism of rutin is through inhibition of MAPK–NF-κB activation and upregulation of antioxidative enzymes.     

Resolvin D1 attenuates inflammation in lipopolysaccharide-induced acute lung injury through a process involving the PPARγ/NF-κB pathway

Background

Docosahexaenoic acid (DHA) and DHA-derived lipid mediators have recently been shown to possess anti-inflammatory and pro-resolving properties. In fact, DHA can down-regulate lipolysaccharide (LPS)-induced activation of NF-κB via a PPARγ-dependent pathway. We sought to investigate the effects of the novel DHA-derived mediator resolvin D1 (RvD1) on LPS-induced acute lung injury and to determine whether these effects occur via a PPARγ-dependent pathway.

Methods

BALB/c mice aged 6–8 weeks were randomly divided into seven groups: two control groups receiving saline or RvD1 (600 ng) without LPS; a control group receiving LPS only; an experimental group receiving RvD1 (300 ng) or RvD1 (600 ng), followed by LPS; a group receiving the PPARγ antagonist GW9662; and a group receiving GW9662, then RvD1 (600 ng) and finally LPS. LPS (50 μM) and saline were administered intratracheally. RvD1 was injected intravenously 24 h and 30 min before LPS, while GW9662 was injected intravenously 30 min before RvD1. Mice were killed at 6, 12, and 24 h. Samples of bronchoalveolar lavage fluid (BALF) were analyzed for cell counts and cytokine analysis. Lung tissues were collected for histology, Western blotting and electrophoretic mobility shift assays (EMSAs).

Results

At all three time points, groups receiving either dose of RvD1 followed by LPS had significantly lower total leukocyte counts and levels of TNF-α and IL-6 levels in BALF than did the group given only LPS. RvD1 markedly attenuated LPS-induced lung inflammation at 24 h, based on hematoxylin-eosin staining of histology sections. RvD1 activated PPARγ and suppressed IκBα degradation and NF-κB p65 nuclear translocation, based on Western blots and EMSAs. The PPARγ inhibitor GW9662 partially reversed RvD1-induced suppression of IκBα degradation and p65 nuclear translocation.

Conclusions

These results suggest that RvD1 may attenuate lung inflammation of LPS-induced acute lung injury by suppressing NF-κB activation through a mechanism partly dependent on PPARγ activation.     

Reduning injection ameliorates paraquat-induced acute lung injury by regulating AMPK/MAPK/NF-κB signaling

Reduning injection (RDN), a patented Chinese medicine, is broadly used for common cold and lung infection in clinic, but the mechanism underlying its effects on inflammation-related pulmonary injury remains unclear. Paraquat (PQ, bolus 15 mg/kg dose, ip) was administered for acute lung injury induction in mice, which were orally administered dexamethasone (2 mg/kg) or RDN (50 and 100 mg/kg/day) for 5 days. After treatment, plasma and lung tissue samples from the euthanized animals were obtained and analyzed by histological, biochemical and immunoblot assays. Histological observation demonstrated RDN alleviated PQ-induced lung damage. Meanwhile, RDN suppressed myeloperoxidase (MPO) activity, reduced the wet/dry (W/D) ratio and decreased the amounts of total leukocytes and neutrophils. Treatment also markedly decreased the amounts of malondialdehyde, MPO, and inflammatory cytokines while increasing superoxide dismutase activity in comparison with the PQ group. In immunoblot, RDN blocked the phosphorylation levels of adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK), JNK, ERK, p38, inhibitor of nuclear factor κB kinase and nuclear factor-κB (NF-κB) in lung tissue specimens in PQ-challenged animals, which was further verified in vitro. The above data indicated protective effects for RDN in PQ-induced lung damage, possibly through inhibition of the AMPK/MAPK/NF-κB pathway.     

Wogonin alleviates liver injury in sepsis through Nrf2-mediated NF-κB signalling suppression

Sepsis is a life-threatening organ dysfunction syndrome, and liver is a susceptible target organ in sepsis, because the activation of inflammatory pathways contributes to septic liver injury. Oxidative stress has been documented to participate in septic liver injury, because it not only directly induces oxidative genotoxicity, but also exacerbates inflammatory pathways to potentiate damage of liver. Therefore, to ameliorate oxidative stress is promising for protecting liver in sepsis. Wogonin is the compound extracted from the medicinal plant Scutellaria baicalensis Geogi and was found to exert therapeutic effects in multiple inflammatory diseases via alleviation of oxidative stress. However, whether wogonin is able to mitigate septic liver injury remains unknown. Herein, we firstly proved that wogonin treatment could improve survival of mice with lipopolysaccharide (LPS)- or caecal ligation and puncture (CLP)-induced sepsis, together with restoration of reduced body temperature and respiratory rate, and suppression of several pro-inflammatory cytokines in circulation. Then, we found that wogonin effectively alleviated liver injury via potentiation of the anti-oxidative capacity. To be specific, wogonin activated Nrf2 thereby promoting expressions of anti-oxidative enzymes including NQO-1, GST, HO-1, SOD1 and SOD2 in hepatocytes. Moreover, wogonin-induced Nrf2 activation could suppress NF-κB-regulated up-regulation of pro-inflammatory cytokines. Ultimately, we provided in vivo evidence that wogonin activated Nrf2 signalling, potentiated anti-oxidative enzymes and inhibited NF-κB-regulated pro-inflammatory signalling. Taken together, this study demonstrates that wogonin can be the potential therapeutic agent for alleviating liver injury in sepsis by simultaneously ameliorating oxidative stress and inflammatory response through the activation of Nrf2.     

Dexmedetomidine improves acute stress-induced liver injury in rats by regulating MKP-1, inhibiting NF-κB pathway and cell apoptosis

Acute stress is a frequent and unpredictable disease for many animals. Stress is widely considered to affect liver function. However, the underlying mechanism by which dexmedetomidine (DEX) attenuates acute stress-induced liver injury in rats remains unclear. In this study, we used forced swimming for 15 min and acute 3-hr restraint stress model. Behavioral tests and changes in norepinephrine levels confirmed the successful establishment of the acute stress model. Acute stress-induced liver injury, evidenced by hematoxylin and eosin-stained pathological sections and increased serum aminotransferase and aspartate aminotransferase levels, was reduced in DEX-treated livers. Reactive oxygen species and oxidative stress levels were dramatically decreased with DEX treatment compared with acute stress-induced liver injury. DEX significantly reduced acute stress-induced liver inflammation and apoptosis, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and inflammation and apoptosis-related protein levels. DEX treatment also effectively inhibited acute stress-induced c-Jun N-terminal kinase (JNK), P38, and BAD signaling pathway activation, and significantly induced MKP-1 activation. Thus, DEX has a protective effect on acute-stress-induced liver injury by reducing inflammation and apoptosis, which suggests a potential clinical application for DEX in stress syndrome.     

Ginkgetin exerts anti-inflammatory effects on cerebral ischemia/reperfusion-induced injury in a rat model via the TLR4/NF-κB signaling pathway

Ginkgo biloba, a natural biflavonoid isolated from Ginkgo biloba leaves, is reported to have strong anti-inflammatory and immunosuppressive properties. The aim of this study is to investigate the potential anti-inflammatory mechanisms of ginkgo flavonoids on cerebral ischemia/reperfusion (I/R) injury. Inflammatory-associated cytokines in cerebral ischemic hemispheres were determined by immunohistochemical staining, Western blot and enzyme-like immunosorbent assay (ELISA). Our results indicated that treatment with Ginkgetin significantly restored rat brain I/R-induced neurological deficit scores. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in Ginkgetin treatment group (100 mg/kg) also significantly reduced. The expression inflammation-related protein prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-8 (IL-8) was also decreased in Ginkgetin treatment group. However, the expression of interleukin-10 (IL-10) was remarkably increased. Thus, this study demonstrates that Ginkgetin protects neurons from I/R-induced rat injury by down-regulating pro-inflammatory cytokines and blocking the TLR4/NF-κB pathway.     

NF-κB involvement in hyperoxia-induced myocardial damage in newborn rat hearts

Premature newborns are frequently exposed to hyperoxia ventilation and some literature data indicate the possibility of hyperoxia-induced myocardial damage. Since nuclear factor κB (NF-κB) is a crucial signaling molecule involved in physiological response to hyperoxia in different cell types as well as in various tissues, our attention has been focused on the role played by NF-κB pathway in response to moderate and severe hyperoxia exposure in rat neonatal heart tissue. Akt and IκBα levels, involved in NF-κB activation, along with the balance between apoptotic and survival pathways have also been investigated. Experimental design of the study has involved exposure of newborn rats to room air (controls), 60 % O₂ (moderate hyperoxia), or 95 % O₂ (severe hyperoxia) for the first two postnatal weeks. Morphological analysis shows a less compact tissue in rat heart exposed to moderate hyperoxia and a decreased number of nuclei in samples exposed to severe hyperoxia. A significant increase of NF-κB positive nuclei percentage and p-IκBα expression in samples exposed to 95 % hyperoxia compared to control and to 60 % hyperoxia is evidenced; in parallel, an increase of pAkt/Akt ratio in both samples exposed to 95 and 60 % hyperoxia is shown. Furthermore, a more evident cytochrome c/Apaf-1 immunocomplex and a decreased Bcl2 expression in 95 % hyperoxia-exposed sample compared to 60 % exposed one is evidenced. In conclusion, our findings suggest the involvement of the NF-κB pathway and Akt signaling in the mechanisms of myocardial hyperoxic damage in the newborns, with particular reference to the induction of oxidative stress-related apoptosis.     

NF-κB acts downstream of EGFR in regulating low dose cadmium induced primary lung cell proliferation

Apart from cytotoxicity cadmium has no special attributes towards cell’s physiological function. The role of cadmium with respect to cell growth is still under debate. Mitogen activated protein kinase and Ca²⁺/calmodulin dependent protein kinase dependent pathways are the two elaborately studied concerning cadmium induced cell proliferation. Low concentration of cadmium chloride (2.5 μM) was applied to mice primary lung epithelial cells and cell proliferation was measured both by cell cycle analysis and Brdu incorporation assay. Effects of differential dose of cadmium chloride on lung epithelial cells were evaluated morphologically by atomic force microscopy. RT-PCR and western blot altogether corroborated the specific signalling pathways concerning cadmium induced lung cell proliferation. Cadmium induced lung epithelial cells which over-expressed EGFR, were transfected with siEGFR, revealed downstream molecules and RNAi induced EGFR silencing. Use of siEGFR effectively prevents expression of proinflammatory and cell proliferative markers. Moreover N-acetyl cysteine and ascorbic acid mediated inhibition of EGFR and downstream signalling molecules indicate the involvement of reactive oxygen species. Exposure to low concentration of cadmium promotes the growth of primary mice lung epithelial cell by EGFR signalling. We have also transfected the primary lung epithelial cell with siRNA against the regulatory subunit of nuclear factor-κB (NF-κB) and the data shows that cadmium induced lung cell proliferation is the effect of EGFR mediated NF-κB activation.     

NF-κB signaling in fetal lung macrophages disrupts airway morphogenesis

Bronchopulmonary dysplasia is a common pulmonary complication of extreme prematurity. Arrested lung development leads to bronchopulmonary dysplasia, but the molecular pathways that cause this arrest are unclear. Lung injury and inflammation increase disease risk, but the cellular site of the inflammatory response and the potential role of localized inflammatory signaling in inhibiting lung morphogenesis are not known. In this study, we show that tissue macrophages present in the fetal mouse lung mediate the inflammatory response to LPS and that macrophage activation inhibits airway morphogenesis. Macrophage depletion or targeted inactivation of the NF-κB signaling pathway protected airway branching in cultured lung explants from the effects of LPS. Macrophages also appear to be the primary cellular site of IL-1β production following LPS exposure. Conversely, targeted NF-κB activation in transgenic macrophages was sufficient to inhibit airway morphogenesis. Macrophage activation in vivo inhibited expression of multiple genes critical for normal lung development, leading to thickened lung interstitium, reduced airway branching, and perinatal death. We propose that fetal lung macrophage activation contributes to bronchopulmonary dysplasia by generating a localized inflammatory response that disrupts developmental signals critical for lung formation.     

Role of NF-κB in the skeleton

Since the discovery that deletion of the NF-κB subunits p50 and p52 causes osteopetrosis in mice, there has been considerable interest in the role of NF-κB signaling in bone. NF-κB controls the differentiation or activity of the major skeletal cell types - osteoclasts, osteoblasts, osteocytes and chondrocytes. However, with five NF-κB subunits and two distinct activation pathways, not all NF-κB signals lead to the same physiologic responses. In this review, we will describe the roles of various NF-κB proteins in basal bone homeostasis and disease states, and explore how NF-κB inhibition might be utilized therapeutically.     

Adverse effects of α-ketoglutarate/malate in a rat model of acute kidney injury

Acute kidney injury (AKI) is the most common kidney disease in hospitalized patients with high mortality. Ischemia and reperfusion (I/R) is one of the major causes of AKI. The combination of α-ketoglutarate+malate (αKG/MAL) showed the ability to reduce hypoxia-induced damage to isolated proximal tubules. The present study utilizes a rat model of I/R-induced AKI accompanied by intensive biomonitoring to examine whether αKG/MAL provides protection in vivo. AKI was induced in male Sprague-Dawley rats by bilateral renal clamping (40 min) followed by reperfusion (240 min). αKG/MAL was infused continuously for 60 min before and 45 min after ischemia. Normoxic and I/R control groups received 0.9% NaCl solution. The effect of αKG/MAL was evaluated by biomonitoring, blood and plasma parameters, histopathology, and immunohistochemical staining for kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), as well as by determination of tissue ATP and nonesterified fatty acid concentrations. Intravenous infusion of αKG/MAL at a cumulative dose of 1 mmol/kg each (146 mg/kg αKG and 134 mg/kg MAL) did not prevent I/R-induced increases in plasma creatinine, histopathological alterations, or cortical ATP depletion. On the contrary, the most notable adverse affect in animals receiving αKG/MAL was the decrease in mean arterial blood pressure, which was also accompanied by a reduction in heart rate. Supplementation with αKG/MAL, which is very protective against hypoxia-induced injury in isolated proximal tubules, does not protect against I/R-induced renal injury in vivo, possibly due to cardiovascular depressive effects.