الفهرس 
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Abstract
Bronchial asthma is one of the chronic inflammatory lung illnesses with a high prevalence all over the world. Despite several categories of effective therapies had been well defined, asthma is still poorly controlled. The airway inflammatory - oxidative reactions and associated airflow obstruction are the principal features driving asthma pathogenesis.
Accordingly, it will be an impetus to seek a drug that can effectively play various roles in relieving airway inflammation, protecting against induced oxidative injury, and counteracting the developed bronchoconstriction. Among the unfamiliar anti-inflammatory agents receiving much attention in the recent decades and at the same time, has been scientifically proven to also behave as a good antioxidant and as a stimulant of cGMP-mediated smooth muscle relaxation, is the anti-diabetic drug; Dapagliflozin (DAPA).
The present study represented the first attempt to determine the preventive and therapeutic effects of DAPA on asthma. It was designated to clarify the potential therapeutic effect that DAPA may exert either alone or when administered with Dexamethasone (DEXA) in the ovalbumin (OVA)-sensitized rat asthma model.
Based on these findings, the current study was carried out to investigate the following:
The effectiveness of DAPA alone in ameliorating the airway oxidative-inflammatory cascade and associated bronchospasm.
The possible airways anti-inflammatory and antioxidant properties of DAPA against the OVA-sensitized rat asthma.
The outcome of treatment with DAPA in terms of preventing bronchoconstriction induced by OVA in an asthma rat model.
The therapeutic behavior especially when combined with the conventional drug used in managing asthma, i.e. dexamethasone (DEXA).
The mechanisms by which DAPA alone or with DEXA could exert its/their pharmacological effects, if any.
In order to investigate these goals, bronchial asthma was induced in rats by 2 phases involving;
Summary & conclusion
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i. Ovalbumin sensitization and treatment phase
Rats were sensitized by intraperitoneal administration (I.P.) of 0.2 mg OVA-alum /kg/day, which was prepared by adding 20 μg of OVA to 1.96 mg of aluminum hydroxide,
dissolving in 0.1 mL sterile normal saline (NS; 0.9% NaCl) and then, gently mixing for 4 h at 4 °C to produce a white solution.
ii. Ovalbumin challenge phase
Rats were challenged with 20 μg OVA dissolved in 50 μl sterile NS via an intranasal administration (i.n.) DROP by DROP using sterile tips. Then, rats had to be kept in a vertical position for at least 1 min.
A total of 50 male rats were used in the present study. On days 0, 7, and 14, only ten rats were sensitized with 1 mL NS/kg/day I.P. and maintained as a “Normal control group”, while other forty rats were sensitized with 1 mL OVA-alum/kg/day I.P. from 15th to 30th day, all treatments were taken orally once a day, as follows;
Normal control group
Each rat was treated with 1 mL/kg/day vehicle (distilled water; DW)
Asthmatic group
Each rat was treated with 1 mL DW/kg/day
Dexamethasone group
Each rat was treated with 1 mg/kg/day of dexamethasone (DEXA)
Dapagliflozin group
Each rat was treated with 1 mg/kg/day of dapagliflozin (DAPA)
Dapagliflozin and dexamethasone group
Each rat was treated with 1 mg/kg/day of DAPA, 2 hr before 1 mg/kg/day of DEXA
Finally, all rats were challenged intranasally with OVA in each nostril on 22nd and 31st days.
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Days 0 7 14
Sensitization phase (i.p.)
OVA (20 μg) + Alum (1.96 mg)
15 30
Challenge phase (i.n.)
OVA (20 μg)
22 31
Start End
- DEXA (1 mg)
- DAPA (1 mg)
- DAPA, 2 h before DEXA
Treatment phase (p.o.)
32
All rats were
anesthetized
ketamine (50 mg)/
xylazine (3.3 mg)
Blood, BALF, and
lungs were collected
At 24 hr after the day of each allergen sensitization and challenge, rats’ body weights and
blood sugar levels were measured. Twenty-four hours following the last OVA challenge, all
animals were anesthetized with I.P. injection of ketamine (50 mg)/xylazine (3.3 mg)/kg.
Blood samples were collected by heart puncture and divided into two portions. One part
was used for determining the concentration of OVA-specific immunoglobulin E (IgE). While,
the other portion was used for determinating total and differential white blood cell count
(neutrophil, eosinophil, lymphocyte, and monocyte).
After blood sample collection, rats chest were opened and their lungs were carefully
dissected. All left lungs were isolated and the right were lavaged with sterile saline. Then, the
bronchoalveolar lavage fluid (BALF) was collected and centrifuged. The cell pellet was used
for enumerating inflammatory cells, while the supernatant was used for determining:
- Airways inflammatory markers; interleukin-17 (IL-17), tumor necrosis factor-alpha
(TNFα), interleukin-1 beta (IL-1β), monocyte chemotactic protein 1 (MCP1), and S100
calcium-binding protein A4 (S100A4) concentrations
- Lung oxidant/antioxidant status; malondialdehyde (MDA) concentration and total
antioxidant capicity (TAC)
- Bronchodilation indicators; nitric oxide (NO) and cyclic guanosine monophosphate
(cGMP)
Scheme 1. Experimental design
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The left lung tissue samples from each group were divided into two parts:
The first parts were used for the immunoblot analysis of the following parameters:
- Soluble guanylyl cyclase (sGC) subunits; alpha subunit (sGC-α1), and beta subunit (sGC-β1)
The second parts were used for assessing:
- Immunohistochemical detection of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)
- Lung lesion scoring
- Histopathologic examination
The effect of ovalbumin sensitization (1 mL OVA-alum/kg, I.P.) on assessed parameters compared to the normal control group can be summarized as follows;
- A significant decrease in body weight on the 32nd day, BALF TAC, BALF cGMP level, as well as in relative protein levels of sGC-α1 and sGC-β1
- A significant increase in blood sugar level on the 23rd and 32nd days, serum OVA-specific IgE, BALF levels of IL-17, TNF-α, IL-1β, MCP-1, and S100A4, total and deferential WBCs count (neutrophil, eosinophil, lymphocyte, and monocyte), BALF total and deferential inflammatory cell recruitment (neutrophil and eosinophil), BALF MDA and NO levels, lesion score, % area expression of eNOS and iNOS, No. of goblet cells as well as % area of smooth muscle cells
- Strong positive immunostaining signals for iNOS in the lung tissues
- Strong positive immunostaining signals for eNOS in the lung tissues
- Significant changes in the normal histological architecture as compared to the normal control group. It revealed thickening of bronchial wall with a high degree of corrugation of the bronchiolar epithelium and obliteration of their lumen, alveoli are emphysematous and fused with each other, severe vasculitis with thickening of pulmonary blood vessels wall. In addition, it showed severe hyperplasia of bronchiolar epithelium and smooth muscle cells with severe bronchoconstriction