The contribution of the WNT pathway to the therapeutic effects of montelukast in experimental murine airway inflammation induced by ovalbumin and lipopolysaccharide.

The wingless/integrase-1 (WNT) pathway involved in the pathogenesis of inflammatory airway diseases has recently generated considerable research interest. Montelukast, a leukotriene receptor antagonist, provides therapeutic benefits in allergic asthma involving eosinophils. We aimed to investigate the role of the WNT pathway in the therapeutic actions of montelukast (MT) in a mixed type of allergic-acute airway inflammation model induced by ovalbumin (OVA) and lipopolysaccharide (LPS) in mice. Female mice were sensitized with intraperitoneal OVA-Al(OH)3 administration in the initiation phase and intranasal OVA followed by LPS administration in the challenge phase. The mice were divided into eight groups: control, asthmatic, and control/asthmatic treated with XAV939 (inhibitor of the canonical WNT pathway), LGK-974 (inhibitor of the secretion of WNT ligands), or MT at different doses. The inhibition of the WNT pathway prevented tracheal 5-HT and bradykinin hyperreactivity, while only the inhibition of the canonical WNT pathway partially reduced 5-HT and bradykinin contractions compared to the inflammation group. Therefore, MT treatment hindered 5-HT and bradykinin hyperreactivity associated with airway inflammation. Furthermore, MT prevented the increases in the phosphorylated GSK-3ß and WNT5A levels, which had been induced by airway inflammation, in a dose-dependent manner. Conversely, the MT application caused a further increase in the fibronectin levels, while there was no significant alteration in the phosphorylation of the Smad-2 levels in the isolated lungs of the mice. The MT treatment reversed the increase in the mRNA expression levels of interleukin-17A. An increase in eosinophil and neutrophil counts was observed in bronchoalveolar lavage fluid samples obtained from the mice in the inflammation group, which was hampered by the MT treatment. The inhibition of the WNT pathway did not alter inflammatory cytokine expression or cell infiltration. The WNT pathway mediated the therapeutic effects of MT due to the inhibition of GSK-3ß phosphorylation as well as the reduction of WNT5A levels in a murine airway inflammation model.

The effect of mitochondria-targeted slow hydrogen sulfide releasing donor AP39-treatment on airway inflammation.

Mitochondrial dysfunction has been shown to contribute to the pathophysiology of airway diseases. Therefore, mitochondria are targeted in the development of new therapeutic approaches. Hydrogen sulfide (H2S) has been shown to be involved in the pathophysiological processes of airway inflammation. We aimed to evaluate the effect of mitochondria-targeted slow H2S releasing donor AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl)triphenylphosphoniumbromide)] on lipopolysaccharide (LPS)-induced airway inflammation in mice. LPS was applied to female Balb/c mice by intranasal (i.n.) route to induce airway inflammation and the subgroups of mice were treated with i.n. AP39 (250-1000 nmol/kg). 48 h after LPS administration airway reactivity was evaluated in vivo, then bronchoalveolar lavage (BAL) fluid and lungs were collected. LPS application led to bronchial hyperreactivity and neutrophil infiltration into the lung tissues along with increased TNF-α, IL-1ß and IL-6 levels in BAL fluid. LPS also induced an increase in the rate of glycolysis, glycogenolysis and Krebs-cycle. AP39 treatment prevented the LPS-induced bronchial hyperreactivity and reversed the increase in TNF-α and IL-6 levels in BAL fluid. The increase in neutrophil numbers in BAL fluid was also prevented by AP39 treatment at the highest dose. Our results indicate that AP39 can prevent bronchial hyperreactivity and decrease airway inflammation. Targeting H2S to the mitochondria may be a new therapeutic approach in airway inflammation.

The inhibitory effect of trimetazidine on detrusor contractility - a potential repositioning of trimetazidine for the treatment of overactive bladder.

OBJECTIVES: This study aimed to identify the effect of trimetazidine (TMZ), an antianginal drug, on detrusor smooth muscle (DSM) contractility and its possible mechanisms of action. METHODS: We performed in-vitro contractility studies on isolated mouse DSM strips and investigated the effect of TMZ on Ca2+ levels in fura-2-loaded A7r5 cells. KEY FINDINGS: TMZ (300 or 1000 µM) inhibited carbachol (CCh)- and KCl-induced contractions and produced a concentration-dependent (10-1000 µM) relaxation in KCl-precontracted DSM strips. TMZ-induced relaxation was markedly decreased by BaCl2, an inward-rectifying K+ channel blocker, but was not altered by preincubation with tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. TMZ (300 or 1000 µM) reduced both the CaCl2-induced contraction of depolarized DSM strips under Ca2+-free conditions and the CCh-induced contraction of DSM strips preincubated with nifedipine in Ca2+-containing Krebs solution. Furthermore, TMZ (1000 µM) significantly decreased the Ca2+ levels in fura-2-loaded A7r5 cells. CONCLUSIONS: TMZ decreased DSM contractility and caused a concentration-dependent relaxation of the tissue possibly through its actions on Ca2+ transients and K+ channels. Our results provide preclinical evidence that TMZ would be a potential candidate to treat disorders related to the overactivity of the bladder.

Alpha-lipoic acid: A promising adjuvant for nonsteroidal anti-inflammatory drugs therapy with improved efficacy and gastroprotection.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in a wide variety of diseases due to their analgesic and anti-inflammatory effects, but their usage have been limited due to significant ulcerogenic side effects. In the present study, we aimed to evaluate the effect of α-lipoic acid (ALA) treatment on the anti-inflammatory activity of indomethacin (Indo) as well as the possible therapeutic effect of ALA on high dose Indo-induced gastropathy in female mice. Mice were treated with Indo (5 or 30 mg/kg, p.o) alone or in combination with ALA (50, 100 or 200 mg/kg, i.p). in vivo anti-inflammatory effect was evaluated by formalin-induced paw edema measured as paw thickness and edema. Gastric damage was evaluated macroscopically and histologically by scoring mucosal hemorrhage, erosion, edema and inflammation. To our results, Indo was ineffective at 5 mg/kg, but co-treatment with Indo and ALA significantly reduced paw edema, implying that ALA augmented the anti-inflammatory effect of subtherapeutic dose of Indo. However, ALA was not able to induce a further increase in the anti-inflammatory effect of Indo at 30 mg/kg. Unlike the treatment with Indo at 5 mg/kg, Indo at 30 mg/kg caused severe gastric damage that prevented by co-treatment with ALA. These results suggest that combination of ALA with NSAIDs can both increase anti-inflammatory effect and prevent NSAIDs-induced gastric damage. ALA would be promising adjuvant that can reduce dose for effective NSAID therapy, which improves safety profile of NSAIDs especially in cases long-term administration of high dose needed.

Hydrogen sulfide donors prevent lipopolysaccharide-induced airway hyperreactivity in an in vitro model of chronic inflammation in mice.

We aimed to investigate and compare the effects of rapid (NaHS) and slow (GYY4137 and AP39) hydrogen sulfide (H2 S) releasing donors on LPS-induced tracheal hyperreactivity and pro-inflammatory cytokine levels in lung tissues of mice. Tissues were isolated from male BALB/c mice and incubated with LPS (10 µg/mL) in tissue culture. The subgroups were incubated with NaHS, GYY4137 and mitochondria-targeted donor AP39. LPS incubation did not alter contraction response to carbachol, but enhanced 5-HT and bradykinin-induced contractions in tracheal rings, and elevated IL-1ß, IL-6 and TNF-α levels in lung homogenates. NaHS at 300 µmol/L and 1000 µmol/L, GYY4137 at 30 µmol/L and 100 µmol/L, and AP39 at 30 nmol/L concentrations inhibited the tracheal hyperreactivity to 5-HT, whereas none of these donors affected the enhanced contraction to bradykinin. GYY4137 was also effective to inhibit 5-HT hyperreactivity acutely. In lung tissues, NaHS prevented the elevation of IL-1ß level at 1000 µmol/L, and IL-6 and TNF-α levels at 100 µmol/L concentrations. Incubation with GYY4137 (100 µmol/L) and AP39 (30 nmol/L and 300 nmol/L) inhibited the increase in IL-6 and TNF-α levels, but not IL-1ß at concentrations that they affected tracheal hyperreactivity. These results indicate that H2 S donors can decrease inflammation and prevent airway hyperreactivity.

Trimebutine maleate relaxes the isolated rat thoracic aorta: The role of nitric oxide and L-type calcium channels.

Trimebutine maleate (TMB), a widely prescribed drug for functional gastrointestinal disorders, has been reported to regulate smooth muscle contractility by modulating multiple ion channel activities in the gastrointestinal tract. However, its action on isolated aorta has not yet been reported. The aim of the present study was to evaluate in vitro vasorelaxant properties and the underlying pharmacological mechanisms of TMB in isolated rat thoracic aortic rings. Vascular activity experiments were performed on thoracic aorta isolated from Sprague-Dawley rats in vitro, including endothelium-intact and endothelium-denuded aortic rings. TMB (10-10 -10-5  mol/L) induced relaxation in endothelium-intact aortic rings precontracted by phenylephrine with a potency similar to that of carbachol. TMB-induced relaxation was not altered by glibenclamide and atropine in endothelium-intact aortic rings. However, L-NAME and endothelium denudation significantly reduced but not completely reversed the vasorelaxant effect of TMB. Also, TMB-induced relaxation wasn't affected by diclofenac in endothelium-intact aortic rings. TMB at 10-5  mol/L significantly reduced the CaCl2 -induced contractions in endothelium-intact aortic rings stimulated with KCl, but not stimulated with phenylephrine under Ca2+ free conditions. Moreover, TMB at 10-5  mol/L effectively attenuated Bay-K8644-induced contractions in aortic rings. These results suggest that TMB-induced relaxation was mediated by both endothelium-dependent and endothelium-independent manner in isolated rat thoracic aorta. The mechanism of TMB-induced relaxation at low concentrations is partially related to NO- and endothelium-dependent but unrelated to prostanoids formation. However, inhibition of Ca2+ influx through voltage-operated calcium channels and L-type Ca2+ channel blocking effect appears to be involved in the mechanism of vasorelaxant effect of TMB at high concentrations.

Effects of intranasal treatment with slow (GYY4137) and rapid (NaHS) donors of hydrogen sulfide in lipopolysaccharide-induced airway inflammation in mice.

We have investigated the effects of slow (GYY4137) and rapid (NaHS) hydrogen sulfide (H2S) releasing donors in lipopolysaccharide (LPS)-induced airway inflammation in mice. LPS (0.1 mg/ml) in 60 µl PBS was administered by the intranasal (i.n.) route and control group received vehicle, whereas the subgroups of mice were treated with i.n. GYY4137 or NaHS. The tracheal reactivity, inflammatory cell count in bronchoalveolar lavage (BAL) fluid and lung histopathology were evaluated in all groups 48 h after LPS/PBS applications. 5-Hydroxytryptamine (5-HT)-induced contraction response in isolated tracheas was enhanced after LPS treatment but carbachol response was not altered. Incubation with atropine (10-6 M), 5-HT2A receptor antagonist ketanserin (10-9-10-7 M) and 5-HT3 receptor antagonist alosetron (10-8 and 10-7 M) prevented 5-HT-induced hyperreactivity whereas 5-HT4 receptor antagonist GR113808 (10-7 M, 10-6 M) did not have any effect in LPS-treated group. Electrical field stimulation (EFS) of isolated tracheas elicited frequency-dependent contractile response, which was not altered by LPS treatment alone but was enhanced in the presence of 5-HT (10-9-10-4 M). This data indicated that 5-HT2A and 5-HT3 receptors, and acetylcholine released from cholinergic nerves were contributing to 5-HT-induced hyperreactivity in the present experiments. The increase in neutrophil count along with cytokine (IL-1ß, TNF-α) levels in bronchoalveolar lavage (BAL) fluid and histopathological changes like paranchymal inflammation and interalveolar thickening were determined in LPS-treated mice. H2S production in lung homogenates were determined by the methylene blue assay, and found to be similar in both LPS and control groups. The experiments conducted after i.n. treatment with H2S donors has shown that only GYY4137 (1 mg/kg) inhibited 5-HT-induced hyperreactivity, and both GYY4137 and NaHS (1 mg/kg) prevented the neutrophil increase in BAL fluid in LPS-induced airway inflammation. IL-1ß increase in BAL fluid was abolished by both GYY4137 and NaHS treatments whereas TNF-α levels remained unchanged. Furthermore, GYY4137 treatment did not have any effect in LPS-induced changes of lung pathology whereas NaHS prevented the paranchymal inflammation. The different H2S releasing pattern of these donors may explain the difference of their effects in this model. Compounds that provide stable H2S levels via local application may be a new therapeutic approach in airway inflammation.