Rosuvastatin Attenuated Elastic Fiber Degradation in Chronic Obstructive Pulmonary Disease Sprague-Dawley Rats Rosuvastatin

Chronic obstructive pulmonary disease (COPD) is an incurable disease which causes disability and death. The main pathogenesis of COPD is oxidative stress due to cigarette smoke which initiates various reactions and lead to lung elastic fibers destruction. Statins are known to have antioxidant effects and reduce mortality in COPD. We studied the effects of cigarette smoke exposure cessation and rosuvastatin on oxidative stress and the level of elastic fiber destruction in COPD model rats. Thirty 10-week old male Sprague-Dawley rats were divided into 2 groups: Control (n=6, did not received fumigation nor treatment) and Smoking (n=24, received fumigation for 70 days) groups. Afterwards, the smoking group was divided into 4 groups: Sham, R2, R5,R10, and received 0.9% NaCl, 2.5, 5 and 10 mg/kg/day of rosuvastatin, respectively. Examination of malondialdehyde (MDA) and desmosine serum were conducted to measure oxidative stress and elastic fiber degradation level, respectively. After smoke exposure, MDA and desmosine levels of COPD rats were found to be significantly higher (p=0.000 and 0.000) than controls. The MDA level in Sham, R2, R5 and R10 groups decreased significantly after therapy (p=0.000; 0.033; 0.015; 0.002). However, the post-treatment desmosine level was increase significantly in Sham and R2 groups (p=0.006 dan 0.012) and insignificantly (p=0.117 dan 0.278) in the R5 and R10 groups. It can be concluded that the cessation of exposure to cigarette smoke can reduce oxidative stress, but not elastic degradation process. The administration of rosuvastatin of 5 or 10 mg/kg/day attenuated elastic degradation process.


INTRODUCTION METHODS
Smoker prevalence in many countries in the world, Animals including Indonesia is increasing every year (1). Cigarette Ten weeks old male Sprague-Dawley rats (n=30) were smoking is the most important risk factor for purchased from Integrated Research and Testing cardiovascular disease and chronic obstructive pulmonary Laboratory, Universitas Gadjah Mada, Yogyakarta, disease (COPD), which cause progressive airflow Indonesia. Rats were maintained in cages with light-dark limitation (2). The pathogenesis of COPD starts from free cycle every 12 hours. Food and water were given ad radicals in cigarette smoke that triggers the activation of libitum. alveolar macrophage cells (MA) as the first-line defense cells. The activated alveolar macrophages release a variety of inflammatory mediators that attract neutrophils and monocytes, which subsequently increase the Induction of COPD production of Reactive Oxygen Species (ROS) such as . superoxide, hydrogen peroxide and hydroxyl radicals ( OH) The rats were divided into 2 groups: Control (n=6) and (3).
Smoking groups (n=24). The Control group did not receive fumigation, while Smoking group received cigarette smoke In a physiological state, human body has its own exposure at 8 cigarettes/day. Each cigarette used in this mechanism to balance the level of oxidants by the activity study contained 38 mg of tar and 2.4 mg of nicotine. of endogenous antioxidants. However, ROS from cigarette Cigarette smoke exposure was given using the whole body smoke could reduce this activity. Oxidant burden from exposure method. Every day, the rats from Smoking group cigarette smoke and inflammatory cell's product can lead were moved into the smoking apparatus. The smoking to oxidant-antioxidant imbalance or oxidative stress. The apparatus consist of three major parts. Cigarette burn level of oxidative stress can be measured indirectly by chamber in the bottom, smoke exposure chamber in the measuring the levels of malondialdehyde (MDA) as the middle and ventilation in the top of the apparatus. The product of lipid peroxidation (3).
smoke exposure chamber was a 50 cm x 50 cm x 50 cm of glass chamber. Burn chamber and smoke exposure In addition to ROS, inflammatory cells also produce a chamber were separated by a metal nets. This nets allowed variety of protease, which is a proteolytic enzyme that can the smoke from the cigarette entering the smoke exposure degrade proteins, including lung elastin. Under normal chamber. For smoke exposure, cigarettes were burned in circumstances, proteases are essential for the defense the burn chamber, and the smoke from the cigarette mechanism and antiproteases are present to protect the entered the box (Figure 1). Since all the smoke was surrounding tissue from the destructive effects of released from the tip of the cigarette, all the smoke proteases. However, the ROS burden could lead to α1-AT produced was side stream smoke. The rats were exposed deficiency in the body (4). Increased production of to cigarette smoke for 70 consecutive days, as described by proteases accompanied by the inactivation of Zhang et al, with a slight modification (smoking exposure antiproteases promotes elastin degradation which results was given everyday instead of 6 days/ week) (18). in the COPD condition (3).
When elastin is degraded by proteases, desmosine, which is a component of cross-links in mature elastin is detached. Since the released desmosine cannot be absorbed, reused or catabolized by the body, it will be disposed of in its unchanged form. Desmosine is a unique amino acid which is only found in mature elastin, the degree of elastin damage can be detected by measuring the desmosine level in the blood (5).
The progress of COPD can be reversed by smoking cessation, but there is no available drug to cure the disease (6). Giving an antioxidant to prevent tobacco smoke-induced oxidative stress and COPD has shown promising results but its remedial effect on COPD patients is very limited (7)(8)(9)(10)(11)(12). Statins, such as rosuvastatin are known to have antioxidant effects. A recent study and meta-analysis on many studies of statin concluded that there is a mortality reduction in COPD patients receiving statin therapy (13)(14)(15). However, Criner G et al found no significant difference of mortality rate in COPD patient with statin compare to without statin (16). Compared to other statins, rosuvastatin has the longest half-life (19 hours) as well as rarely causes myopathy, liver function test abnormalities and adverse interactions with other drugs (17). This study aimed to determine the effect of smoke cessation and administration of rosuvastatin in pre and post-treatment in each group were analyzed using paired t-test. All the results were shown in mean + SD. The On Day 71, exposure to cigarette smoke was discontinued.
statistical significance was set at p value of <0.05. The rats from Smoking group were divided further into 4 groups: Sham, R2, R5 and R10 groups (n = 6 for each RESULTS group). Subsequently, each rat of Sham, R2, R5 and R10 groups received treatment for 27 days via oral Serum MDA Level administration. Sham group received 2 ml of NaCl 0.9%; After fumigation, serum MDA levels in the Smoking group R2, R5, R10 groups received 2.5 mg/kgBW, 5 mg/kgBW were significantly higher than Control group (Table 1). This and 10 mg/kgBW of rosuvastatin, respectively. The result indicated an increase of oxidative stress in rats rosuvastatin tablet was crushed into powdered and exposed to cigarette smoke. diluted in NaCl 0.9% solution, and given to the rats through a feeding tube.
Sample Collection Table 1

. Serum MDA and desmosine levels in control and
Blood samples were taken one day after the last cigarette smoking groups after 70 days of cigarette smoke exposure smoke exposure (for pre-treatment measurement) and one day after the last treatment (for post-treatment measurement). The blood was taken from the retroorbital vein using a micro-hematocrit capillary tube. Within one hour after collection, the pre and posttreatment blood were centrifuged at 4000 rpm for 20 minutes to collect the serum.

Oxidative Stress Level Measurement
The level of oxidative stress was determined by measuring serum MDA levels using the thiobarbituric acid-reactive substance (TBARS) method according to the previous One day after smoke cessation, the Smoking group was study (19,20). Briefly, 1 ml serum was mixed with 4 ml 'TBA o further divided into 4 groups (Sham, R2, R5, and R10). At reagent', incubated in water bath at 90 C for 80 minutes the end of the study, 29 rats were survived, but one rat and cooled in ice. After being mixed with butanol from R2 group died in the middle of the study. After 27 days extraction solution and centrifuged, the pink color of rosuvastatin treatment, serum MDA level was measured co m p l ex i n t h e s u p e r n ata nt wa s m e a s u re d again. Paired t-test analysis showed that post-treatment spectrophotometrically at 510, 532 and 560 nm. The serum MDA levels in Sham, R2, R5, and R10 groups were actual absorbance was determined by the following significantly lower than the pre-treatment level (Table 2) groups using one way ANOVA showed no significant difference. This suggested that smoking cessation with or TBARS concentration (µmol/l solution) was read using 1,1,3-3-tetramethoxypropane as a standard.
without rosuvastatin reduced MDA level.

Elastin Degradation Level Measurement Serum Desmosine Level
The determination of elastin destruction level was Following cigarette smoke exposure, serum desmosine performed by measuring the level of desmosine serum levels in the Smoking groups were significantly higher than using ELISA. The procedure was based on the that of the Control group (Table 1). This suggested that manufacturer's protocol (MyBiosource, catalog number: smoke exposure induced elastic fiber degradation in rats. MBS 748709).
Twenty-seven days after the administration of NaCl 0.9% Statistical Analysis or rosuvastatin, the level of serum desmosine was measured again. We found that the post-treatment The statistical analyze was performed using SPSS desmosine level increase in all treatment groups (Statistical Product and Service Solutions). After compared to pre-treatment levels. Paired t-test analysis fumigation, the MDA and desmosine level between between pre and post-treatment within groups showed Control and Smoking groups were analyzed using sigificantly higher post-treatment desmosine level than unpaired t-test. After treatment, the post-treatment the pre-treatment level in Sham and R2 groups. However serum MDA levels and desmosine levels between Control, there was no significant difference between pre and post-Sham, R2, R5, and R10 groups were compared using One Way ANOVA. Serum MDA and desmosine levels between treatment desmosine level in R5 and R10 groups.

DISCUSSION
smoke are potent to inactivate α1-AT, which is a major antiprotease, by oxidating the active side of this enzyme Cigarette smoke exposure is known to be the main risk (30). This mechanism induces a protease-antiprotease factor for COPD. Cigarette smoke consists of many toxic imbalance that leads to elastin degradation in cigarette particles, including ROS (3). Lipid content in the cell exposure. membrane is the principal target molecule of oxidation by ROS (21). The increase of MDA level after smoke exposure Smoke cessation was expected to reduce the serum observed in the present study was consistent with the desmosine. The antioxidant effects of rosuvastatin as an . findings in other studies on animals and humans (22) This adjuvant therapy was expected to restore the function of α1-AT which in turn supress the elastin degradation rate. finding suggests that cigarette smoke exposure increases However, this was not the case of our study. Our study oxidative stress. Some studies revealed that the increase found a significant increase of desmosine levels in the of oxidative stress in smoking subjects were caused by the Sham and R2 groups following treatment, but not higher concentration of ROS derived from cigarette smoke significant in R5 and R10 groups. This result may suggest as well as activated neutrophils and macrophages.
that the elastin degradation process continued after Moreover, cigarette smoke also leads to the depletion of smoking cessation. Furthermore, there is a possibility that endogenous antioxidants level such as vitamin E, vitamin elastin degradation was affected by other factors besides C, superoxide dismutase (SOD), Catalase (CAT) and oxidative stress. glutathione peroxidase (GPX). This in turn worsens the oxidative stress condition (22,23).
One of the factors that could possibly increase elastin degradation after smoking cessation is inflammation.

Serum MDA Levels
Studies in humans showed that the inflammatory process To date, pharmacology therapies that are available for in COPD subjects was persistent and even increased after COPD are for reducing the symptoms, but not for curing smoking cessation (31). Since inflammatory cells such as the disease. Therapy that is known to inhibit the neutrophils and macrophages are capable to produce a progressivity of COPD is smoking cessation. In this study, variety of proteases (3), possibly further destruction of smoke cessation has been shown to reduce MDA levels.
elastin continue to occur. However, the administration of 5 Other studies also demonstrated that smoking cessation mg/kgBW or 10 mg/kgBW of rosuvastatin was shown to reduced the levels of MDA (24). However, the present attenuate the destruction process, since there was no study found that rosuvastatin did not reduce the levels of significant difference in desmosine levels between pre and serum MDA greater than smoke cessation only. Statistical post-treatment. However, this possibility needs to be analysis using one-way ANOVA did not show any confirmed. In the future, it is important to measure the significant difference between groups in post-therapy inflammation level of the subjects. Furthermore, to ensure MDA levels. that the increase of desmosine level comes from the lungs, the measurement of the desmosine protein levels and Some studies reported that rosuvastatin reduced the level mRNA expression in the lungs are needed. of MDA and increased antioxidant levels in subjects with stroke, atherosclerosis, dyslipidemia, and obesity (25-27).
In conclusion, this study demonstrated that cigarette On the other hand, one previous study showed that smoke cessation decreased oxidative stress as although antioxidant administration was able to reduce characterized by the reduction of serum MDA levels. the level of serum MDA on rats which were exposed to However, the antioxidant effect of rosuvastatin could not cigarette smoke, its administration on normal rats did not be observed. This study found an increase of desmosine show any effect on MDA levels (28). Therefore, there is a level following smoke cessation, suggests that elastic possibility that upon the optimum redox balance of the degradation continued after smoke cessation. The oxidant-antioxidant system, the body will maintain this administration of 5 or 10 mg/kgBW/day of rosuvastatin balance. Overall, our study shows that cigarette smoke has been shown to attenuate elastic degradation process. cessation is capable of reducing the serum MDA level in COPD model rats. However, the ability of rosuvastatin to ACKNOWLEDGEMENT reduce the level of oxidative stress in COPD model rats was