An experimental model of asthma in rats using ovalbumin and lipopolysaccharide allergens.

Asthma is chronic and multi-factorial inflammatory disease hence single allergen induced asthma in an animal is not identical to clinical asthma. Therefore, we developed a novel experimental model of asthma in rats using ovalbumin (OVA) and lipopolysaccharide (LPS) allergens. Rats were divided into four groups; normal (NC), OVA, LPS, and OVA-LPS treated. Rats were sensitized with OVA (100 µg/kg, adsorbed in 100 mg/mL aluminum hydroxide, i.p.), LPS (10 µg/kg, i.p.) and both (OVA-LPS) on 7th, 14th, 21st days and was followed by challenge with OVA (1%w/v), LPS (1%w/v), OVA (0.5%w/v) and LPS (0.5%w/v) for 30 min thrice/week for three weeks in the OVA, LPS and OVA-LPS groups, respectively. On 41 day, lung function parameters (respiration rate, tidal volume, and airflow rate), total and differential leukocytes count in the blood as well as BALf and inflammatory cytokines (IL-4, IL-5, and IL-13) in serum were measured. Histology of lungs was performed. The results suggested that the tidal volume and airflow rate were significantly decreased while respiration rate, total and differential leukocytes count in blood as well as BALf and serum cytokines level were significantly increased in the OVA-LPS as compared to NC, OVA, and LPS. In conclusion, the combination of OVA and LPS induced phenotypes of severe asthma with eosinophilic, neutrophilic and lymphocytic inflammation.

Protective effects of Gαq-RGS2 signalling inhibitor in aminophylline induced cardiac arrhythmia.

An over activation of GPCR mediated Gαq dependent signalling pathway is widely associated with the development of cardiovascular abnormalities. The objective of study was to evaluate the effects of (1-(5-chloro-2-hydroxyphenyl)-3-(4-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-5(4H)-one) Gαq-RGS2 signalling inhibitor on aminophylline induced cardiac arrhythmia in rats. Rats were divided into four groups; normal rats, disease control (DC, aminophylline treated 100 mg/kg/d, i.p., 7 days), Gαq-RGS2 signalling inhibitor (1 and 10 mg/kg/d, p.o., 7 days) treated arrhythmic rats. Gαq-RGS2 signalling inhibitor was administered 1 hour prior to the administration of aminophylline from 1st day. At the end of study, heart rate (HR), QRS complex, QT and RR interval were measured by electrocardiogram (ECG) of anesthetized rats. Systolic and diastolic blood pressure (SBP, DBP) by invasive method, cardiac damage markers (CK-MB, LDH) in the serum, antioxidant enzymes (SOD, catalase, glutathione) and cAMP level were measured. The treatment of Gαq-RGS2 signalling inhibitor (10 mg/kg) significantly abolished the aminophylline induced increase of heart rate, prolongation of RR and QT interval as compared to DC rats. Gαq-RGS2 signalling inhibitor (1 and 10 mg/kg) significantly attenuated the prolongation in QRS complex, increase of SBP, DBP and cardiac damage markers as compared to DC. Gαq-RGS2 signalling inhibitor treatment (10 mg/kg) significantly reduced the cAMP level and increased the antioxidant enzyme level as compared to DC. Gαq-RGS2 signalling inhibitor (10 mg/kg) showed the protective effect against the aminophylline induced cardiac arrhythmia and it might be due to improvement in cAMP level and antioxidant enzymes.

An anti-asthmatic activity of natural Toll-like receptor-4 antagonist in OVA-LPS-induced asthmatic rats.

Toll-like receptor-4 (TLR4) is a key component of the innate immune system and activation of TLR4 signaling has a significant role in the pathogenesis of asthma. Therefore, our objective was to identify the natural TLR4 antagonist and evaluate its activity in experimentally induced asthma. Soya lecithin origin phosphatidylcholine (soya PC) was identified as a natural TLR4 antagonist by computational study. Based on the computational study, TLR4 antagonist activity of soya PC was confirmed in in vitro lipopolysaccharide (LPS)-induced neutrophil adhesion assay. In the in vivo study, rats were sensitized with ovalbumin (OVA) (100 µg/kg, i.p.) on the 7th, 14th and 21st days and challenged intranasally with OVA (100 µg/100 µL) and LPS (10 ng/100 µL), 4 days/wk for 3 weeks. At the end of the experiment, we performed lung function parameters (respiratory rate, tidal volume, airflow rate), inflammatory cytokines (interleukin [IL]-4, IL-5, IL-13), total and differential leukocytes in blood as well as bronchoalveolar lavage fluid (BALf) and histological examinations. The computational study indicated that TLR4 antagonist activity of soya PC is due to linoleic acid (18:2) fatty acid chain. Soya PC significantly suppressed the LPS-induced neutrophil adhesion in a concentration-dependent manner to 1 µg/mL. The treatment of soya PC (5 and 10 mg/kg, 18 days, i.p.) significantly improved the lung function parameters, total and differential leukocyte counts in blood and BALf in asthmatic rats. This efficacy of soya PC was in extent similar to dexamethasone (2.5 mg/kg, 18 days, i.p.). However, soya PC was superior to dexamethasone in terms of benefits. The protective action of soya PC may be due to TLR4 antagonist activity and linoleic acid composition.

Anti-leukemic and anti-angiogenic effects of d-Limonene on K562-implanted C57BL/6 mice and the chick chorioallantoic membrane model.

BACKGROUND: d-Limonene, a monoterpene from citrus fruit has been found to have chemopreventive and chemotherapeutic activities in various types of cancers. In this study, we evaluated the in vivo effect of d-Limonene on a K562-induced model of chronic myeloid leukemia (CML) in C57BL/6 mice. METHOD: The tail vein injection model of K562 cells in immunocompromised C57BL/6 mice was developed and evaluated for characteristics of the disease. The mice were treated with d-Limonene and evaluated for haematological parameters. We also evaluated the effect of d-Limonene on angiogenesis using the chick chorioallantoic membrane (CAM) assay. RESULTS: In a complete blood count, a significant dose-dependent reduction in white blood cell, neutrophil and lymphocyte counts, but an elevation in red blood cell count and haemoglobin content was observed with d-Limonene treatment compared to the disease control or untreated group. In the CAM assay, d-Limonene produced a significant dose-dependent reduction in number of blood vessels in treatment groups compared to the vehicle-treated group. CONCLUSION: These studies suggest promising anti-leukemic and anti-angiogenic effects of d-Limonene in the treatment of CML.

Myocardial connexin-43 is upregulated in response to acute cardiac injury in rats.

We aimed to explore whether myocardial intercellular channel protein connexin-43 (Cx43) along with PKCε and MMP-2 might be implicated in responses to acute cardiac injury induced by 2 distinct sublethal interventions in Wistar rats. Animals underwent either single chest irradiation at dose of 25 Gy or subcutaneous injection of isoproterenol (ISO, 120 mg/kg) and were compared with untreated controls. Forty-two days post-interventions, the hearts were excised and left ventricles were used for analysis. The findings showed an increase of total as well as phosphorylated forms of myocardial Cx43 regardless of the type of interventions. Enhanced phosphorylation of Cx43 coincided with increased PKCε expression in both models. Elevation of Cx43 was associated with its enhanced distribution on lateral surfaces of the cardiomyocytes in response to both interventions, while focal areas of fibrosis without Cx43 were found in post-ISO but not post-irradiated rat hearts. In parallel, MMP-2 activity was decreased in the former while increased in the latter. Cardiac function was maintained and the susceptibility of the hearts to ischemia or malignant arrhythmias was not deteriorated 42 days after interventions when compared with controls. Altogether, the findings indicate that myocardial Cx43 is most likely implicated in potentially salutary responses to acute heart injury.

Vitamin D improves corticosteroid efficacy and attenuates its side-effects in an animal model of asthma.

The subacute use of corticosteroids has side-effects such as glucose intolerance, dyslipidemia, anxiety, and depression, which could be halted with vitamin D, which is an immunomodulatory vitamin. Thus, we aimed to study the anti-asthmatic efficacy and side-effects profile of vitamin D, the corticosteroid dexamethasone, and their combination on ovalbumin-induced airway inflammation in rats. For this, 2 different doses of vitamin D (50 IU/kg, daily for 2 weeks, or and 60000 IU/kg, bolus dose, by intraperitoneal injection (i.p.)) were administered in combination with dexamethasone (2.5 mg/kg, i.p., for 2 weeks) prior to challenge with ovalbumin. At the end of the therapy, the asthmatic parameters such as differential white blood cell counts, serum levels of immunoglobulin E, bronchoalveolar lavaged fluid, and interleukin-5, as well as serum levels of nitric oxide were significantly increased after allergen challenges in asthmatic rats as compared with the controls. Such increases were significantly attenuated by monotherapy with vitamin D and with combination therapy of vitamin D and dexamethasone, where the combination therapy was superior to the monotherapy. Dexamethasone-induced hyperglycemia, hyperlipidemia, and behavioral abnormalities in the allergic rats were attenuated with vitamin D. The daily dose was better for controlling serum levels of immunoglobulin E than the bolus dose, whereas the bolus was superior for reducing dexamethasone-induced psychotropic abnormalities. There were no significant changes in other parameters between the daily and the bolus dose. In conclusion, a daily dose of vitamin D in combination with dexamethasone is more efficacious for treating asthma in allergic rats than monotherapy.

Effect of atorvastatin on the angiogenic responsiveness of coronary endothelial cells in normal and streptozotocin (STZ) induced diabetic rats.

Atorvastatin, a lipid lowering agent, possesses various pleiotropic vasculoprotective effects, but its role in coronary angiogenesis is still controversial. Our objective was to study the effects of atorvastatin on the angiogenic responsiveness of coronary endothelial cells (cEC) from normal and diabetic rats. Male Wistar rats were distributed among 9 groups; (i) normal rats, (ii) 30 day diabetic rats, (iii) 60 day diabetic rats, (iv) normal rats administered a low dose of atorvastatin (1 mg/kg body mass, per oral (p.o.), for 15 days); (v) 30 day diabetic rats administered a low dose of atorvastatin; (vi) 60 day diabetic rats administered a low dose of atorvastatin; (vii) normal rats administered a high dose of atorvastatin (5 mg/kg, p.o., for 15 days); (viii) 30 day diabetic rats administered a high dose of atorvastatin; (ix) 60 day diabetic rats administered a high dose of atorvastatin. Each group was further divided into 2 subgroups, (i) sham ischemia-reperfusion and (ii) rats hearts that underwent ischemia-reperfusion. Angiogenic responsiveness the and nitric oxide (NO) releasing properties of the subgroups of cECs were studied using a chorioallantoic membrane assay and the Griess method, respectively. Atorvastatin treatment significantly increased VEGF-induced angiogenic responsiveness and the NO-releasing properties of cECs from all of the subgroups, compared with their respective non-treated subgroups except for the late-phase diabetic rat hearts that underwent ischemia-reperfusion, and the high dose of atorvastatin treatment groups. These effects of atorvastatin were significantly inhibited by pretreatment of cECs with l-NAME, wortmannin, and chelerythrine. Thus, treatment with a low dose of atorvastatin improves the angiogenic responsiveness of the cECs from normal and diabetic rats, in the presence of VEGF, via activation of eNOS-NO release.

Effect of telmisartan on VEGF-induced and VEGF-independent angiogenic responsiveness of coronary endothelial cells in normal and streptozotocin (STZ)-induced diabetic rats.

Telmisartan possesses endothelial protective effects due to angiotensin II type 1 receptor antagonist, peroxisome proliferator-activated receptor γ (PPARγ) agonist and antioxidant action. Therefore, our objective was to study effect of telmisartan on angiogenic responsiveness of coronary endothelial cells (cECs) of normal and diabetic rats. Male Wistar rats were divided into six groups, normal rats, diabetic rats 30 d. (30 days after administration of STZ), diabetic rats 60 ds. (60 days after administration of STZ), telmisartan-treated normal rats (2 mg/kg, p.o., for 15 days before isolation of hearts), telmisartan-treated diabetic rats 30 ds, and telmisartan-treated diabetic rats 60 ds. Each group was further divided into two subgroups, sham rat hearts and ischemia-reperfused rat hearts. After isolation of cEC from each subgroup, angiogenic responsiveness and nitric oxide releasing properties were studied using chorioallantoic membrane (CAM) assay and Griess method, respectively. cEC of normal rats showed significant increase in angiogenic responsiveness in presence of vascular endothelial growth factor (VEGF) but not in absence of it. This activity was attenuated by pretreatment of cEC with l-NAME, wortmannin and chelerythrine. Diabetes and ischemia reperfusion injury suppressed angiogenic responsiveness of cEC. Telmisartan treatment showed significant increase in VEGF-induced angiogenic responsiveness and nitric oxide releasing properties of cECs of all subgroups as compared to their respective non-treated subgroups. These effects of telmisartan were significantly inhibited by pretreatment of cECs with L-NAME and wortmannin but not with chelerythrine. Our data suggest that telmisartan improves VEGF-induced coronary angiogenic activity in normal and diabetic rats via stimulation of PI3K/eNOS/NO pathway.