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Abstract

Chronic Obstructive Pulmonary Disease (COPD) is a dangerous prevalent smoking-related disease characterized by abnormal inflammation and oxidative stress and expected to be the third cause of death in the world next decade. Corticosteroids have low effects in decreasing numbers of inflammatory mediators specifically in long-term use. Our study designed to investigate the possible protective effects of combined dexamethasone (Dex) (2mg/kg) and losartan (Los) (30mg/kg angiotensin receptor blocker, it possesses antioxidant and anti-inflammatory properties in lung injury in mice) against cigarette -smoke (CS) induced COPD in rats compared with dexamethasone and losartan. Male Sprague Dawley rats (N = 40) divided into five groups (n = 8): control group, CS group, Dex group, Los group, and Dex +Los group. COPD induced in rats by CS exposure twice daily for 10 weeks. After the specified treatment period, bronchoalveolar lavage fluid (BALF) and lung tissue were collected for measurement of SOD, NO, MDA, ICAM-, MMP-9, CRP, NF-κB and histopathology scoring. Our results indicated that Los+Dex significantly prevent CS-induced COPD emphysema, congested alveoli, and elevation of lung injury parameters in BALF. They also showed a significant decrease in MDA, ICAM-1, MMP-9, CRP, and NF-κB and a significant increase in SOD and NO. In conclusion, adding Los to Dex potentiating their activity in inhibition the progression of COPD based on its activity on oxidative stress, inflammation, and NF-κB protein expression.

Keywords

Chronic obstructive pulmonary disease losartan dexamethasone bronchoalveolar lavage fluid

Introduction

Chronic obstructive pulmonary disease (COPD) is a universal problem especially in developing countries.^1,2 It is expected to be the third cause of death in the world next decade.³ COPD is defined as a progressive decline in airways and shortness of breath, which is linked to oxidative stress and chronic inflammation which can cause weight loss and tissue depletion.^4,5 Cigarette smoking is responsible for the bulk of COPD cases.⁶ However the existing therapy helps in modifying the quality of life of COPD patients by only decreasing the catenation of this disease.⁷ Corticosteroids have several benefits in controlling shortness of breath but have low effects in decreasing inflammation specifically in a long term use.⁸ Corticosteroids do not have a role in exacerbation even in large doses.^9,10 Besides, the long term use of systematic corticosteroids can case hypertension and other several side effects.¹¹ Also, cigarette smoke (CS) is correlated with glucocorticoid resistance.¹² Therefore, we require more researches about COPD management drugs.

Losartan (Los) (Angiotensin receptor type 1 blocker) is widely used in clinics for the management of hypertension, heart failure, and diabetic nephropathy. It also possesses antioxidant effect and anti-inflammatory properties in cigarette smoke-induced lung injury in mice.¹³ To the best of our knowledge, the effect of the combination of dexamethasone and losartan in protection from CS-induced COPD has not yet been explored. Accordingly, the present study aimed to investigate the possible protective effects of combined dexamethasone and losartan against cigarette smoke-induced COPD in rats. We also attempted to clarify the related molecular mechanisms possibly involved in any potential therapeutic effects.

Methods and materials

Animals

Male Sprague Dawley rats (weight range 200–220 g; 8 weeks of age) were purchased from the unit of experimental animal production at (VACSERA), Giza, Egypt. The rats were maintained under controlled temperature (23 ± 2°C) and relative humidity (60 ± 10%) conditions, with a 12/12 hour light/dark cycle. They were kept 1 week in the laboratory before the experiment for acclimatization. At all times, excluding the smoke exposure period, water and diets were provided ad libitum. All experimental procedures described in this study comply with the ethical principles and guidelines for care and the use of laboratory animals adopted by the ‘‘ Research Ethics Committee’’ Faculty of Pharmacy, Tanta University.

Drugs and chemicals

Losartan (Cozaar®) was purchased as a powder from Merck Sharp & Dohme, Cairo, Egypt. Dexamethasone (Dex) was purchased from Sigma, Cairo, Egypt, as a solution for injection each 2 ml contains 8 mg Dex. Cigarettes (Cleopatra) were purchased from the Eastern, Giza; Egypt. Each cigarette containing 15 mg tar (equivalent to 1.56-fold of tar quantity in the Kentuchy Reference Cigarette 2R4F) and 1 mg nicotine (equivalent to 1.14-fold of nicotine quantity in the Kentuchy Reference Cigarette 2R4F) per cigarette were used in this study. Los was freshly dissolved and prepared in saline immediately before use with final concentration 15 mg/ml.

Experimental protocol

Forty adult rats were randomly divided into five groups (n = 8): Group 1 (control): rats were received saline and rats were exposed to room air simultaneously. Group 2 (CS): rats were exposed to CS of 8 cigarettes/day for 7 day/week for 10 weeks according to the method published before with minor modification.¹⁴ Group 3 (Dex): rats were received Dex (2mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks.¹⁵ Group 4 (Los): rats were received Los (30mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for 10 weeks.¹³ Group 5(Dex+Los): rats were received Dex (2mg/kg, i.p) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks and also rats were received Los (30mg/kg, i.p), 30 minutes before exposure to CS for 10 weeks.^13,15 Smoking apparatus consists of four major parts including a power supply, a cigarette burn box, a circulation fan, and handling cigarette. The smoking chamber in a standard smoking apparatus was adapted for three rats as whole-body exposure. The smoke concentration in the exposure chamber controlled by the air control holes and cigarette burning speed.¹⁴

Bronchoalveolar lavage fluid (BALF) and lung tissue samples collection

One day after the last challenge, rats weighed and anesthetized with thiopental (30mg/kg IP). Then, the lungs were inflated and the left main bronchus was tied and left lung from all rats was removed and kept in 10%neutral formalin for histopathological and immune-histochemical evaluations. The right lung from all rats was lavaged with 6-mL aliquots of sterile saline for 5 times and the BAL was collected. BAL fluids centrifuged at 800 g for 10 min.¹⁶ BAL cells suspended in 1 ml of saline, tained with Giemsa stain solution, and counted using a hemocytometer (Neubauer, Marienfeld, Germany) for cell differentiation.¹⁷ BAL fluid was immediately stored at -80°C for the determination of superoxide dismutase activity (SOD), malondialdehyde concentration (MDA) and nitric oxide content (NO), matrix metalloproteinase 9 (MMP-9), intracellular adhesion molecule (ICAM-1) and C-reactive protein (CRP).

Measurement of oxidative stress, lipid peroxidation, Nitric oxide content and inflammation parameters

Superoxide dismutase activity (SOD), malondialdehyde concentration (MDA) and nitric oxide content (NO) in BALF were measured by using the corresponding Biodiagnostic colorimetric kits (Cairo, Egypt). The concentration of matrix metalloproteinase 9 (MMP-9) and intracellular adhesion molecule (ICAM-1) were analyzed also in BALF using rat-specific ELISA kit (Shanghai Sunredbio technology, Ltd, China). Finally, the content of C-reactive protein (CRP) was also analyzed in BALF via using the corresponding Biosystem colorimetric kits (Cairo, Egypt).

Histopathology examination and determination of NF-κB protein expression

For histopathology examination, the mid-zone of the left lung was embedded in paraffin wax. From each lung sample, 3–4 µm sections were cut and stained with hematoxylin and eosin (H&E) for histopathological examination. Slides were scanned with a microscope at 10x magnification.¹⁴ An index in representative images to quantify these pathological changes was blindly scored. According to the scope and severity of the lung, it was graded on a scale of 0.5–4: 0.5 = minor, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe.¹⁵

Nuclear factor kapp B/p65 was detected by immunostaining of sections prepared from formalin-fixed, paraffin-embedded lung samples, based on a standard indirect immunoperoxidase procedure (Peroxidase/Antiperoxidase (PAP) technique) using Lab Vision Kit.¹⁸ The nuclear and or cytoplasmic immunostaining intensity for NF-κB subunits was graded using the following scoring criteria: The number of cells stained was scored as either no cells stained (0), less than 10% of cells stained (1), 10– 50% of cells stained (2), 50– 80% of cells stained (3) or more than 80% of cells stained (4).¹⁹

Statistical analysis

Data expressed as mean ± SD. Results assayed using a one-way analysis of variance test (one-way ANOVA) followed by Tukey-Kramer multiple comparison test. Statistical analysis was carried using Graph Pad Prism software version 7; a probability level of less than 0.05 was accepted as statistically significant.

Results

Pre-treatment effects of dexamethasone and losartan on body weight of rats

Although initial body weight was non-significant in between groups, the rats in CS group weighed significantly less than those in control group by 37% after 10 weeks exposure period. In contrast, the rats which exposed to clean air, gained normal body weight. Dex+Los group showed a significant increase in body weight of rats at week 10 by 58.13% compared with the CS group and by (55.25% and 9.19%, respectively) compared with the Dex and Los groups. On the other hand, no significant difference between CS and Des groups was observed. As in Figure 1. Pre-treatment with Dex+Los can increase the body weight in COPD rats.

Figure 1.

Pre-treatment effects of dexamethasone and losartan on body weight of rats in 12 cigarette-smoke induced COPD. Rats were received saline and exposed to room air simultaneously (CTRL) group; rats were exposed to CS of 8 cigarettes/day for 7 day/week for 10 weeks (CS) group; rats were received Dex (2mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks (Dex) group; rats were received Los (30mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for 10 weeks(Los) group; rats were received Dex (2 mg/kg, i.p) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks and also received Los (30mg/kg, i.p), 30 minutes before exposure to CS for 10 weeks (Dex+Los) group. Data expressed as the mean ± SD, n = 8. ^a (p < 0.05) compared with CTRL group, ^b (p < 0.05) compared with CS group, ^c (p < 0.05) compared with Dex group, ^d (p < 0.05) compared with Los group. We used one-way ANOVA followed by Tukey-Kramer multiple comparison test. CTRL: control; CS: cigarette smoke; Dex: dexamethasone; Los: losartan.

Pre-treatment effects of dexamethasone and losartan on oxidative stress parameters in BALF

Superoxide dismutase activity exerts protective activity against oxidative stress that keeps a balance between the generation and scavenging of ROS. While, MDA concentration has been usually used as an indication of lipid peroxidation, indicating the oxidative injury of the cell plasma membrane. As shown in Figure 2, the CS group significantly reduced levels of SOD by 48.86% fold and increased levels of MDA by 3.8 fold in BALF compared with the control group. The results showed that Dex+Los group significantly increased the levels of SOD by 45.22% and decreased levels of MDA by 50.72% in rats compared with the CS group. In addition, Dex+Los group in comparison with Dex and Los groups showed a significant elevation in SOD by (10. 82% and 7.48%, respectively) and a significant reduction in MDA by (23.42% and 18.3% respectively). Pre-treatment with Dex+Los can increase the antioxidant capacity of SOD and decrease MDA in COPD rats.

Figure 2.

Pre-treatment effects of dexamethasone and losartan on BALF oxidative stress in cigarette-smoke induced COPD in male rats. (A) SOD (U/ml); (B)MDA (nmol/ml). Rats were received saline and exposed to room air simultaneously (CTRL) group; rats were exposed to CS of 8 cigarettes/day for 7 day/week for 10 weeks (CS) group; rats were received Dex (2mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks (Dex) group; rats were received Los (30mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for 10 weeks(Los) group; rats were received Dex (2mg/kg, i.p) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks and also received Los (30mg/kg, i.p),30 minutes before exposure to CS for 10 weeks (Dex+Los) group. Data expressed as the mean ± SD, n = 8. ^a (p < 0.05) compared with CTRL group, ^b (p < 0.05) compared with CS group, ^c (p < 0.05) compared with Dex group, ^d (p < 0.05) compared with Los group. We used one-way ANOVA followed by Tukey-Kramer multiple comparison test. CTRL: control; CS: cigarette smoke; Dex: dexamethasone; Los: losartan; SOD: superoxide dismutase activity; MDA: malondialdehyde.

Pre-treatment effects of dexamethasone and losartan on inflammatory parameters in BALF

As depicted in Figure 3, the CS group displayed a marked increase in the levels of CRP, MMP-9, and ICAM-1by (8.94-fold, 90.22% and 2.07-fold, respectively) compared with the control group. Dex+Los group significantly reduced the levels of CRP by 98.35%, MMP-9 by 66.12%, and ICAM-1 by 38.01% compared with the CS group. Moreover, Dex+Los group in comparison with Dex and Los groups showed a significant reduction in levels of CRP by (97.93% and 94.67%, respectively), MMP-9 by (64.22% and 23.15%, respectively), and ICAM-1 by (36.26% and 23.59%, respectively). Dex was non-significant in reducing the levels of MMP-9, ICAM-1, and CRP compared with the CS group. The finding demonstrated that Dex when co-administrated with Los could suppress CS-stimulated inflammation by inhibiting the generations of inflammatory cytokines in COPD rats.

Figure 3.

Pre-treatment effects of dexamethasone and losartan on BALF inflammatory mediators in cigarette-smoke induced COPD in male rats. (A) ICAM-1 (ng/mL); (B) CRP (mg/ml); (C) MMP-9 (ng/ml). Rats were received saline and exposed to room air simultaneously (CTRL) group; rats were exposed to CS of 8 cigarettes/day for 7 day/week for 10 weeks (CS) group; rats were received Dex (2mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks (Dex) group; rats were received Los (30mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for 10 weeks(Los) group; rats were received Dex (2mg/kg, i.p) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks and also received Los (30mg/kg, i.p),30 minutes before exposure to CS for 10 weeks (Dex+Los) group. Data expressed as the mean ± SD, n = 8. ^a (p < 0.05) compared with CTRL group, ^b (p < 0.05) compared with CS group, ^c (p < 0.05) compared with Dex group, ^d (p < 0.05) compared with Los group. We used one-way ANOVA followed by Tukey-Kramer multiple comparison test. CTRL: control; CS: cigarette smoke; Dex: dexamethasone; Los: losartan; ICAM-1: intracellular adhesion molecule-1; CRP: C-reactive protein; MMP-9: matrix metalloproteinase 9.

Pre-treatment effects of dexamethasone and losartan on BALF analysis

As shown in Figure 4, a significant decrease in the percentage of viable cells by 5.9-fold and a significant increase in the total numbers of lymphocytes and macrophages in BALF by (73.76 and 23.28 folds, respectively) of the CS group compared with the control group. Dex+Los group significantly increased the total numbers of viable cells by 5.72 fold and significantly decreased total numbers of macrophages by 74.17% and lymphocytes by 95.02% compared with the CS group. In comparison with the Dex and Los groups we found that Dex+Los group showed a significant elevation in percentage of viable cells by (29.62% and 5.13%, respectively) and a significant decrease in total numbers of lymphocytes by (68.75% and 40.48%, respectively). Dex+ Los significantly decreased the total numbers of macrophages by 93.4% compared with Los group. However, Dex+Los group compared with Dex group showed non-significant decrease in the total numbers of macrophages. Pre-treatment with Dex in combination with Los significantly suppressed lymphocytes and macrophage cells in BALF.

Figure 4.

Pre-treatment effects of dexamethasone and losartan on BALF analysis in cigarette-smoke induced COPD in male rats.(A) viable cells (%); (B)lymphocytes (cells/mL);(C) macrophages (cells/ml). Rats were received saline and exposed to room air simultaneously (CTRL) group; rats were exposed to CS of 8 cigarettes/day for 7 day/week for 10 weeks (CS) group; rats were received Dex (2mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks (Dex) group; rats were received Los (30mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for 10 weeks(Los) group; rats were received Dex (2mg/kg, i.p) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks and also received Los (30mg/kg, i.p),30 minutes before exposure to CS for 10 weeks (Dex+Los) group. Data expressed as the mean ± SD, n = 8. ^a (p < 0.05) compared with CTRL group, ^b (p < 0.05) compared with CS group, ^c (p < 0.05) compared with Dex group, ^d (p < 0.05) compared with Los group. We used one-way ANOVA followed by Tukey-Kramer multiple comparison test. CTRL: control; CS: cigarette smoke; Dex: dexamethasone; Los: losartan.

Pre-treatment effects of dexamethasone and losartan on Nitric oxide content in BALF

Endothelial cells release NO which activates smooth muscle relaxation. As shown in Figure 5, the CS group significantly reduced levels of NO by 3.76 fold in BALF compared with the control group. The results showed that Dex+Los group significantly increased the levels of NO by 3.2 fold in rats compared with the CS group. However, Dex+Los group showed a significant elevation in NO by 2.07-fold compared with the Dex group and by 30.08% compared with the Los group. Pre-treatment with Dex plus Los can increase the content of NO in COPD rats.

Figure 5.

Pre-treatment effects of dexamethasone and losartan on BALF Nitric oxide (NO) in cigarette-smoke induced COPD in male rat. Rats were received saline and exposed to room air simultaneously (CTRL) group; rats were exposed to CS of 8 cigarettes/day for 7 day/week for 10 weeks (CS) group; rats were received Dex (2mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks (Dex) group; rats were received Los (30mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for 10 weeks(Los) group; rats were received Dex (2mg/kg, i.p) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks and also received Los (30mg/kg, i.p),30 minutes before exposure to CS for 10 weeks (Dex+Los) group. Data expressed as the mean ± SD, n = 8. ^a (p < 0.05) compared with CTRL group, ^b (p < 0.05) compared with CS group, ^c (p < 0.05) compared with Dex group, ^d (p < 0.05) compared with Los group. We used one-way ANOVA followed by Tukey-Kramer multiple comparison test. CTRL: control; CS: cigarette smoke; Dex: dexamethasone; Los: losartan; NO: Nitric oxide.

Pre-treatment effects of dexamethasone and losartan on histopathological examination and NF-κB expression of lung tissue

To evaluate the protective role of Dex+Los on pulmonary pathological changes, hematoxylin and eosin (H&E) staining performed. As observed in Figure 6, CS group showed epithelial cells injury of bronchi, goblet epithelium cells metaplasia, inflammatory cells infiltration, mucus secretion and cell blocking in bronchial lumen. Besides, necrosis of epithelial cells and dilated heavily congested alveoli with destructed wall in between (blue arrows) (emphysema). Dex group showed moderate lymphocytic infiltrate (red arrow) around dilated bronchus with focal squamous metaplasia (blue arrow). Los group showed dilated bronchi lined with goblet cellular hyperplasia (blue arrow) surrounded by few chronic inflammatory cells (red arrow). Dex+ Los group showed dilated bronchi lined with mild goblet cellular hyperplasia (blue arrow). We found that the indices of Dex+Los group showed a significant downward trend compared with the CS, Dex and Los groups. However, the Dex-treated group showed a non-significant difference compared with the CS group. Finally, the results of histopathology indicated that CS group was successfully established and the ability of Dex+Los in improving the pathological injury of COPD.

Figure 6.

Pre-treatment effects of dexamethasone and losartan on histopathological examination of lung tissue in cigarette-smoke induced COPD in male rats. Photomicrographs were taken at x100. We blindly scored an index in representative images to quantify these histopathological changes. According to the scope and severity of the lung, it was graded on a scale of 0.5–4: 0.5 = minor, 1 = mild, 2 = moderate, 3 = severe, 4 = very sever. CTRL group showed normal bronchiole with intact lining of respiratory epithelium (blue arrow). CS group showed epithelial cells injury of bronchi, goblet epithelium cells metaplasia, inflammatory cells infiltration, mucus secretion and cell blocking in bronchial lumen. Beside, necrosis of epithelial cells and dilated heavily congested alveoli with destructed wall in between (blue arrows) (emphysema). Dex group showed moderate lymphocytic infiltrate (red arrow) around dilated bronchus with focal squamous metaplasia (blue arrow). Los group showed dilated bronchi lined with goblet cellular hyperplasia (blue arrow) surrounded by few chronic inflammatory cells (red arrow). Dex+ Los group showed dilated bronchi lined with mild goblet cellular hyperplasia (blue arrow). Rats were received saline and exposed to room air simultaneously (CTRL) group; rats were exposed to CS of 8 cigarettes/day for 7 day/week for 10 weeks (CS) group; rats were received Dex (2mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks (Dex) group; rats were received Los (30mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for 10 weeks(Los) group; rats were received Dex (2mg/kg, i.p) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks and also received Los (30mg/kg, i.p),30 minutes before exposure to CS for 10 weeks (Dex+Los) group. Data expressed as the mean ± SD, n = 8. ^a (p < 0.05) compared with CTRL group, ^b (p < 0.05) compared with CS group, ^c (p < 0.05) compared with Dex group, ^d (p < 0.05) compared with Los group. We used one-way ANOVA followed by Tukey-Kramer multiple comparison test. CTRL: control; CS: cigarette smoke; Dex: dexamethasone; Los: losartan.

As shown in Figure 7 the results showed a significant difference between the CS and the control group, and showed a non-significant difference between the CS and the Dex-treated group. Dex+Los group showed a significant reduction in NF-κB protein expression compared with CS, Dex and Los groups. Pre-treatment with Dex in combination with Los reduced NF-κB expression of lung tissue induced by cigarette smoke.

Figure 7.

Pre-treatment effects of dexamethasone and losartan on NF-κB expression of lung tissue in cigarette-smoke induced COPD in male rats. Photomicrographs were taken at Streptavidin biotin X 100. The nuclear and or cytoplasmic immunostaining intensity for NF-κB subunits was graded using the following scoring criteria: The number of cells stained was scored as either no cells stained (0), less than 10% of cells stained (1), 10– 50% of cells stained (2), 50– 80% of cells stained (3) or more than 80% of cells stained (4). Rats were received saline and exposed to room air simultaneously (CTRL) group; rats were exposed to CS of 8 cigarettes/day for 7 day/week for 10 weeks (CS) group; rats were received Dex (2mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks (Dex) group; rats were received Los (30mg/kg, i.p.) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for 10 weeks(Los) group; rats were received Dex (2mg/kg, i.p) as single dose, 30 minutes before exposure to CS of 8 cigarettes/day for 7 day/week for the last 5 weeks and also received Los (30mg/kg, i.p),30 minutes before exposure to CS for 10 weeks (Dex+Los) group. Data expressed as the mean ± SD, n = 8. ^a (p < 0.05) compared with CTRL group, ^b (p < 0.05) compared with CS group, ^c (p < 0.05) compared with Dex group, ^d (p < 0.05) compared with Los group. We used one-way ANOVA followed by Tukey-Kramer multiple comparison test. CTRL: control; CS: cigarette smoke; Dex: dexamethasone; Los: losartan.

Disscussion

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease associated with airway obstruction and pulmonary abnormal inflammatory reaction.²⁰ Chronic exposure to CS known as the main cause of chronic inflammation leading to air remodeling, destruction of alveolar tissue, progressive mucus hypersecretion, and decline lung function.^21,22 Steroid therapy may be beneficial for COPD patients, but its results are limited by uncertain and variable clinical responses in some patients. However, the use of large doses of steroids may have little role in a rapid decline in lung function in COPD¹³ and showed a lot of side effects.¹¹ In our research COPD model is well established by histopathological examination (as CS group showed epithelial cells injury of bronchi, goblet epithelium cells metaplasia, inflammatory cells infiltration, mucus secretion and cell blocking in bronchial lumen. Besides, necrosis of epithelial cells and dilated heavily congested alveoli with destructed wall in between (emphysema)), BALF analysis and weight loss. Our research also showed that the use of dexamethasone in combination with losartan was effective in prevention from COPD via decreasing oxidative stress, inflammatory mediators, NF-κB expression and histopathological studies. However, the use of Dex alone had no beneficial roles in inflammation, NF-kB protein expression and histopathological examination. Several reports widely used the well-established experimental animal model of CS-induced COPD, namely CS-induced emphysema to evaluate the results if selected targets against COPD.²³ Although the several experimental models of CS-induced COPD, the limited data is still poor. So we require more studies to fully understand the CS-induced COPD animal model.

The pathogenesis of COPD is poorly discussed.²⁴ Oxidative stress can exacerbate airway inflammation.²⁵ Oxidant in smoke has direct damage to airway epithelial cell and inflammatory response as it contains free radicals and reactive oxygen species (ROS).²⁶ Because of pulmonary chronic inflammation and bronchial epithelial cell damage, the synthetic ability of SOD is reduced and the level of MDA increased, which results in body decompensation.²⁷ The increase in SOD activity following losartan administration may be related to its ability to decrease the production of superoxide radicals through inhibiting 5-lipoxygenase and cyclooxygenase and decreasing the number of circulating neutrophils. Moreover, losartan may eliminate superoxide radicals by a free radical scavenging effect and induce gene expression of SOD enzyme through inhibiting TNF-1 and interleukin-1β. The role of losartan in decreasing MDA concentration was through its antioxidative and free radical scavenging properities.^28,29 NO is obtained from L-arginine by NO synthase (NOS). Both neuronal NOS (nNOS) and endothelial NOS (eNOS) are considered constitutive and are involved in vasodilation and bronchodilation. Many studies have shown that NO is an important factor contributing to muscle relaxation and the attenuation of airway hyper-responsiveness to bronchoconstrictor factors.³⁰ The inactivation of endothelial-derived NO is linked with increased oxidative stress, particularly increased production of superoxide radicals.³¹ Our study showed that the concentration of MDA was significantly decreased, and activities of SOD and content of NO were significantly increased in Dex+Los group compared with Dex and Los groups. Therefore, the combination might have possible protective activities to CS-induced COPD via the anti-oxidant property and might cause bronchodilation by activation of smooth muscle cell-soluble guanylate cyclase and the formation of cGMP(Cyclic guanosine monophosphate), which is mediated by NOS-derived NO.³² The use of Dex with Los may prevent COPD occurrence and may potentiate the activity of each other.

Inflammation is the most important reason is the development of COPD which involves the infiltration of inflammatory cells including macrophages, neutrophils, lymphocytes, and eosinophils. Inflammatory cells also play critical rule through their potent release of pro-inflammatory cytokines.^33,34 C-reactive protein (CRP) can activate NF-kB in endothelial cells and mononuclear cells to induce proteases and pro-inflammatory cytokines. CRP may be useful as a marker to monitor inflammation during COPD treatment.³⁵ The use of Losartan, as angiotensin receptor type 1 blocker, can inhibit Interleukin-6 (IL-6) that cause induction of CRP release.^36,37 The imbalance between aggressive and protective defense systems in the lungs (proteases and antiproteases) might play a major role in the pathogenesis of COPD. There are three important groups of proteinases including matrix metalloproteinases (MMPs(that are believed to play a key role in the development of the characteristic COPD inflammatory process.³⁸ Several studies measured MMP-9 concentration in sputum as a sign of local inflammation in COPD patients.³⁹ The use of Losartan, as angiotensin receptor type 1 blocker, can inhibit Tumor growth factor –β (TGB-β), Tumor necrosis factor-1 (TNF-1), NF-kB activation that cause induction of MMP9 expression.^13,40,41 ICAM-1 is an important adhesion molecule involved in several inflammatory processes, such as neutrophil and lymphocyte trafficking and it acts as an accessory molecule in antigen presentation.⁴² Several studies have pointed to the role of ICAM-1 expression in the pathogenesis of COPD. ICAM-1 concentrations in the bronchoalveolar lavage of COPD patients are increased and cigarette smoke extract increases ICAM-1 production by macrophages.^43,44 The action of Losartan, as angiotensin receptor type 1 blocker, in decreasing ICAM-1 is mediated by inhibition of TNF-1, NF-kB activation and MMPs.^13,40,41 In the present study, Dex+Los significantly reduced the levels of CRP, MMP-9, and ICAM-1, which indicated the reduction in the symptoms of COPD. This is due to the inhibition of the inflammatory reaction.^13,45 Dex had no rule in the reduction of inflammatory mediators like CRP, MMP-9, and ICAM-1. The administration of Dex+Los may protect from COPD and augmenting the efficacy of each other.

A nuclear transcription factor (NF-kB), has intimate relativity with several physiologically and pathologically processes such as inflammatory response, immune reaction, cell proliferation, transformation and apoptosis.⁴⁶ NF-κB activation is stimulated by cytokines activation, which in turn regulates the expression of immunomodulatory and inflammatory mediators. Many NF-κB-regulated responses are insensitive to the actions of glucocorticoids.⁴⁷ Of note; Dex+Los significantly reduced the NF-κB expression, which indicated that the reduction in the severity of COPD is due to the prevention of apoptosis.⁴⁸ On the other hand, Dex had no role in suppression of the activation of NF-κB expression, supporting the beneficial use of Dex+Los in COPD and the additive effects of the use of both drugs.

Decreasing NO, increasing oxidative stress and inflammation resulted in higher protein content in BALF, increased lung cell necrosis and emphysema. In our study, CS rats group developed necrosis and emphysema in histopathological studies which can be related to decreased levels of NO and SOD, and increased levels of MDA, MMP-9, ICAM-1 and NF-kB expression. However, Dex+los group showed significant increasing in levels of NO and SOD, and decreasing levels of MDA, MMP-9, ICAM-1 and NF-kB expression.^49
–51 Moreover, the proliferation of human lung fibroblasts and induction of alveolar epithelial cell apoptosis were apparently increased by the binding of angiotensin II to the AT1 receptor. Losartan prevents fibrosis mainly via inhibition of binding of angiotensin II to the AT1 receptor.³²

Finally losartan can prevent weight loss in COPD disease by protecting from oxidative stress which is linked to normal process of aging that cause loss of muscle mass.⁵² In addition, losartan can decrease release of many cytokines particularly TNF-1 and prevent NF-kB activation that lead to weight loss in COPD patients.^52,53 Dex+Los administration can significantly prevent weight loss in COPD and can enhance the activity of each other.

Conclusion

The protective effects of combined dexamethasone and losartan in CS-induced COPD appear to be mediated via their antioxidant, anti-inflammatory, and anti-apoptotic properties, revealing a clinical potential of their therapeutic applications. Accordingly, adding Los to Dex potentiating their activity against COPD. In addition, dexamethasone can cause hypertension as a side effect which can be prevented by the use of losartan. Further clinical research is necessary before clinical application.

Footnotes

Acknowledgments

The authors extend their sincere appreciation to the research facility provided by Chairperson, Department of Pharmacology and toxicology, Faculty of pharmacy, Tanta University. We would like to acknowledge Dr. Youmna Zamzam (Ph.D., department of clinical pathology, Tanta University) for her help on histopathological and immunohistochemical examination.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Esraa H Afify

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Dexamethasone and losartan combination treatment protected cigarette smoke-induced COPD in rats

Abstract

Keywords

Introduction

Methods and materials

Animals

Drugs and chemicals

Experimental protocol

Bronchoalveolar lavage fluid (BALF) and lung tissue samples collection

Measurement of oxidative stress, lipid peroxidation, Nitric oxide content and inflammation parameters

Histopathology examination and determination of NF-κB protein expression

Statistical analysis

Results

Pre-treatment effects of dexamethasone and losartan on body weight of rats

Pre-treatment effects of dexamethasone and losartan on oxidative stress parameters in BALF

Pre-treatment effects of dexamethasone and losartan on inflammatory parameters in BALF

Pre-treatment effects of dexamethasone and losartan on BALF analysis

Pre-treatment effects of dexamethasone and losartan on Nitric oxide content in BALF

Pre-treatment effects of dexamethasone and losartan on histopathological examination and NF-κB expression of lung tissue

Disscussion

Conclusion

Footnotes

Acknowledgments

Declaration of conflicting interests

Funding

ORCID iD

References