Histamine oxidation in mouse adipose tissue is controlled by the AOC3 gene-encoded amine oxidase

Introduction

Histaminergic status can modify adipose tissue (AT) development: histamine-free mice exhibit visceral obesity, and treatments with H3-antagonists reduce body weight gain. However, direct histamine effects on AT remain poorly documented: it has been observed that histamine stimulates lipolysis in rodent adipocytes when its oxidation by amine oxidases (AOs) is blocked by inhibitors such as semicarbazide.

Objective

The aim of this work was to study the influence of AOC3 gene invalidation, encoding for semicarbazide-sensitive AO (SSAO), on histamine oxidation and on histamine lipolytic activity in AT.

Materials and methods

Expression of AOC- and MAO-encoding genes was determined by real-type PCR in wild-type (WT) and SSAO-deficient (AOC3-KO) mice. Lipolysis was assessed by glycerol release in isolated adipocytes and AO activity by substrate-induced hydrogen peroxide formation in kidney, ileum and AT.

Results

The expression levels of the genes encoding AOC1, AOC2 or MAOA and MAOB were not modified in the AT of AOC3-KO mice. In WT mice, histamine oxidation was lower than that of the reference SSAO-substrate benzylamine in AT, but not in ileum. The order of magnitude regarding benzylamine oxidation was AT > ileum >> kidney. In AOC3-KO mice, benzylamine oxidation was abolished in all tissues, while histamine oxidation was abolished in AT but not in ileum. Histamine was inactive on lipolysis in WT but stimulated lipolysis in fat cells from AOC3-KO mice, without reaching the maximal intensity of beta-adrenergic stimulation.

Conclusion

Histamine was mainly oxidized by diamine oxidase (AOC1 product) in intestine, but by SSAO (AOC3 product) in AT. When protected from its oxidation by SSAO in AT, histamine moderately activated lipolysis in adipocytes in AOC3-KO mice.

     

TLR4 regulates insulin-resistant proteins to increase apoptosis in the mouse retina

Objective and design

Work in multiple organs has suggested that toll-like receptor 4 (TLR4) may play a role in insulin resistance. Additional studies have shown a negative role for TLR4 on retinal health. We have previously reported that β-adrenergic receptors can regulate both TLR4 signal transduction, as well as insulin signaling in the retina and in retinal endothelial cells. Thus, we hypothesized that TLR4 would regulate retinal insulin signaling.

Materials and methods

We used endothelial cell-specific TLR4 knockout mice, as well as TLR4-overexpressing mice for these studies.

Methods

Western blotting and ELISA analyses were done for investigations of insulin receptor, insulin receptor substrate 1 (IRS-1) serine 307, and Akt phosphorylation, as well as cleaved caspase 3 levels in the mouse retina.

Results

We found that loss of TLR4 led to increased insulin receptor and Akt phosphorylation, as well as decreased IRS-1^Ser307 levels. In support of these results, TLR4 overexpression decreased insulin signaling and the cleavage of caspase 3.

Conclusions

Therefore, these results suggest that TLR4 plays a key role in insulin signaling in the retina. Reduction of TLR4 levels may be protective to the retina.

     

Deficiencies in elements involved in TLR4-receptor signalling in RBL-2H3 cells

Objective

RBL-2H3 cells express Toll-like receptors, including TLR4. This study aims to assess various aspects of the TLR4 pathway.

Methods

RBL-2H3 cells were indirectly stained for cell surface TLR4, 25 CD14 and intracellular MyD88 proteins and analysed through flow cytometry for single-colour staining.

Results

While TLR4-receptors are expressed in RBL-2H3 cells, associated elements involved in the signaling pathway, CD14 and MyD88, are not.

Conclusion

Care should be taken if RBL-2H3 cells are used to study aspects of the innate immune system in mast cells.

     

Endocytosis is required for exocytosis and priming of respiratory burst activity in human neutrophils

Objective and design

Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47^phox phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis.

Materials and methods

Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47^phox phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey–Kramer multiple-comparison test.

Results

Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47^phox phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis.

Conclusions

Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.

     

Nuciferine alleviates LPS-induced mastitis in mice via suppressing the TLR4-NF-κB signaling pathway

Background

Nuciferine, a major bioactive component from the lotus leaf, has been reported to have notable anti-inflammatory activities such as renal inflammation and acute lung injury in previous studies. Mastitis is one of the most prevalent diseases in the dairy cattle, which causes large economic losses for the dairy industry. However, the effects of nuciferine on lipopolysaccharide (LPS)-induced mastitis have not been reported.

Methods and results

Here, we investigated the anti-inflammatory effects of nuciferine on LPS-induced mastitis in mice and illuminated its potential mechanism on the TLR4-mediated signaling pathway in mouse mammary epithelial cells (mMECs). Histopathological changes and myeloperoxidase (MPO) activity assay showed that nuciferine treatment significantly alleviated the LPS-induced injury of mammary gland flocculus, inflammatory cells infiltration. qPCR and ELISA assays indicated that nuciferine dose-dependently reduced the levels of TNF-α and IL-1β, which indicated that nuciferine might have therapeutic effects on mastitis. Furthermore, nuciferine treatment significantly decreased the expression of TLR4 in a dose-dependent manner. Besides, nuciferine was also found to suppress LPS-induced NF-κB activation.

Conclusion

These findings indicate that nuciferine potently ameliorates LPS-induced mastitis by inhibition of the TLR4-NF-κB signaling pathway.

     

Inhibitory effects and related molecular mechanisms of total flavonoids in <Emphasis Type="Italic">Mosla chinensis</Emphasis> Maxim against H1N1 influenza virus

Objective

The Shixiangru (Mosla chinensis Maxim) total flavonoids (STF) mainly contain luteolin and apigenin. The study aims to examine the inhibitory effects of STF on anti-H1N1 influenza virus and its related molecular mechanisms in pneumonia mice.

Methods

The viral pneumonia mice were treated with Ribavirin or various doses of STF. We observed histological changes of lung by immunohistochemistry and measured lung index to value anti-influenza virus effects of STF. The concentrations of inflammatory cytokines and anti-oxidant factors were detected by ELISA. RT-PCR and western blot assays were used to determine the expression level of TLR pathway’s key genes and proteins in lung tissues.

Results

We found that the pathological changes of lung in the viral pneumonia mice obviously alleviated by STF treatments and the STF (288 or 576 mg/kg) could significantly decrease lung indices. Moreover, the up-regulation (IL-6, TNF-α, IFN-γ, and NO) and down-regulation (IL-2, SOD and GSH) of inflammatory cytokines and anti-oxidant factors were associated with higher clearance of virus and reduction of inflammatory lung tissue damage. Meanwhile, the expression levels of TLR3, TLR7, MyD88, TRAF3 and NF-κB p65 of the TLR pathway were reduced by STF treatment.

Conclusions

This study suggested that STF may be a promising candidate for treating H1N1 influenza and subsequent viral pneumonia.

     

Macrophages in trigeminal ganglion contribute to ectopic mechanical hypersensitivity following inferior alveolar nerve injury in rats

Background

Accidental mandibular nerve injury may occur during tooth extraction or implant procedures, causing ectopic orofacial pain. The exact mechanisms underlying ectopic orofacial pain following mandibular nerve injury is still unknown. Here, we investigated the role of macrophages and tumor necrosis factor alpha (TNFα) in the trigeminal ganglion (TG) in ectopic orofacial pain following inferior alveolar nerve transection (IANX).

Methods

IANX was performed and the mechanical head-withdrawal threshold (MHWT) in the whisker pad skin ipsilateral to IANX was measured for 15 days. Expression of Iba1 in the TG was examined on day 3 after IANX, and the MHWT in the whisker pad skin ipsilateral to IANX was measured following successive intra-ganglion administration of the macrophage depletion agent liposomal clodronate Clophosome-A (LCCA). TNFα expression in the TG and the MHWT in the whisker pad skin ipsilateral to IANX following successive intra-ganglion administration of the TNFα blocker etanercept were measured on day 3 after IANX, and tumor necrosis factor receptor-1 (TNFR1) immunoreactive (IR) cells in the TG were analyzed immunohistochemically on day 3.

Results

The MHWT in the whisker pad skin was significantly decreased for 15 days, and the number of Iba1-IR cells was significantly increased in the TG on day 3 after IANX. Successive intra-ganglion administration of the macrophage depletion agent LCCA significantly reduced the increased number of Iba1-IR cells in the TG and reversed the IANX-induced decrease in MHWT in the whisker pad skin. TNFα expression was increased in the TG on day 3 after IANX and was reduced following successive intra-ganglion administration of the TNFα inhibitor etanercept. The decreased MHWT was also recovered by etanercept administration, and TNFR1-IR cells in the TG were increased on day 3 following IANX.

Conclusions

These findings suggest that signaling cascades resulting from the production of TNFα by infiltrated macrophages in the TG contributes to the development of ectopic mechanical allodynia in whisker pad skin following IANX.

     

Tetraspanin 1 inhibits TNFα-induced apoptosis via NF-κB signaling pathway in alveolar epithelial cells

Objective

Tetraspanin family plays an important role in the pathogenesis of cancer, but its role in lung fibrosis is unknown. To determine whether tetraspanin 1 (TSPAN1), a member of the family, may be involved in the pathogenesis of pulmonary fibrosis.

Methods

TNFα -stimulated human alveolar epithelial (A549) and alveolar epithelial type II cell (AT2) were treated in vitro. Murine pulmonary fibrosis model was generated by injection of bleomycin (BLM). The expression of TSPAN1 was examined in vivo using the bleomycin-induced lung fibrosis model and tissue sample of IPF patients. Then we transfected the cells with TSPAN1 siRNA or plasmid and detected the expression changes of related proteins and cell apoptosis.

Results

In our study, we found that TSPAN1 was markedly down-regulated in lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and in bleomycin-induced pulmonary fibrosis in mice. We also found that TSPAN1 was significantly down-regulated in A549 and primary (AT2) cells following exposure to TNFα. Meanwhile, TSPAN1 inhibited p-IκBα, which attenuated nuclear NF-κB translocation and activation and inhibited apoptosis. We demonstrated that TSPAN1 reduced Bax translocation and caspase-3 activation, inhibited the apoptosis by regulating the NF-κB pathway in response to TNFα.

Conclusions

We conclude that TSPAN1 mediated apoptosis resistance of alveolar epithelial cells by regulating the NF-κB pathway. TSPAN1 may be a potential therapeutic target for pulmonary fibrosis or acute lung injury.

     

Dexmedetomidine protects against cisplatin-induced acute kidney injury in mice through regulating apoptosis and inflammation

Objective and design

Cisplatin-based chemotherapy has been widely used in the perioperative period of cancer surgery, which exacerbates the risk of renal injury. In this study, we examined whether dexmedetomidine (DEX), a commonly used anesthetic adjuvant, shows a protective effect against cisplatin-induced acute kidney injury.

Materials

Acute kidney injury in mice was induced by cisplatin.

Treatments

Mice were administered with DEX 25 μg/kg or atipamezole 250 μg/kg (once a day, for 3 days) after cisplatin treatment.

Methods

The renal function and tubular damage score were evaluated at 72 h following cisplatin administration. Apoptotic tubular cells were detected by TUNEL assay. Caspase-3, p53, Bax, F4/80⁺ macrophages, CD3⁺ T cells, and NF-κB were examined by immunohistochemistry staining or Western blot. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein (MCP)-1 in kidney were measured using real-time polymerase chain reaction.

Results

DEX treatment preserved renal function and reduced tubular damage score of mice after cisplatin administration. Mice treated with DEX exhibited less apoptotic tubular cells in response to cisplatin insult, which was associated with decreased Bax and reduced activation of p53 and caspase-3. DEX suppressed the infiltration of macrophages and T cells into the kidneys following cisplatin treatment, which was involved in the inhibition of NF-κB activation and decreased expression of TNF-α, IL-1β, IL-6, and MCP-1. Furthermore, we showed that the renoprotective effect conferred by DEX may be related to α₂ adrenoceptor-dependent pathway.

Conclusion

We demonstrate that DEX protects the kidney against cisplatin-induced AKI by the regulation of apoptosis and inflammatory response.

     

Mesenchymal stem cells cannot affect mRNA expression of toll-like receptors in different tissues during sepsis

Objective and design

Experimental animal models and human clinical studies support a crucial role for TLRs in infectious diseases. The aim of this study was to test the ability of MSCs, which have immunomodulatory effects, of altering the mRNA expression of toll-like receptors during a experimental model of sepsis in different tissues.

Materials and methods

Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 10⁶ cells/animal). Lungs, cortex, kidney, liver and colon tissue were dissected after 12 h of sepsis induction and TLR2/3/4/9 mRNA were evaluated by RT-qPCR.

Results

We observed a decrease of TLR2 and 9 mRNA expression in the liver of the sepsis group, while TLR3 was decreased in the lung and liver. No change was found between the sepsis group and the sepsis + MSC group.

Conclusions

In this model of experimental sepsis the MSCs were unable to modify the mRNA expression of the different toll-like receptors evaluated.

     

Atherogenic high cholesterol/high fat diet induces TLRs-associated pulmonary inflammation in C57BL/6J mice

Objective

To explore the effects of high cholesterol/high fat diet-induced hypercholesterolemia on pulmonary homeostasis of wild-type C57BL/6J mice.

Materials and methods

Six- to eight-week-old male C57BL/6J mice were randomly divided into two groups and treated with either high cholesterol/high fat diet (HCD, containing 20 % fat, 1.25 % cholesterol and 0.5 % sodium cholate) or a matching regular diet (RD, containing 4 % fat with no cholesterol and cholate added) for 12–16 weeks.

Results

Twelve to sixteen weeks after HCD diet feeding, hypercholesterolemia and pulmonary lipid accumulation were progressively exacerbated in C57BL/6J mice. Meanwhile, the HCD-fed mice showed distinctive signs of inflammation in the lung, which includes macrophage accumulation in alveolar lumen and lymphocyte infiltration around perivascular area. Simultaneously, the mRNA and protein expression of TLR2 and TLR4 were significantly up-regulated, and the translocation of NFκB into nucleus was activated in HCD-fed mice lung. In vitro, oxidized low-density lipoprotein (oxLDL) could directly up-regulate the expression of TLR2 and TLR4 in both A549 and MLE-12 lung epithelial cell lines.

Conclusions

These findings suggested that high cholesterol/high fat diet-induced hypercholesterolemia could result in TLRs/NFκB pathway-associated low-grade pulmonary inflammation in C57BL/6J mice, which might alter the lung’s immune responsiveness to a variety of environmental exposures.

     

Kinin B<Subscript>1</Subscript> receptors mediate depression-like behavior response in stressed mice treated with systemic <Emphasis Type="Italic">E. coli</Emphasis> lipopolysaccharide

Background

Kinin B₁ receptors are inducible molecules up-regulated after inflammatory stimuli. This study evaluated the relevance of kinin B₁ receptors in a mouse depression behavior model.

Methods

Mice were exposed to a 5-min swimming session, and 30 min later they were injected with E. coli lipopolysaccharide (LPS). Depression-like behavior was assessed by determining immobility time in a tail suspension test. Different brain structures were collected for molecular and immunohistochemical studies. Anhedonia was assessed by means of a sucrose intake test.

Results

Our protocol elicited an increase in depression-like behavior in CF1 mice, as assessed by the tail-suspension test, at 24 h. This behavior was significantly reduced by treatment with the selective B₁ receptor antagonists R-715 and SSR240612. Administration of SSR240612 also prevented an increase in number of activated microglial cells in mouse hippocampus, but did not affect a reduction in expression of mRNA for brain-derived neurotrophic factor. The increased immobility time following LPS treatment was preceded by an enhancement of hippocampal and cortical B₁ receptor mRNA expression (which were maximal at 1 h), and a marked production of TNFα in serum, brain and cerebrospinal fluid (between 1 and 6 h). The depression-like behavior was virtually abolished in TNFα p55 receptor-knockout mice, and increased B₁ receptor mRNA expression was completely absent in this mouse strain. Furthermore, treatment with SSR240612 was also effective in preventing anhedonia in LPS-treated mice, as assessed using a sucrose preference test.

Conclusion

Our data show, for the first time, involvement of kinin B₁ receptors in depressive behavioral responses, in a process likely associated with microglial activation and TNFα production. Thus, selective and orally active B₁ receptor antagonists might well represent promising pharmacological tools for depression therapy.

     

The isoprenoid end product N6-isopentenyladenosine reduces inflammatory response through the inhibition of the NFκB and STAT3 pathways in cystic fibrosis cells

Objective

N6-isopentenyladenosine (iPA) is an intermediate of the mevalonate pathway that exhibits various anti-cancer effects. However, studies on its anti-inflammatory activity are scarce and underlying molecular mechanisms are unknown. Therefore, we aimed to investigate the ability of iPA to exert anti-inflammatory effects in the human cystic fibrosis (CF) cell model of exacerbated inflammation.

Materials and methods

TNFα-stimulated CF cells CuFi-1 and its normal counterpart NuLi-1 were pre-treated with increasing concentrations of iPA and cell viability and proliferation were assessed by MTT and BrdU assays. The effect of iPA on IL-8 and RANTES secretion was determined by ELISA, and the activation and expression of signaling molecules and selenoproteins were studied by Western blot. To assess the direct effect of iPA on NFκB activity, luciferase assay was performed on TNFα-stimulated HEK293/T cells transfected with a NFκB reporter plasmid.

Results

We demonstrated for the first time that iPA prevents IL-8 and RANTES release in TNFα-stimulated CF cells and this effect is mediated by increasing the expression of the direct NFκB inhibitor IκBα and decreasing the levels of STAT3. Consistent with this, we showed that iPA inhibited TNFα-mediated NFκB activation in HEK/293T cells. Finally, we also found that iPA improved the levels of glutathione peroxidase 1 and thioredoxin reductase 1 only in CF cells suggesting its ability to maintain sufficient expression of these anti-oxidant selenoproteins.

Conclusions

Our findings indicate that iPA can exert anti-inflammatory activity especially in the cases of excessive inflammatory response as in CF.

     

Oxygen tension modulates the effects of TNFα in compressed chondrocytes

Objective and design

Oxygen tension and biomechanical signals are factors that regulate inflammatory mechanisms in chondrocytes. We examined whether low oxygen tension influenced the cells response to TNFα and dynamic compression.

Materials and methods

Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1–100 ng/ml) and cultured at 5 and 21 % oxygen tension for 48 h. In separate experiments, constructs were subjected to dynamic compression (15 %) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5 and 21 % oxygen tension using an ex vivo bioreactor for 48 h. Markers for catabolic activity (NO, PGE₂) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t test were used to analyse data.

Results

TNFα dose-dependently increased NO, PGE₂ and MMP activity (all p < 0.001) and induced MMP-13 (p < 0.05) and ADAMTS-5 gene expression (pp < 0.01) with values greater at 5 % oxygen tension than 21 %. The induction of catabolic mediators by TNFα was reduced by dynamic compression and/or L-NIO (all p < 0.001), with a greater inhibition observed at 5% than 21 %. The stimulation of GAG synthesis by dynamic compression was greater at 21 % than 5 % oxygen tension and this response was reduced with TNFα or reversed with L-NIO.

Conclusions

The present findings revealed that TNFα increased production of NO, PGE₂ and MMP activity at 5 % oxygen tension. The effects induced by TNFα were reduced by dynamic compression and/or the NOS inhibitor, linking both types of stimuli to reparative activities. Future therapeutics should develop oxygen-sensitive antagonists which are directed to interfering with the TNFα-induced pathways.

     

Structural organization of mammalian copper-containing amine oxidase genes

Objective

Analysis of sequence conservation and structural organization of mammalian genes encoding copper-containing amine oxidases (CAO).

Methods

Sequences of previously characterized genes encoding CAO proteins were used to identify homologous mammalian genes in the NCBI genome sequence databases and to analyze sequence and structural conservation of these genes.

Results

Mammals possess four AOC genes encoding diamine oxidase (AOC1), retina-specific amine oxidase (AOC2), vascular adhesion protein-1 (AOC3), and serum amine oxidase (AOC4), with a defective AOC4 gene present in humans, mice, and rats. In addition to the common structure of all AOC genes, there is a high degree of interspecies sequence conservation for each of the four genes.

Conclusions

Sequence and structural conservation of mammalian AOC genes implies a common evolutionary origin and functional diversification after gene duplication events.