Nuclear factor-kappaB P65 Activation Research Articles

Development of chimeric peptides to facilitate the neutralisation of lipopolysaccharides during bactericidal targeting of multidrug-resistant Escherichia coli

Pathogenic Escherichia coli can cause fatal diarrheal diseases in both animals and humans. However, no antibiotics or antimicrobial peptides (AMPs) can adequately kill resistant bacteria and clear bacterial endotoxin, lipopolysaccharide (LPS) which leads to inflammation and sepsis. Here, the LPS-targeted smart chimeric peptides (SCPs)-A6 and G6 are generated by connecting LPS-targeting peptide-LBP14 and killing domain-N6 via different linkers. Rigid and flexible linkers retain the independent biological activities from each component. SCPs-A6 and G6 exert low toxicity and no bacterial resistance, and they more rapidly kill multiple-drug-resistant E. coli and more effectively neutralize LPS toxicity than N6 alone. The SCPs can enhance mouse survival more effectively than N6 or polymyxin B and alleviate lung injuries by blocking mitogen-activated protein kinase and nuclear factor kappa-B p65 activation. These findings uniquely show that SCPs-A6 and G6 may be promising dual-function candidates as improved antibacterial and anti-endotoxin agents to treat bacterial infection and sepsis.

Effects of matrine on apoptosis and nuclear factor kappaBp65 activity of HL-60 cells induced by pirarubicin

Objective To explore the effects of matrine on apoptosis and nuclear factor(NF)-κBp65 activity of HL-60 cells induced by pirarubicin(THP). Methods Effects of the apoptosis: the HL-60 cells in blank control group were cultured 14 hours with RPMI 1640; groups of different concentration drugs: matrine: 0.1 μmol/L, 1.0 μmol/L, 10.0 μmol/L and THP: 1.0 μmol/L, 10.0 μmol/L, 100.0 μmol/L.The apoptosis of HL-60 cells were tested by fluorescence double colour painting and flow cytometry(FCM). The NF-κBp65 activity of HL-60 cells were determined by FCM. Results The rates of apoptosis of HL-60 cells were (7.14±2.95)%, (12.34±2.55)%, (19.3±2.31)% and (31.78±4.31)%, (47.25±5.27)%, (56.49±1.59)% in matrine 0.1 μmol/L, 1.0 μmol/L, 10.0 μmol/L and THP 1.0 μmol/L, 10.0 μmol/L, 100.0 μmol/L groups, compared to blank control group(6.46±1.45)%, there were significant difference (F=257.72, 677.19; all P<0.001); (70.17±5.68)% in matrine 0.1 μmol/L + THP 1.0 μmol/L group[vs the THP 1.0 μmol/L group (31.78±4.31)%, (t=38.94, P<0.001)]. The rates of NF-κBp65 activity of HL-60 cells in matrine 0.1 μmol/L, 1.0 μmol/L, 10.0 μmol/L and THP 1.0 μmol/L, 10.0 μmol/L, 100.0 μmol/L groups were(8.34±1.52)%, (7.11±1.29)%, (4.78±0.31)% and (16.21±1.20)%, (23.98±3.21)%, (32.44±2.89)%, with the control group( 8.44±2.20)%, there were significant difference(F=65.35, P<0.001; F=674.11, P<0.001); (12.01±2.27)% in matrine 0.1 μmol/L + THP 1.0 μmol/L group [with (16.21±1.20)% of THP 1.0 μmol/L group(t=21.42, P<0.001)]. Conclusions The apoptosis of HL-60 cell is induced by matrine and pirarubicin in a dose-dependent manner.NF-κBp65 activity of HL-60 cell is inhibited by matrine and increased by pirarubicin in a dose-dependent manner.Matrine can enhannce the effect of induction by pirarubicin of apoptosis of HL-60 cell, and decrease activity of NF-κBp65 by pirarubicin. Key words: Matrine; Pirarubicin; Drug combination; Apoptosis; Nuclear factor-κBp65 activity

Anesthetic Isoflurane Posttreatment Attenuates Experimental Lung Injury by Inhibiting Inflammation and Apoptosis

We investigated the effect of 1.4% isoflurane (ISO) on the development of inflammation and apoptosis caused by zymosan (ZY) in mice. We found that ZY-challenged mice exhibited significant body weight loss, markedly high mortality, and significant lung injury characterized by the deterioration of histopathology, histologic scores, and wet-to-dry ratio after ISO treatment. ISO dramatically attenuated ZY-induced lung neutrophil recruitment and inflammation, as evidenced by the reduced levels of total cells, neutrophils, and proinflammatory cytokines (i.e., tumor necrosis factor-α, interleukin- (IL-) 1β, IL-6, and macrophage inflammatory protein-2) in bronchoalveolar lavage fluid and of their mRNA expression in lung tissues. ISO also inhibited ZY-induced expression and activation of nuclear factor-kappaB p65 and inducible nitric oxide synthase in pulmonary tissue. ZY administration also resulted in the upregulation of heme oxygenase-1 expression and activity in the lung, which was further enhanced by ISO treatment. Moreover, ISO markedly prevented ZY-induced pulmonary cell apoptosis in mice, as reflected by the decrease in expression of procaspase-8, procaspase-3, cleaved caspase-8, and cleaved caspase-3, as well as in caspase-3 activity and Bcl-2-associated X/B-cell lymphoma 2 ratio. These results indicate that ISO is a potential therapeutic drug for treating ZY-induced lung injury, and further investigations are warranted.

RESUSCITATION WITH HYDROXYETHYL STARCH SOLUTION PREVENTS CD4+ T-LYMPHOCYTE APOPTOSIS AND MODULATES THE BALANCE OF T HELPER TYPE 1 AND T HELPER TYPE 2 RESPONSES IN THE RAT WITH TRAUMATIC VIRGULE/SHILL HEMORRHAGIC SHOCK

Trauma/hemorrhagic shock (TH/S) has been associated with inflammation and immunodisorders, leading to immunosuppression, multiorgan dysfunction, and death. However, little is known about the effect of resuscitation with different solutions on the immunological function. To address this issue, groups of male Sprague-Dawley rats were induced with TH/S by fracture in the left femur and continual bleeding to keep the MAP of 30 +/- 5 mmHg for 30 min, followed by resuscitation with 6% hydroxyethyl starch solution (HES), Ringer's lactate solution (RS), or 5% albumin (ALB), and the impact of resuscitation on the activation, differentiation, and survival of CD4 T cells was longitudinally examined after TH/S and resuscitation. After resuscitation, the MAP, as expected, gradually increased regardless of the type of fluids transfused. The percentage of CD4+ T cells decreased to 20% to 25%, and the ratio of T helper type 1 (TH1)/TH2 responses was significantly reduced in all TH/S rats, however, resuscitation with HES alone reversed the trends (49.4% +/- 9.7% vs. 55.2% +/- 2.6% in sham for CD4 T cells; 0.64 +/- 0.23 vs. 0.71 +/- 0.16 in sham for the ratio of TH1/TH2, P > 0.05 for both). Treatment with HES or ALB, but not RS, prevented CD4 T-cell apoptosis (sham, 7.23% +/- 3.4%; HES, 10.2% +/- 4.1%; RS, 15.2% +/- 5.4%; ALB, 10.6% +/- 4.3%; 48 h) and nuclear factor-kappaB p65 activation (sham, 0.17 +/- 0.04; HES, 0.34 +/- 0.05; RS, 0.41 +/- 0.09; ALB, 0.25 +/- 0.09; 48 h) induced by TH/S early after resuscitation. These data demonstrated that HES resuscitation modulated the balance of TH1 and TH2 responses and inhibited TH/S-related nuclear factor-kappaB activation and CD4 T-cell apoptosis in TH/S rats. Our findings provide new insights into understanding the TH/S-related immunodisorders and may aid in the design of new therapy for intervention of TH/S.

Concomitant Reduction of Low-Density Lipoprotein-Cholesterol and Biomarkers of Inflammation with Low-Dose Simvastatin Therapy in Patients with Type 1 Diabetes

Cardiovascular disease is a major cause of mortality in type 1 diabetes (TIDM). TIDM is a proinflammatory state. Whereas there is consensus on lipid management in type 2 diabetes, there is a lack of data in type 1 diabetes. In addition to benefits on the lipid profile, statin therapy is antiinflammatory. There are scant data on statin therapy in T1DM. Thus, we tested the effect of simvastatin, compared with placebo, on biomarkers of inflammation and monocyte function in TIDM patients. This was a double-blind, randomized, placebo-controlled study of T1DM patients, randomized to placebo or simvastatin, 20 mg/d for 3 months. The study was conducted at the University of California, Davis, Medical Center. Participants included patients with T1DM. Analytes measured at baseline and 3 months included liver function tests, creatinine, hemoglobin AIC, high-sensitivity C-reactive protein, soluble CD40 ligand, monocyte O(2)(-), cytokines, nuclear factor-kappaB. Simvastatin therapy resulted in significant reduction in low-density lipoprotein and non-high-density lipoprotein cholesterol, high-sensitivity C-reactive protein (18% reduction, P < 0.001) and soluble CD40 ligand (22% reduction, P < 0.05), compared with placebo. Simvastatin therapy significantly inhibited lipopolysaccharide-activated monocyte release of O(2)(-) (P < 0.0005), IL-8 (P < 0.03), and TNF (P < 0.02). Simvastatin therapy significantly inhibited monocyte IL-6 release, compared with baseline (P = 0.02). Simvastatin therapy also significantly reduced monocytic nuclear factor-kappaB p65 activity, compared with placebo (P < 0.01). This study demonstrates that simvastatin (20 mg/d) is safe in T1DM patients and has concomitant benefits on the lipid profile and biomarkers of inflammation. These novel findings could have implications for developing policy guidelines for statin therapy in forestalling vascular complications in young T1DM.

Insulin reduces the multiple organ injury and dysfunction caused by coadministration of lipopolysaccharide and peptidoglycan independently of blood glucose: Role of glycogen synthase kinase-3β inhibition*

Insulin reduces morbidity and mortality among critically ill patients, but the molecular mechanisms of its effect remain unknown. Insulin is a well-known inhibitor of glycogen synthase kinase-3, which may play an important role in systemic inflammation and shock. Here we investigate the role of blood glucose and glycogen synthase kinase-3beta inhibition in the protective effect of insulin on the organ injury/dysfunction associated with excessive systemic inflammation. Prospective, randomized study. University-based research laboratory. Eighty-five anesthetized Wistar rats. Rats received Escherichia coli lipopolysaccharide (1 mg/kg) and Staphylococcus aureus peptidoglycan (0.3 mg/kg) or vehicle intravenously. Insulin (1.4 units/kg intravenously) was administered in the absence or presence of continuous glucose administration (4.5 mg/kg/hr intravenously) either prophylactically or therapeutically. The potent and selective glycogen synthase kinase-3beta inhibitor TDZD-8 (1 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered either prophylactically or therapeutically. Coadministration of lipopolysaccharide and peptidoglycan resulted in increases in the serum levels of creatinine (indicator of renal dysfunction), alanine aminotransferase, and aspartate aminotransferase (indicators of liver injury) at 6 hrs. Insulin or TDZD-8 similarly attenuated the organ injury/dysfunction caused by lipopolysaccharide and peptidoglycan when given either prophylactically or therapeutically. Continuous glucose administration had no effect on blood glucose levels or organ injury/dysfunction at 6 hrs. Treatment with insulin or TDZD-8 reduced the plasma levels of the proinflammatory cytokine interleukin-1beta. In vitro, insulin or TDZD-8 caused similar reductions in the nuclear factor-kappaB p65 activity and similar increases in the phosphorylation of Ser9 of glycogen synthase kinase-3beta. Therapy with insulin or the potent and selective glycogen synthase kinase-3beta inhibitor TDZD-8 reduced the organ injury/dysfunction caused by lipopolysaccharide and peptidoglycan in the rat. We propose that the inhibitory effect of insulin on the activity of glycogen synthase kinase-3beta contributes to the protective effect of insulin against the organ injury/dysfunction caused by excessive systemic inflammation independently of any effects on blood glucose.

Cilostazol Suppresses Superoxide Production and Expression of Adhesion Molecules in Human Endothelial Cells via Mediation of cAMP-Dependent Protein Kinase-Mediated Maxi-K Channel Activation

This study shows whether increased intracellular cAMP level by cilostazol is directly coupled to its maxi-K channel activation in human endothelial cells. Cilostazol (1 microM) increased the K+ currents in the human endothelial cells by activating maxi-K channels, which was abolished by iberiotoxin (100 nM), a maxi-K channel blocker. On incubation of human coronary artery endothelial cells with tumor necrosis factor-alpha (TNF-alpha) (50 ng/ml), monocyte adhesion significantly increased with increased superoxide generation and expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) accompanied by increased degradation of inhibitory kappaBalpha in cytoplasm and activation of nuclear factor-kappaB p65 in nucleus. All these variables were significantly suppressed by cilostazol (10 microM), which was antagonized by iberiotoxin (1 microM) and (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) (300 nM, cAMP-dependent protein kinase inhibitor), but not by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindo-lo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT 5823) (300 nM, cGMP-dependent protein kinase inhibitor). In the human endothelial cells transfected with siRNA-targeting maxi-K channels, cilostazol did not suppress the superoxide generation, VCAM-1 and MCP-1 expressions, and monocyte adhesion as contrasted with the wild-type cells. These findings were similarly evident with (3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one (BMS-204352), a maxi-K channel opener, and forskolin and dibutyryl cAMP. In conclusion, increased cAMP level by cilostazol is directly coupled to its maxi-K channel opening action via protein kinase activation in human endothelial cells, thereby suppressing TNF-alpha-stimulated superoxide production and expression of adhesion molecules.

GSK-3β inhibitors attenuate the organ injury/dysfunction caused by endotoxemia in the rat*

Serine-threonine protein kinase glycogen synthase kinase (GSK)-3 is involved in regulation of many cell functions, but its role in regulation of inflammatory response is unknown. Here we investigate the effects of GSK-3beta inhibition on organ injury/dysfunction caused by lipopolysaccharide or coadministration of lipopolysaccharide and peptidoglycan in the rat. Prospective, randomized study. University-based research laboratory. Ninety-nine anesthetized male Wistar rats. Study 1: Rats received either intravenous Escherichia coli lipopolysaccharide (6 mg/kg) or vehicle (1 mL/kg; saline). Study 2: Rats received either intravenous E. coli lipopolysaccharide (1 mg/kg) and Staphylococcus aureus peptidoglycan (0.3 mg/kg) or vehicle. The potent and selective GSK-3beta inhibitors TDZD-8 (1 mg/kg intravenously), SB216763 (0.6 mg/kg intravenously), and SB415286 (1 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered 30 mins before lipopolysaccharide or lipopolysaccharide and peptidoglycan. Endotoxemia resulted in increases in the serum levels of creatinine (indicator of renal dysfunction), aspartate aminotransferase, alanine aminotransferase (markers for hepatocellular injury), lipase (indicator of pancreatic injury), and creatine kinase (indicator of neuromuscular injury). Coadministration of lipopolysaccharide and peptidoglycan resulted in hepatocellular injury and renal dysfunction. All GSK-3beta inhibitors attenuated the organ injury/dysfunction caused by lipopolysaccharide or lipopolysaccharide and peptidoglycan. GSK-3beta inhibition reduced the Ser536 phosphorylation of nuclear factor-kappaB subunit p65 and the messenger RNA expression of nuclear factor-kappaB-dependent proinflammatory mediators but had no effect on the nuclear factor-kappaB/DNA binding activity in the lung. GSK-3beta inhibition reduced the increase in nuclear factor-kappaB p65 activity caused by interleukin-1 in human embryonic kidney cells in vitro. The potent and selective GSK-3beta inhibitors TDZD-8, SB216763, and SB415286 reduced the organ injury/dysfunction caused by lipopolysaccharide or lipopolysaccharide and peptidoglycan in the rat. We propose that GSK-3beta inhibition may be useful in the therapy of the organ injury/dysfunction associated with sepsis, shock, and other diseases associated with local or systemic inflammation.