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 inhibitory effect of escitalopram on mouse detrusor contractility: The role of L-type calcium channels.

Selective serotonin reuptake inhibitors (SSRIs) are associated with urinary problems attributed to their central effects. ESC is a preferred SSRI and several case reports described that ESC is related to urinary retention. However, the direct effect of ESC on detrusor contractility is still not completely elucidated. Thus, we investigated the effect of ESC on detrusor contractility and mechanism(s) of its action in isolated mouse detrusor strips. Molecular docking and measurement of intracellular calcium were performed to determine the possible calcium channel blocking effect of ESC. The contractile responses to carbachol (CCh), KCl and electrical field stimulation of detrusor strips were significantly abolished by ESC (10 or 100 µM). ESC relaxed KCl-precontracted detrusor strips concentration-dependently, which was not affected by tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. ESC (10 or 100 µM) reduced both the CaCl2- and CCh-induced contractions under calcium-free conditions, indicating the role of calcium-involved mechanisms in ESC-mediated relaxation. Furthermore, ESC significantly decreased Bay K8644-induced contraction and the cytosolic calcium level in fura-2-loaded A7r5 cells. Molecular docking study also revealed the potential of ESC to bind L-type calcium (Cav1) channels. Our results demonstrate that ESC inhibits detrusor contractility via blocking Cav1 channels, which provides evidence for the direct effect of ESC on detrusor contractility and its mechanism.

Riluzole, a neuroprotective agent, preserves erectile function following bilateral cavernous nerve injury in male rats.

Neurogenic erectile dysfunction is a highly prevalent complication in men undergoing radical prostatectomy. The underlying mechanisms remain incompletely defined and the effective therapy has been limited. This study aimed to evaluate the protective effect of riluzole and the role of PKC ß and excitatory amino acid transporters (EAATs) mediating this effect in a rat model of bilateral cavernous injury (BCNI). A total of 48 male Sprague-Dawley rats were divided into sham, BCNI (at 7, 15 days post-injury) and BCNI treated with riluzole (8 mg/kg/day) groups. Erectile function was measured as maximum intracavernosal pressure (mICP)/mean arterial pressure (MAP) and total ICP/MAP. Changes in protein expressions of phospho (p)-PKC ß IIser660 and EAATs were analysed in penis and major pelvic ganglion with western blotting. BCNI decreased erectile function at 7 and 15 days post-injury (mICP/MAP at 4 V: 0.45 ± 0.06 vs 0.84 ± 0.07; 0.34 ± 0.04 vs 0.77 ± 0.04 respectively; p < 0.001) whereas riluzole treatment (for 15 days) preserved erectile function (mICP/MAP at 4 V: 0.62 ± 0.03 vs 0.34 ± 0.04; p < 0.01). The decline in the expression of p-PKC ß IIser660 was observed in penis at 7 and 15 days post-injury (p = 0.0003, p = 0.0033), which was prevented by riluzole treatment for 15 days (p = 0.0464). While expressions of EAAT-1 and EAAT-2 decreased in major pelvic ganglion following BCNI (p = 0.0428, p = 0.002), riluzole treatment for 15 days prevented the decrease only in EAAT-2 expression (p = 0.0456). Riluzole improved erectile function via possibly interacting with PKC ß II and glutamatergic pathways, as a potential therapeutic candidate for erectile dysfunction.

Trimetazidine attenuates cyclophosphamide-induced cystitis by inhibiting TLR4-mediated NFκB signaling in mice.

AIM: Cyclophosphamide (CP)-induced cystitis is a challenging clinical problem involving inflammation and dysfunction of bladder. Trimetazidine (TMZ) is an anti-anginal drug with anti-oxidant and anti-inflammatory properties. We aimed to investigate the protective effects of TMZ in CP-induced cystitis via inhibiting TLR4/NFκB signaling. MAIN METHODS: Balb/c mice were administrated TMZ (10 or 20 mg/kg/day) intraperitoneally (i.p.) for 5 consecutive days before CP. On day 6, cystitis was induced by a single dose of CP (300 mg/kg, i.p.). Mesna (2-mercaptoethane sulfonate sodium; 30 mg/kg, i.p.) was administered 20 min before and at 4 and 8 h after the CP injection. After 24 h of cystitis induction, the bladders were removed for histopathological evaluation, contractility studies, biochemical analysis and western blotting. MTT assay was performed in a cancer cell line (MDA-MB-231) to evaluate the effect of TMZ on the cytotoxicity of CP. KEY FINDINGS: CP-induced severe cystitis was confirmed by histological disturbances and the decrease in carbachol-evoked contractions of detrusor strips, which was partially improved by TMZ (20 mg/kg/day). SOD activity and GSH content were decreased whereas TNF-α and IL-1ß levels were increased in the bladders of CP-treated mice, which were restored by TMZ or mesna. TMZ reduced the CP-induced increase in the protein expressions of caspase-3, TLR4 and phosphorylated-NFκB in bladder tissues. TMZ alone decreased the cell viability and TMZ also enhanced the cytotoxicity of CP. SIGNIFICANCE: Our study provides the first preclinical evidence that TMZ attenuates CP-induced urotoxicity by enhancing anti-oxidant capacity and suppressing inflammation possibly via downregulating TLR4-mediated NFκB signaling while augmenting the cytotoxicity of CP.

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.

Improved Endothelium-Dependent Relaxation of Thoracic Aorta in Niclosamide-Treated Diabetic Rats.

Diabetes-induced endothelial dysfunction is critical for the development of diabetic cardiovascular complications. The aim of this study was to investigate the effect of niclosamide (Nic) on vascular endothelial dysfunction in streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley rats were injected with a single intraperitoneal injection of STZ (75 mg/kg) to induce type 1 diabetes, and Nic (10 mg/kg) was intraperitoneally administered per day for 4 weeks. Endothelial function was evaluated as carbachol (CCh, an endothelium-dependent vasodilator)-evoked relaxation in the experiments performed on isolated thoracic aortas. The changes in the protein expressions of phosphorylated eNOS at serine 1177 (p-eNOSSer1177) and phosphorylated VASP at serine 239 (p-VASPSer239) of the rat aortas were analyzed by western blotting to determine whether NO/cGMP signaling is involved in the mechanism of Nic. STZ-injected rats had higher fasting blood glucose and less body weight compared to control rats (p < 0.05). Nic treatment did not affect blood glucose levels or body weights of the rats. CCh-induced endothelium-dependent relaxation of the aortic rings was significantly decreased in diabetic rats compared to control (Emax = 66.79 ± 7.41% and 90.28 ± 5.55%, respectively; p < 0.05). CCh-induced relaxation response was greater in Nic-treated diabetic rats compared to diabetic rats (Emax = 91.56 ± 1.20% and 66.79 ± 7.41%, respectively; p < 0.05). Phosphorylation of eNOS and VASP in aortic tissues was significantly reduced in diabetic rats, which were markedly increased by Nic treatment (p < 0.05). We demonstrated that Nic improved endothelial dysfunction possibly through the activation of NO/cGMP signaling without affecting hyperglycemia in diabetic rats. Our results suggesting that Nic has potential of repurposing for diabetic cardiovascular complications.

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.

Double benefit of metformin treatment: improved bladder function in cyclophosphamide-induced cystitis and enhanced cytotoxicity in cancer cells.

Cyclophosphamide (CP) is a widely used anti-neoplastic drug; however, it leads to bladder dysfunction in the form of hemorrhagic cystitis that is a serious dose-limiting complication in cancer patients. We aimed to evaluate the protective effects of metformin (MET) in a mouse model of CP-related cystitis in parallel with its effect on CP-induced cytotoxicity in a breast cancer cell line, MDA-MB-231. Cystitis was induced by a single intraperitoneal injection of CP (300 mg/kg), and mice were administered MET, mesna, or vehicle treatment. 24 hours after cystitis induction, the bladders were removed for histopathological analysis and ex vivo evaluation of detrusor muscle contractility. The effect of MET on the cytotoxicity of CP in MDA-MB-231 cells was evaluated as the viability of the cells via MTT assay. Histopathological evaluation confirmed that CP induced a severe cystitis, and MET partially inhibited CP-induced bladder damage. Carbachol-evoked cholinergic contractions were significantly decreased in detrusor strips of mice injected with CP only compared to control (Emax=293.67± 20.00 vs. 497.79± 21.78 mg tension/mg tissue, respectively). In CP-injected mice, treatment with 100 mg/kg MET restored cholinergic contractions (Emax=473.72±62.61 mg tension/mg tissue). In MDA-MB-231 cells, MET decreased their viability, and the combination of MET and CP caused more decrease in cell viability as compared to CP alone (p<0.05), demonstrating that MET enhances the cytotoxicity of CP in these cancer cells. Our results indicate that MET has a strong potential as a therapeutic adjuvant to prevent CP-induced cystitis while enhancing the efficacy of CP.

Photodynamic therapy effect of morpholinium containing silicon (IV) phthalocyanine on HCT-116 cells.

In this study, we investigated the in vitro potential of axially 1-morpholiniumpropan-2-ol disubstituted silicon (IV) phthalocyanine (SiPc) which was synthesized previously, on HCT-116 cells as a photodynamic therapy (PDT) agent. The singlet oxygen and photodegradation quantum yields of SiPc were calculated using UV-vis spectrophotometer. The cytotoxic and phototoxic effects of SiPc were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Annexin V-FITC/PI double staining kit, cell cycle kit, and mitochondria membrane potential (ΔΨm) assay kit with JC-1 were used to indicate the cell death pathway. Caspase-3 and ß-catenin protein expressions were evaluated by western blotting. The singlet oxygen and photodegradation quantum yields of SiPc were calculated as 0.73 and 3.64 × 10-4 in DMSO. The cell viability assays showed that IC50 value of SiPc did not reach to 100 µM without irradiation. However, excellent phototoxicity was observed in the presence of SiPc upon light irradiation. The cells undergoing early/late apoptosis significantly increased in the presence SiPc at 5 µM upon light irradiation. Besides, the proportion of cells at S and G2/M phase increased. Moreover, mitochondria membrane potentials significantly decreased at 1 and 5 µM of SiPc with light irradiation. While caspase-3 expression increased, ß-catenin expression significantly decreased on HCT-116 in the presence of SiPc (p < 0.01). The results indicated that the PDT could be related to apoptosis and Wnt/ß-catenin signaling pathway. Based on our findings, SiPc exhibited a significant PDT effect on HCT-116 cells therefore, worthy of more detailed study.

Uroprotective effect of pantoprazole against cyclophosphamide-induced cystitis in mice.

PURPOSE: The aim of the present study was to evaluate the potential uroprotective effect of pantoprazole (PPZ) in a mouse model of cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) due to its antioxidant and anti-inflammatory properties. METHODS: Balb/c mice received a single intraperitoneal (i.p.) injection of CP (300 mg/kg) to induce HC. PPZ (20, 50, and 100 mg/kg/day;i.p.) was administered for 3 consecutive days before the induction of HC. Mesna (30 mg/kg;i.p.) was administered 20 min before, 4 and 8 h after CP injection to compare the protective effects of PPZ. After 24 h of HC induction, the bladders were removed for functional studies, biochemical analyses, and histopathological examination. RESULTS: In vitro contractility studies demonstrated that CP-induced HC decreased the responsiveness of detrusor muscle strips to acetylcholine (ACh), which was reversed by PPZ pretreatment at all doses tested. However, mesna treatment was not able to improve responsiveness to ACh. Biochemical analyses showed that CP caused significant elevation of malondialdehyde (MDA), reduction of total glutathione (GSH), and increment of proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) level, which were measured in bladder homogenates. PPZ pretreatment at three doses found to be effective in reducing the CP-induced elevation of MDA and TNF-α levels. The highest dose of PPZ (100 mg/kg) caused a significant increase in GSH level. CP induced severe HC with marked bladder edema and histological disturbances which were partially abolished by PPZ pretreatment. CONCLUSIONS: Our results indicate that PPZ pretreatment could attenuate CP-induced HC by interfering with oxidative stress and modulating proinflammatory cytokines.

Uroprotective effect of ambroxol in cyclophosphamide-induced cystitis in mice.

PURPOSE: Hemorrhagic cystitis (HC) is defined as any types of acute or chronic inflammation of urinary bladder with several reasons. One of the most common causes of HC is cyclophosphamide (CYP), an effective antineoplastic agent, due to its urotoxic potential. Ambroxol (AMB) is a mucoactive drug that has been used for numerous respiratory diseases. Besides its mucolytic activity, AMB is a potent antioxidant and antiinflammatory agent that is becoming more attractive for the treatment of several oxidative/inflammatory disorders. The aim of this study was to evaluate the uroprotective potential of AMB in CYP-induced HC. METHOD: Male Balb/c mice were pretreated with AMB (30, 70, and 100 mg/kg) once a day for 3 consecutive days before HC induction with CYP (300 mg/kg). Mesna (30 mg/kg;i.p.), only drug in the management of CYP-induced HC, was administered 20 min before; 4 and 8 h after cystitis induction. The urinary bladders were harvested and evaluated in functional, biochemical, and histological studies. RESULTS: CYP-induced HC markedly reduced acetylcholine (ACh)-induced contractions in detrusor strips and AMB at 100 mg/kg caused a significant increase in the responsiveness to ACh. Pretreatment with AMB prevented the elevation of malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) level, reduction of total glutathione (GSH) that induced by CYP. However, treatment with AMB did not improve the bladder weight and some histological parameters. CONCLUSION: These results suggest that AMB pretreatment could improve CYP-induced HC via antioxidant and antiinflammatory activities.

Investigation of the Antioxidant, α-Glucosidase Inhibitory, Anti-inflammatory, and DNA Protective Properties of Vaccinium arctostaphylos L.

OBJECTIVES: The scope of this study was to investigate the total phenolic, anthocyanin, and flavonoid contents and the biological properties of ethanol extract (EE), methanol extract (ME), and aqueous extract (AE) from Vaccinium arctostaphylos L. MATERIALS AND METHODS: EE, ME, and AE of V. arctostaphylos were prepared. Various biological activities such as total phenolic, anthocyanin, and flavonoid contents, and antioxidant (2,2'-diphenyl-1-picrylhydrazyl ferrous ion-chelating, and ferric reducing antioxidant power assays), α-glucosidase inhibitory, anti-inflammatory, and DNA protective properties of these extracts were studied. RESULTS: EE exhibited the highest total phenolic, anthocyanin, and flavonoid contents with 44.42±1.22 mg gallic acid equivalents/g dry weight, 8.46±0.49 mg/Cyaniding-3-glucoside equivalents/g dry weight, and 9.22±0.92 mg quercetin equivalents/g dry weight, respectively. The antioxidant activities of the extracts followed the order: EE>ME>AE. EE and ME inhibited α-glucosidase enzyme and their IC50 values were 0.301±0.002 mg/mL and 0.477±0.003 mg/mL, respectively. In addition, EE and ME were determined as noncompetitive inhibitors with inhibitory constant (Ki ) values of 0.48±0.02 mg/mL and 0.46±0.01 mg/mL, respectively. EE in 100 and 300 mg/kg doses caused a significant reduction in formalin-induced edema in mice, demonstrating the anti-inflammatory effect of EE. In DNA protective studies, all of the extracts protected supercoiled plasmid pBR322 DNA against damage caused by Fenton's reagents due to their radical scavenging activities. CONCLUSION: Our results demonstrated that EE of V. arctostaphylos L. had strong antioxidant, anti-inflammatory, α-glucosidase inhibitory, and DNA protective effects, suggesting that it might be an effective medical plant to prevent or treat diseases associated with oxidative damage and inflammation.

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.