Rimonabant inhibits TNF-α-induced endothelial IL-6 secretion via CB1 receptor and cAMP-dependent protein kinase pathway

Aim:

To investigate whether rimonabant, a cannabinoid receptor antagonist, had inhibitory effects on inflammatory reactions in human umbilical vein endothelial cells (HUVEC).

Methods:

TNF-α-induced IL-6 production was measured by ELISA and effects on related signaling pathways were investigated by immunoblot analysis. Cellular cAMP level was measured using kinase-coupled luciferase reaction.

Results:

Rimonabant at 1 and 10 μmol/L significantly inhibited TNF-α-induced IL-6 production when added 15, 30 and 60 minutes before TNF-α treatment. Rimonabant also inhibited TNF-α-induced phosphorylation of IκB kinase (IKK) α/β and IκB-α degradation. ACEA, a cannabinoid receptor subtype 1 (CB1) agonist, added before rimonabant abolished the former effects of rimonabant. H-89, an inhibitor of cAMP-dependent protein kinase (PKA), abolished the inhibitory effects of rimonabant on TNF-α induced IL-6 production. Rimonabant also increased the phosphorylation of PKA regulatory subunit II (PKA-RII), implying the essential role of PKA activation in the inhibitory effects of rimonabant. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin did not abolish the inhibitory effects of rimonabant on TNF-α induced IL-6 production.

Conclusion:

Rimonabant had anti-inflammatory effects on endothelial cells and inhibited TNF-α-induced IKKα/β phosphorylation, IκB-α degradation and IL-6 production in HUVEC. This effect was related to CB1 antagonism and PKA activation.     

TNF-α-induces airway hyperresponsiveness to cholinergic stimulation in guinea pig airways

BACKGROUND AND PURPOSE

TNF-α is an inflammatory cytokine implicated in the pathogenesis of asthma and it causes airway inflammation, bronchoconstriction and airway hyperresponsiveness to a number of spasmogens following inhalation.

EXPERIMENTAL APPROACH

We compared contractions of guinea pig isolated trachea incubated with saline or TNF-α for 1, 2 or 4 days to electrical field stimulation (EFS), 5-HT or methacholine. In addition, we compared bronchoconstriction in anaesthetized guinea pigs 6 h after intratracheal instillation of saline or TNF-α to vagal nerve stimulation, i.v. 5-HT or methacholine. Differential counts were performed on the bronchoalvelolar lavage fluid (BALF).

KEY RESULTS

Maximum contractions to methacholine, 5-HT and EFS were not different between freshly prepared and saline-incubated tissues. Exposure to TNF-α concentration-dependently potentiated contractions to 5-HT and EFS, but not methacholine. All contractions were atropine-sensitive, but not hexamethonium-sensitive. 5-HT-evoked contractions were inhibited by ketanserin or epithelial denudation. Only EFS-evoked contractions were tetrodotoxin-sensitive. Vagal stimulation, i.v. 5-HT or MCh caused a significant atropine-sensitive, frequency- and dose-dependent bronchoconstriction and decreased blood pressure similarly in both saline and TNF-α pre-treated animals. TNF-α potentiated the bronchoconstriction to vagal stimulation and 5-HT, but not MCh. The BALF from saline-treated animals contained predominantly macrophages, whereas that from TNF-α–treated animals contained neutrophils.

CONCLUSIONS AND IMPLICATIONS

TNF-α caused airway hyperresponsiveness to nerve stimulation in vivo and increased contractility in vitro. However, responsiveness to MCh was unchanged, suggesting a pre-synaptic action of TNF-α on parasympathetic nerves. TNF-α-induced airway hyperresponsiveness to 5-HT suggested an increased 5-HT_2A receptor-mediated acetylcholine release from epithelial cells.     

Kukoamine B, a novel dual inhibitor of LPS and CpG DNA, is a potential candidate for sepsis treatment

BACKGROUND AND PURPOSE

Lipopolysaccharides (LPS) and oligodeoxynucleotides containing CpG motifs (CpG DNA) are important pathogenic molecules for the induction of sepsis, and thus are drug targets for sepsis treatment. The present drugs for treating sepsis act only against either LPS or CpG DNA. Hence, they are not particularly efficient at combating sepsis as the latter two molecules usually cooperate during sepsis. In this study, a natural alkaloid compound kukoamine B (KB) is presented as a potent dual inhibitor for both LPS and CpG DNA.

EXPERIMENTAL APPROACH

The affinities of KB for LPS and CpG DNA were assessed using biosensor technology. Direct interaction of KB with LPS and CpG DNA were evaluated using neutralization assays. Selective inhibitory activities of KB on pro-inflammatory signal transduction and cytokine expression induced by LPS and CpG DNA were analysed by cellular assays. Protective effects of KB in a sepsis model in mice were elucidated by determining survival and circulatory LPS and tumour necrosis factor-alpha (TNF-α) concentrations.

KEY RESULTS

KB had high affinities for LPS and CpG DNA. It neutralized LPS and CpG DNA and prevented them from interacting with mouse macrophages. KB selectively inhibited LPS- and CpG DNA-induced signal transduction and expression of pro-inflammatory mediators without interfering with signal pathways or cell viability in macrophages. KB protected mice challenged with heat-killed Escherichia coli, and reduced the circulatory levels of LPS and TNF-α.

CONCLUSIONS AND IMPLICATIONS

This is the first report of a novel dual inhibitor of LPS and CpG DNA. KB is worthy of further investigation as a potential candidate to treat sepsis.     

TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways

Aim： Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-a has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro. Methods： Human umbilical vein endothelial cells （HUVECs） were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-a-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTTassay. Monocyte migration was measured with transwell migration assay. Results： Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α （2, 5 ng/mL） induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α （5 ng/mL） caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor （SP600125）, p38 MAPK inhibitor （SB202190） or PI-3K inhibitor （LY294002） significantly suppressed TNF-a-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-a-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone （up to 500 nmol/L） failed to affect TNF-a-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-a-induced CXCL1 from HUVECs attracted human monocyte migration. Conclusion： TNF-a stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.     

Cooperative role of tumour necrosis factor-α, interleukin-1β and neutrophils in a novel behavioural model that concomitantly demonstrates articular inflammation and hypernociception in mice

BACKGROUND AND PURPOSE

Chronic joint inflammation and pain are the hallmarks of disease in patients with inflammatory arthritis, notably rheumatoid arthritis. The aim of the present study was to investigate the relative contribution of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and neutrophil influx for joint inflammation and nociception in a novel murine model of antigen-induced arthritis (AIA).

EXPERIMENTAL APPROACH

AIA was induced by administration of antigen into knee joint of previously immunized mice. Neutrophil accumulation was determined by counting neutrophils in the joints and assessing myeloperoxidase activity in tissues surrounding the joints. TNF-α, IL-1β and CXCL-1 were measured by elisa. Mechanical hypernociception was assessed in parallel, using an electronic pressure meter.

KEY RESULTS

Hypernociception was dependent on antigen dose and the time after its administration; it was prevented by treatment with morphine and associated with neutrophil infiltration and local production of TNF-α, IL-1β and CXCL-1. Administration of a chimeric monoclonal antibody to TNF-α (infliximab) or IL-1receptor antagonist prevented neutrophil influx and hypernociception, and this was comparable to the effects of dexamethasone. Treatment with fucoidin (a leucocyte adhesion inhibitor) greatly suppressed neutrophil influx and local production of TNF-α and IL-1β, and hypernociception.

CONCLUSIONS AND IMPLICATIONS

In conclusion, the present study describes a new model that allows for the concomitant evaluation of articular hypernociception and inflammation. Using this system, we demonstrated that a positive feedback loop involving neutrophil influx and the pro-inflammatory cytokines TNF-α and IL-1β is necessary for articular hypernociception after antigen challenge of immunized mice.     

Nicorandil ameliorates ischaemia-reperfusion injury in the rat kidney

BACKGROUND AND PURPOSE

Nicorandil, an ATP-sensitive potassium (K_ATP) channel opener and nitric oxide donor, is used in the treatment of angina and acute heart failure. Here we investigated the effects of two K_ATP channel openers, nicorandil and cromakalim on ischaemia reperfusion (I-R) injury in the kidney.

EXPERIMENTAL APPROACH

Right nephrectomy was performed in 8-week-old male Sprague-Dawley rats and they were then divided into six groups: control group; I-R, including 30 min of left renal ischaemia followed by 24 h of reperfusion; I-R groups plus nicorandil 3 or 10 mg·kg⁻¹ i.p.; and I-R groups plus cromakalim 100 or 300 µg·kg⁻¹ i.p. After reperfusion, renal function was estimated by serum creatinine (SCr), urinary albumin : creatinine ratio (ACR) and urinary β2-microglobulin (β2-MG). Levels of K_ATP channel subtypes were investigated by Western blot. Kidney sections were stained for 4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine.

KEY RESULTS

Renal I-R induced significant increases in SCr, ACR and β2-MG levels compared with the control animals. Treatment with K_ATP channel openers reduced urinary β2-MG levels, raised by I-R. Both K_IR6.1 and K_IR6.2 channels were expressed. Expression of K_IR6.2 channels in the I-R group was lower than in the control group, which was restored to normal by treatment with K_ATP channel openers. Histologically, severe acute tubular damage was observed in the I-R kidney and this damage was ameliorated by K_ATP channel openers, dose-dependently.

CONCLUSIONS AND IMPLICATIONS

ATP-sensitive potassium channel openers protected against proximal tubule damage after I-R injury. Nicorandil could represent a powerful additional component in the treatment of patients undergoing partial nephrectomy or renal transplantation.     

Human umbilical cord blood mesenchymal stem cell transplantation suppresses inflammatory responses and neuronal apoptosis during early stage of focal cerebral ischemia in rabbits

Aim： Human umbilical cord blood mesenchymal stem cells （hUCB-MSCs） have been shown to ameliorate cerebral ischemia in animal models. In this study we investigated the effects of hUCB-MSCs on inflammatory responses and neuronal apoptosis during the early stage of focal cerebral ischemia in rabbits. Methods： Focal cerebral ischemia was induced in male New Zealand rabbits by occlusion of MCA for 2 h. The blood samples were collected at different time points prior and during MCAO-reperfusion. The animals were euthanized 3 d after MCAO, and the protein levels of IL-113, IL-6, IL-IO, and TNF-a in the serum and peri-ischemic brain tissues were detected using Western blot and ELISA, respectively. Inflammatory cell infiltration, neuronal apoptosis and neuronal density were measured morphologically, hUCB-MSCs （5x106） were iv injected a few minutes after MCAO. Results： The serum levels of IL-113, IL-6, and TNF-~ were rapidly increased, and peaked at 2 h after the start of MCAO. hUCB-MSC transplantation markedly and progressively suppressed the ischemia-induced increases of serum IL-113, IL-6, and TNF-a levels within 6 h MCAO-reperfusion. Focal cerebral ischemia decreased the serum level of IL-IO, which was prevented by hUCB-MSC transplantation The expression of IL-113, IL-6, IL-IO, and TNF-a in the peri-ischemic brain tissues showed similar changes as in the serum, hUCB-MSC transplantation markedly suppressed the infiltration of inflammatory cells, and increased the neuronal density around the ischemic region. Furthermore, hUCB-MSC transplantation significantly decreased the percentage of apoptosis around the ischemic region. Conclusion： hUCB-MSCs transplantation suppresses inflammatory responses and neuronal apoptosis during the early stage focal cerebral ischemia in rabbits.     

The new antihypertensive drug iptakalim activates ATP-sensitive potassium channels in the endothelium of resistance blood vessels

Aim:

To investigate the mechanisms underlying the activation of ATP-sensitive potassium channels (K_ATP) by iptakalim in cultured rat mesenteric microvascular endothelial cells (MVECs).

Methods:

Whole-cell K_ATP currents were recorded in MVECs using automated patch clamp devices. Nucleotides (ATP, ADP and UDP) were added to the internal perfusion system, whereas other drugs were added to the cell suspension on NPC-1 borosilicate glass chips.

Results:

Application of iptakalim (10 and 100 μmol/L) significantly increased the whole-cell K_ATP currents, which were prevented by the specific K_ATP blocker glibenclamide (1.0 μmol/L). The opening of K_ATP channels by iptakalim depended upon the intracellular concentrations of ATP or NDPs: iptakalim activated K_ATP channels when the intracellular ATP or NDPs were at 100 or 1000 μmol/L, and was ineffective when the non-hydrolysable ATP analogue ATPγS (1000 μmol/L) was infused into the cells. In contrast, the K_ATP opener pinacidil activated K_ATP channels when the intracellular concentrations of ATP or NDPs ranged from 10 to 5000 μmol/L, and even ATPγS (1000 μmol/L) was infused into the cells.

Conclusion:

Iptakalim activates K_ATP channels in the endothelial cells of resistance blood vessels with a low metabolic status, and this activation is dependent on both ATP hydrolysis and ATP ligands.     

Cannabinoid CB(2) receptor attenuates morphine-induced inflammatory responses in activated microglial cells

BACKGROUND AND PURPOSE

Among several pharmacological properties, analgesia is the most common feature shared by either opioid or cannabinoid systems. Cannabinoids and opioids are distinct drug classes that have been historically used separately or in combination to treat different pain states. In the present study, we characterized the signal transduction pathways mediated by cannabinoid CB₂ and µ-opioid receptors in quiescent and LPS-stimulated murine microglial cells.

EXPERIMENTAL APPROACH

We examined the effects of µ-opioid and CB₂ receptor stimulation on phosphorylation of MAPKs and Akt and on IL-1β, TNF-α, IL-6 and NO production in primary mouse microglial cells.

KEY RESULTS

Morphine enhanced release of the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and of NO via µ-opioid receptor in activated microglial cells. In contrast, CB₂ receptor stimulation attenuated morphine-induced microglial proinflammatory mediator increases, interfering with morphine action by acting on the Akt-ERK1/2 signalling pathway.

CONCLUSIONS AND IMPLICATIONS

Because glial activation opposes opioid analgesia and enhances opioid tolerance and dependence, we suggest that CB₂ receptors, by inhibiting microglial activity, may be potential targets to increase clinical efficacy of opioids.     

14-Deoxyandrographolide desensitizes hepatocytes to tumour necrosis factor-alpha-induced apoptosis through calcium-dependent tumour necrosis factor receptor superfamily member 1A release via the NO/cGMP pathway

BACKGROUND AND PURPOSE

Andrographis paniculata (AP) has been found to display hepatoprotective effect, although the mechanism of action of the active compounds of AP in this context still remains unclear. Here, we evaluated the hepatoprotective efficacy of 14-deoxyandrographolide (14-DAG), a bioactive compound of AP, particularly its role in desensitization of hepatocytes to tumour necrosis factor-alpha (TNF-α)-induced signalling of apoptosis.

EXPERIMENTAL APPROACH

TNF-α-mediated ligand receptor interaction in hepatocytes in the presence of 14-DAG was studied in vitro in primary hepatocyte cultures, with the help of co-immunoprecipitation, confocal microscopy and FACS analysis. Events associated with 14-DAG-induced TNFRSF1A release from hepatocytes were determined using immunoblotting, biochemical assay and fluorimetric studies. Pulse-chase experiments with radiolabelled TNF-α and detection of apoptotic nuclei by terminal transferase-mediated dUTP nick-end labelling were performed under in vivo conditions.

KEY RESULTS

14-DAG down-regulated the formation of death-inducing signalling complex, resulting in desensitization of hepatocytes to TNF-α-induced apoptosis. Pretreatment of hepatocytes with 14-DAG accentuated microsomal Ca-ATPase activity through induction of NO/cGMP pathway. This resulted in enhanced calcium influx into microsomal lumen with the formation of TNFRSF1A–ARTS-1–NUCB2 complex in cellular vesicles. It was followed by the release of full-length 55 kDa TNFRSF1A and a reduction in the number of cell surface TNFRSF1A, which eventually caused diminution of TNF-α signal in hepatocytes.

CONCLUSION AND IMPLICATION

Taken together, the results demonstrate for the first time that 14-DAG desensitizes hepatocytes to TNF-α-mediated apoptosis through the release of TNFRSF1A. This can be used as a strategy against cytokine-mediated hepatocyte apoptosis in liver dysfunctions.     

Lack of effects of acemetacin on signalling pathways for leukocyte adherence may explain its gastrointestinal safety

Background and purpose:

Acemetacin is a non-steroidal anti-inflammatory drug which is rapidly bioconverted to indomethacin, but produces significantly less gastric damage than indomethacin. This study was performed to investigate several possible mechanisms that could account for the gastrointestinal tolerability of acemetacin.

Experimental approach:

The gastric and intestinal damaging effects of acemetacin and indomethacin were examined in the rat. Effects of the drugs on blood levels of leukotriene B₄ and thromboxane B₂, on leukocyte-endothelial adherence in post-capillary mesenteric venules, and on gastric expression of tumour necrosis factor-α (TNF-α) were determined. The two drugs were also compared for gastric toxicity in rats pretreated with inhibitors of COX-2 and NOS.

Key results:

Acemetacin induced significantly less gastric and intestinal damage than indomethacin, despite markedly suppressing COX activity. Indomethacin, but not acemetacin, significantly increased leukocyte adherence within mesenteric venules, and gastric expression of TNF-α. Pretreatment with L-nitro-arginine methyl ester or lumiracoxib increased the severity of indomethacin-induced gastric damage, but this was not the case with acemetacin.

Conclusions and implications:

The increased gastric and intestinal tolerability of acemetacin may be related to the lack of induction of leukocyte–endothelial adherence. This may be attributable to the reduced ability of acemetacin to elevate leukotriene-B₄ synthesis and TNF-α expression, compared to indomethacin, despite the fact that acemetacin is rapidly bioconverted to indomethacin after its absorption.     

Over-expression of mitogen-activated protein kinase phosphatase-2 enhances adhesion molecule expression and protects against apoptosis in human endothelial cells

BACKGROUND AND PURPOSE

We assessed the effects of over-expressing the dual-specific phosphatase, mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2), in human umbilical vein endothelial cells (HUVECs) on inflammatory protein expression and apoptosis, two key features of endothelial dysfunction in disease.

EXPERIMENTAL APPROACHES

We infected HUVECs for 40 h with an adenoviral version of MKP-2 (Adv.MKP-2). Tumour necrosis factor (TNF)-α-stimulated phosphorylation of MAP kinase and protein expression was measured by Western blotting. Cellular apoptosis was assayed by FACS.

KEY RESULTS

Infection with Adv.MKP-2 selectively abolished TNF-α-mediated c-Jun-N-terminal kinase (JNK) activation and had little effect upon extracellular signal-regulated kinase or p38 MAP kinase. Adv.MKP-2 abolished COX-2 expression, while induction of the endothelial cell adhesion molecules, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), two NFκB-dependent proteins, was not affected. However, when ICAM and VCAM expression was partly reduced by blockade of the NFκB pathway, Adv.MKP-2 was able to reverse this inhibition. This correlated with enhanced TNF-α-induced loss of the inhibitor of κB (IκB)α loss, a marker of NFκB activation. TNF-α in combination with NFκB blockade also increased HUVEC apoptosis; this was significantly reversed by Adv.MKP-2. Protein markers of cellular damage and apoptosis, H2AX phosphorylation and caspase-3 cleavage, were also reversed by MKP-2 over-expression.

CONCLUSIONS AND IMPLICATIONS

Over-expression of MKP-2 had different effects upon the expression of inflammatory proteins due to a reciprocal effect upon JNK and NFκB signalling, and also prevented TNF-α-mediated endothelial cell death. These properties may make Adv.MKP-2 a potentially useful future therapy in cardiovascular diseases where endothelial dysfunction is a feature.     

Glycyrrhizin attenuates rat ischemic spinal cord injury by suppressing inflammatory cytokines and HMGB1

Aim:

To investigate the neuroprotective effect of glycyrrhizin (Gly) against the ischemic injury of rat spinal cord and the possible role of the nuclear protein high-mobility group box 1 (HMGB1) in the process.

Methods:

Male Sprague-Dawley rats were subjected to 45 min aortic occlusion to induce transient lumbar spinal cord ischemia. The motor functions of the animals were assessed according to the modified Tarlov scale. The animals were sacrificed 72 h after reperfusion and the lumbar spinal cord segment (L2–L4) was taken out for histopathological examination and Western blotting analysis. Serum inflammatory cytokine and HMGB1 levels were analyzed using ELISA.

Results:

Gly (6 mg/kg) administered intravenously 30 min before inducing the transient lumbar spinal cord ischemia significantly improved the hind-limb motor function scores, and reduced the number of apoptotic neurons, which was accompanied by reduced levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the plasma and injured spinal cord. Moreover, the serum HMGB1 level correlated well with the serum TNF-α, IL-1β and IL-6 levels during the time period of reperfusion.

Conclusion:

The results suggest that Gly can attenuate the transient spinal cord ischemic injury in rats via reducing inflammatory cytokines and inhibiting the release of HMGB1.     

Glycyrrhetinic acid inhibits ICAM-1 expression via blocking JNK and NF-KB pathways in TNF-α-activated endothelial cells

Aim:

To investigate the effects of glycyrrhetinic acid (GA), an active component extracted from the root of Glycyrrhizae glabra, on the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVEC).

Methods:

ICAM-1 mRNA and protein levels were detected using RT-PCR and cell enzyme-linked immunosorbent assays. The adherence of human monocytic THP-1 cells labeled with [³H]thymidine to HUVEC was determined by counting radioactivity with a scintillation counter. The activation of mitogen-activated protein kinases as well as the degradation of IκB and nuclear factor-κB (NF-κB) or phospho-c-Jun in the nucleus were detected by western blots. NF-κB binding activity was detected using electrophoretic mobility shift assay.

Results:

GA (50 and 100 μmol/L) significantly inhibits TNF-α-induced ICAM-1 mRNA and protein expressions, as well as THP-1 cell adhesiveness in HUVEC. GA selectively inhibited TNF-α-activated signal pathway of c-Jun N-terminal kinase (JNK), without affecting extracellular signal-regulated kinase 1/2 and p38. Furthermore, GA apparently inhibited IκB/NF-κB signaling system by preventing IκB degradation, NF-κB translocation, and NF-κB/DNA binding activity. Finally, pretreatment with GA or the inhibitors of NF-κB, JNK, and p38 reduced the ICAM-1 protein expression induced by TNF-α.

Conclusion:

GA inhibits TNF-α-stimulated ICAM-1 expression, leading to a decrease in adherent monocytes to HUVEC. This inhibition is attributed to GA interruption of both JNK/c-Jun and IκB/NF-κB signaling pathways, which decrease activator protein-1 (AP-1) and NF-κB mediated ICAM-1 expressions. The results suggest that GA may provide a beneficial effect in treating vascular diseases associated with inflammation, such as atherosclerosis.     

Calcineurin/NFAT pathway mediates wear particleinduced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro

Aim： To investigate the roles of the calcineurin/nuclear factor of activated T cells （NFAT） pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro. Methods： Osteoclasts were induced from mouse bone marrow macrophages （BMMs） in the presence of 100 ng/mL receptor activator of NF-KB ligand （RANKL）. Acridine orange staining and M＇IF assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATcl were examined using RT-PCR and immunofluorescent staining. The production of TNFa was analyzed with ELISA. Results： Titanium （Ti） or polymethylmethacrylate （PMMA） particles （0.1 mg/mL） did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-α production. The TNF-α level in the PMMA group was significantly higher than in the Ti group （96 h）. The expression of NFATcl was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VlVIT peptide （a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 pmol/L） did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VlVlT reduced TNF-α production much more efficiently in the PMMA group than in the Ti group （96 h）. Conclusion： Calcineurin/NFAT pathway mediates wear particles-induced TNF-α release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VlVlT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis.     

Thioredoxin interacting protein is a novel mediator of retinal inflammation and neurotoxicity

BACKGROUND AND PURPOSE

Up-regulation of thioredoxin interacting protein (TXNIP), an endogenous inhibitor of thioredoxin (Trx), compromises cellular antioxidant and anti-apoptotic defences and stimulates pro-inflammatory cytokines expression, implying a role for TXNIP in apoptosis. Here we have examined the causal role of TXNIP expression in mediating retinal neurotoxicity and assessed the neuroprotective actions of verapamil, a calcium channel blocker and an inhibitor of TXNIP expression.

EXPERIMENTAL APPROACH

Retinal neurotoxicity was induced by intravitreal injection of NMDA in Sprague–Dawley rats, which received verapamil (10 mg·kg⁻¹, p.o.) or vehicle. Neurotoxicity was examined by terminal dUTP nick-end labelling assay and ganglion cell count. Expression of TXNIP, apoptosis signal-regulating kinase 1 (ASK-1), NF-κB, p38 MAPK, JNK, cleaved poly-ADP-ribose polymerase (PARP), caspase-3, nitrotyrosine and 4-hydroxy-nonenal were examined by Western and slot-blot analysis. Release of TNF-α and IL-1β was examined by elisa.

KEY RESULTS

NMDA injection enhanced TXNIP expression, decreased Trx activity, causing increased oxidative stress, glial activation and release of TNF-α and IL-1β. Enhanced TXNIP expression disrupted Trx/ASK-1 inhibitory complex leading to release of ASK-1 and activation of the pro-apoptotic p38 MAPK/JNK pathway, as indicated by cleaved PARP and caspase-3 expression. Treatment with verapamil blocked these effects.

CONCLUSION AND IMPLICATIONS

Elevated TXNIP expression contributed to retinal neurotoxicity by three different mechanisms, inducing release of inflammatory mediators such as TNF-α and IL-1β, altering antioxidant status and disrupting the Trx-ASK-1 inhibitory complex leading to activation of the p38 MAPK/JNK apoptotic pathway. Targeting TXNIP expression is a potential therapeutic target for retinal neurodegenerative disease.