Post-Natal Inhibition of NF-κB Activation Prevents Renal Damage Caused by Prenatal LPS Exposure

Prenatal exposure to an inflammatory stimulus has been shown to cause renal damage in offspring. Our present study explored the role of intra-renal NF-κB activation in the development of progressive renal fibrosis in offspring that underwent prenatal exposure to an inflammatory stimulus. Time-dated pregnant rats were treated with saline (control group) or 0.79 mg/kg lipopolysaccharide (LPS) through intra-peritoneal injection on gestational day 8, 10 and 12. At the age of 7 weeks, offspring from control or LPS group were treated with either tap water (Con+Ve or LPS+Ve group) or pyrollidine dithiocarbamate (PDTC, 120mg/L), a NF-κB inhibitor, via drinking water starting (Con+PDTC or LPS+PDTC group), respectively, till the age of 20 or 68 weeks. The gross structure of kidney was assessed by hematoxylin-eosin, periodic acid–Schiff staining and Sirius red staining. The expression levels of TNF-α, IL-6, α-smooth muscle actin (α-SMA) and renin-angiotensin system (RAS) genes were determined by real time polymerase chain reaction and/or immunohistochemical staining. Our data showed that post-natal persistent PDTC administration efficiently repressed intra-renal NF-κB activation, TNF-α and IL-6 expression. Post-natal PDTC also prevented intra-renal glycogen deposition and collagenous fiber generation as evident by the reduced expression of collagen III and interstitial α-SMA in offspring of prenatal LPS exposure. Furthermore, post-natal PDTC administration reversed the intra-renal renin-angiotensin system (RAS) over-activity in offspring of prenatal LPS exposure. In conclusion, prenatal inflammatory exposure results in offspring’s intra-renal NF-κB activation along with inflammation which cross-talked with excessive RAS activation that caused exacerbation of renal fibrosis and dysfunction in the offspring. Thus, early life prevention of NF-κB activation may be a potential preventive strategy for chronic renal inflammation and progressive renal damage.     

Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

Introduction

We have previously reported that bacterial toxins, especially endotoxins such as lipopolysaccharides (LPS), might be important causative agents in the pathogenesis of rheumatoid arthritis (RA) in an in vitro model that simulates the potential effects of residing in damp buildings. Since numerous inflammatory processes are linked with the nuclear factor-κB (NF-κB), we investigated in detail the effects of LPS on the NF-κB pathway and the postulated formation of procollagen-endotoxin complexes.

Methods

An in vitro model of human chondrocytes was used to investigate LPS-mediated inflammatory signaling.

Results

Immunoelectron microscopy revealed that LPS physically interact with collagen type II in the extracellular matrix (ECM) and anti-collagen type II significantly reduced this interaction. BMS-345541 (a specific inhibitor of IκB kinase (IKK)) or wortmannin (a specific inhibitor of phosphatidylinositol 3-kinase (PI-3K)) inhibited the LPS-induced degradation of the ECM and apoptosis in chondrocytes. This effect was completely inhibited by combining BMS-345541 and wortmannin. Furthermore, BMS-345541 and/or wortmannin suppressed the LPS-induced upregulation of catabolic enzymes that mediate ECM degradation (matrix metalloproteinases-9, -13), cyclooxygenase-2 and apoptosis (activated caspase-3). These proteins are regulated by NF-κB, suggesting that the NF-κB and PI-3K pathways are involved in LPS-induced cartilage degradation. The induction of NF-κB correlated with activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation and p65 nuclear translocation. Further upstream, LPS induced the expression of Toll-like receptor 4 (TLR4) and bound with TLR4, indicating that LPS acts through TLR4.

Conclusion

These results suggest that molecular associations between LPS/TLR4/collagen type II in chondrocytes upregulate the NF-κB and PI-3K signaling pathways and activate proinflammatory activity.     

Protection of Ischemic Postconditioning against Neuronal Apoptosis Induced by Transient Focal Ischemia Is Associated with Attenuation of NF-κB/p65 Activation

Background and Purpose

Accumulating evidences have demonstrated that nuclear factor κB/p65 plays a protective role in the protection of ischemic preconditioning and detrimental role in lethal ischemia-induced programmed cell death including apoptosis and autophagic death. However, its role in the protection of ischemic postconditioning is still unclear.

Methods

Rat MCAO model was used to produce transient focal ischemia. The procedure of ischemic postconditioning consisted of three cycles of 30 seconds reperfusion/reocclusion of MCA. The volume of cerebral infarction was measured by TTC staining and neuronal apoptosis was detected by TUNEL staining. Western blotting was used to analyze the changes in protein levels of Caspase-3, NF-κB/p65, phosphor- NF-κB/p65, IκBα, phosphor- IκBα, Noxa, Bim and Bax between rats treated with and without ischemic postconditioning. Laser scanning confocal microscopy was used to examine the distribution of NF-κB/p65 and Noxa.

Results

Ischemic postconditioning made transient focal ischemia-induced infarct volume decrease obviously from 38.6%±5.8% to 23.5%±4.3%, and apoptosis rate reduce significantly from 46.5%±6.2 to 29.6%±5.3% at reperfusion 24 h following 2 h focal cerebral ischemia. Western blotting analysis showed that ischemic postconditioning suppressed markedly the reduction of NF-κB/p65 in cytoplasm, but elevated its content in nucleus either at reperfusion 6 h or 24 h. Moreover, the decrease of IκBα and the increase of phosphorylated IκBα and phosphorylated NF-κB/p65 at indicated reperfusion time were reversed by ischemic postconditioning. Correspondingly, proapoptotic proteins Caspase-3, cleaved Caspase-3, Noxa, Bim and Bax were all mitigated significantly by ischemic postconditioning. Confocal microscopy revealed that ischemic postconditioning not only attenuated ischemia-induced translocation of NF-κB/p65 from neuronal cytoplasm to nucleus, but also inhibited the abnormal expression of proapoptotic protein Noxa within neurons.

Conclusions

We demonstrated in this study that the protection of ischemic postconditioning on neuronal apoptosis caused by transient focal ischemia is associated with attenuation of the activation of NF-κB/p65 in neurons.     

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.     

Prenatal Exposure to Lipopolysaccharide Results in Local RAS Activation in the Adipose Tissue of Rat Offspring

Background

Adult metabolic syndrome may originate in part during fetal or early life. This study was designed to investigate the effects of prenatal exposure to lipopolysaccharide (LPS) on adipose development and local renin-angiotensin system (RAS) activation in rat offspring.

Methods

Pregnant rats were randomly divided into three groups (n = 8 in each), including an NS group (pregnant rats were only treated with 0.5 ml normal saline from the 8^th to the 14^th day of gestation); an LPS group (pregnant rats were injected intraperitoneally with 0.79 mg/kg LPS on the 8^th, 10^th and 12^th days of pregnancy); and an LPS+pyrrolidine dithiocarbamate (PDTC) group (identical to the LPS group except that 100 mg/kg PDTC was administered from the 8^th to the 14^th day of gestation).

Results

Prenatal exposure to LPS resulted in increased blood pressure, adipose coefficient and body weight in rat offspring. Specifically, during the infancy of the offspring rats, the LPS stimulus promoted the differentiation of adipose cells, diminishing their diameters and proportions while simultaneously increasing cell number. In contrast, once the rats were grown, adipose cell differentiation was inhibited, and the diameters and proportions of the cells were increased. Moreover, each component of the RAS was changed and was shown to be activated. PDTC, an inhibitor of NF-κB, could reverse the influence of the stimulus during pregnancy.

Conclusion

Prenatal exposure to LPS in rats results in increased blood pressure, adipose coefficient, body weight and activation of adipose RAS in offspring.     

Desflurane Preconditioning Induces Oscillation of NF-κB in Human Umbilical Vein Endothelial Cells

Background

Nuclear factor kappa B (NF-κB) has been implicated in anesthetic preconditioning (APC) induced protection against anoxia and reoxygenation (A/R) injury. The authors hypothesized that desflurane preconditioning would induce NF-κB oscillation and prevent endothelial cells apoptosis.

Methods

A human umbilical vein endothelial cells (HUVECs) A/R injury model was used. A 30 minute desflurane treatment was initiated before anoxia. NF-κB inhibitor BAY11-7082 was administered in some experiments before desflurane preconditioning. Cells apoptosis was analyzed by flow cytometry using annexin V–fluorescein isothiocyanate staining and cell viability was evaluated by modified tertrozalium salt (MTT) assay. The cellular superoxide dismutases (SOD) activitiy were tested by water-soluble tetrazolium salt (WST-1) assay. NF-κB p65 subunit nuclear translocation was detected by immunofluorescence staining. Expression of inhibitor of NF-κB-α (IκBα), NF-κB p65 and cellular inhibitor of apoptosis 1 (c-IAP1), B-cell leukemia/lymphoma 2 (Bcl-2), cysteine containing aspartate specific protease 3 (caspases-3) and second mitochondrial-derived activator of caspase (SMAC/DIABLO) were determined by western blot.

Results

Desflurane preconditioning caused phosphorylation and nuclear translocation of NF-κB before anoxia, on the contrary, induced the synthesis of IκBα and inhibition of NF-κB after reoxygenation. Desflurane preconditioning up-regulated the expression of c-IAP1 and Bcl-2, blocked the cleavage of caspase-3 and reduced SMAC release, and decreased the cell death of HUVECs after A/R. The protective effect was abolished by BAY11-7082 administered before desflurane.

Conclusions

The results demonstrated that desflurane activated NF-κB during the preconditioning period and inhibited excessive activation of NF-κB in reperfusion. And the oscillation of NF-κB induced by desflurane preconditioning finally up-regulated antiapoptotic proteins expression and protected endothelial cells against A/R.     

Lipopolysaccharide-Induced NF-κB Activation in Human Endothelial Cells Involves Degradation of IκBα but Not IκBβ

We studied the signal transduction pathways involved in NF-κB activation and the induction of the cytoprotective A20 gene by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVEC). LPS induced human A20 mRNA expression with a maximum level 2 h after stimulation. The proteasome inhibitorN-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) and the tyrosine kinase inhibitor herbimycin A (HMA) blocked A20 mRNA expression and partially inhibited NF-κB DNA-binding activity induced by LPS treatment. LPS induced IκBα degradation at 30–60 min after treatment, but did not induce IκBβ degradation up to 120 min. In contrast, TNF-α rapidly induced IκBα degradation within 5 min and IκBβ degradation within 15 min. Cycloheximide did not prevent LPS-induced IκBα degradation, indicating that newly synthesized proteins induced by LPS were not involved in LPS-stimulated IκBα degradation. LPS-induced IκBα degradation was inhibited by ALLN, confirming that ALLN inhibits NF-κB activation by preventing IκBα degradation. Of note, HMA also inhibited LPS-induced IκBα degradation. However, tyrosine phosphorylation of IκBα itself was not elicited by LPS stimulation, suggesting that tyrosine phosphorylation of a protein(s) upstream of IκBα is required for subsequent degradation. We conclude that in HUVEC, LPS induces NF-κB-dependent genes through degradation of IκBα, not IκBβ, and propose that this degradation is induced in part by HMA-sensitive kinase(s) upstream of IκBα.     

DNA-Dependent Protein Kinase Phosphorylation of IκBα and IκBβ Regulates NF-κB DNA Binding Properties

Regulation of the IκBα and IκBβ proteins is critical for modulating NF-κB-directed gene expression. Both IκBα and IκBβ are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both IκBα and IκBβ. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IκBα and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IκBα, murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NF-κB DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IκBα that was phosphorylated by DNA-PK was a more potent inhibitor of NF-κB binding than nonphosphorylated IκBα. These results suggest that DNA-PK phosphorylation of IκBα increases its interaction with NF-κB to reduce NF-κB DNA binding properties.     

Long-term Incubation with Proteasome Inhibitors (PIs) Induces IκBα Degradation via the Lysosomal Pathway in an IκB Kinase (IKK)-dependent and IKK-independent Manner

Proteasome inhibitors (PIs) have been reported to induce apoptosis in many types of tumor. Their apoptotic activities have been suggested to be associated with the up-regulation of molecules implicated in pro-apoptotic cascades such as p53, p21^Waf1, and p27^Kip1. Moreover, the blocking of NF-κB nuclear translocation via the stabilization of IκB is an important mechanism of PI-induced apoptosis. However, we found that long-term incubation with PIs (PS-341 or MG132) increased NF-κB-regulated gene expression such as COX-2, cIAP2, XIAP, and IL-8 in a dose- and time-dependent manner, which was mediated by phosphorylation of IκBα and its subsequent degradation via the alternative route, lysosome. Overexpression of the IκBα superrepressor (IκBα-SR) blocked PI-induced NF-κB activation. Treatment with lysosomal inhibitors (ammonium chloride or chloroquine) or inhibitors of cathepsins (Z-FF-FMK or Z-FA-FMK) or knock-down of LC3B expression by siRNAs suppressed PI-induced IκBα degradation. Furthermore, we found that both IKK-dependent and IKK-independent pathways were required for PI-induced IκBα degradation. Pretreatment with IKKβ specific inhibitor, SC-514, partially suppressed IκBα degradation and IL-8 production by PIs. Blockade of IKK activity using insolubilization by heat shock (HS) and knock-down by siRNAs for IKKβ only delayed IκBα degradation up to 8 h after treatment with PIs. In addition, PIs induced Akt-dependent inactivation of GSK-3β. Inactive GSK-3β accelerated PI-induced IκBα degradation. Overexpression of active GSK-3β (S9A) or knock-down of GSK-3β delayed PI-induced IκBα degradation. Collectively, our data demonstrate that long-term incubation with PIs activates NF-κB, which is mediated by IκBα degradation via the lysosome in an IKK-dependent and IKK-independent manner.     

Gelam Honey Attenuates Carrageenan-Induced Rat Paw Inflammation via NF-κB Pathway

The activation of nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of a number of inflammatory diseases. In this study, we investigated the anti-inflammatory mechanism of Gelam honey in inflammation induced rats via NF-κB signalling pathway. Rats paw edema was induced by subplantar injection of 1% carrageenan into the right hind paw. Rats were pre-treated with Gelam honey at different doses (1 or 2 g/kg, p.o.) and NSAID Indomethacin (10 mg/kg, p.o.), in two time points (1 and 7 days). Our results showed that Gelam honey at both concentrations suppressed the gene expressions of NF-κB (p65 & p50) and IκBα in inflamed rats paw tissues. In addition, Gelam honey inhibited the nuclear translocation and activation of NF-κB and decreased the cytosolic degradation of IκBα dose dependently in inflamed rats paw tissues. The immunohistochemical expressions of pro-inflammatory mediators COX-2 and TNF-α were also decreased in inflamed rats paw tissues when treated with Gelam honey. The results of our findings suggest that Gelam honey exhibits its inhibitory effects by attenuating NF-κB translocation to the nucleus and inhibiting IκBα degradation, with subsequent decrease of inflammatory mediators COX-2 and TNF-α.     

Changes in the Expression of the Toll-Like Receptor System in the Aging Rat Kidneys

Background

The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Toll-like receptors (TLRs) are one of the receptor types of the body''s innate immune system. The function of the TLR system and the mechanisms by which it functions in renal aging remain unclear. In the present study, we, for the first time, systematically investigated the role of the TLR system and the inflammation responses activated by TLRs during kidney aging.

Methods

We used western blot and immunohistochemistry to systematically analyze the changes in the expression and activation of the endogenous TLR ligands HSP70 and HMGB1, the TLRs (TLR1–TLR11), their downstream signaling pathway molecules MyD88 and Phospho-IRF-3, and the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα (NF-κB inhibition factor α), NF-κBp65, and Phospho-NF-κBp65 (activated NF-κB p65) in the kidneys of 3 months old (youth group), 12 months old (middle age group), and 24 months old (elderly group) rats. We used RT-qPCR to detect the mRNA expression changes of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b in the rat renal tissues of the various age groups.

Results

We found that during kidney aging, the HSP70 and HMGB1 expression levels were significantly increased, and the expression levels of TLR1, 2, 3, 4, 5, and 11 and their downstream signaling pathway molecules MyD88 and Phospho-IRF-3 were markedly elevated. Further studies have shown that in the aging kidneys, the expression levels of the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα, NF-κBp65, and Phospho-NF-κBp65 were obviously increased, and those of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b were significantly upregulated.

Conclusions

These results showed that the TLR system might play an important role during the kidney aging process maybe by activating the NF-κB signaling pathway and promoting the high expression of inflammation factors.