br Materials and methods br Results

Materials and methods
Results
Discussion Approximately 90% of pulmonary emphysema causes are related to smoking and its progression in patients and are characterized by severity levels [5]. Experimental models using CS to induce emphysema are widely used because they resemble the emphysema observed in humans [6,7,11,20]. However, these models do not reach a stage equivalent to severe emphysema [9,[13], [14], [15]]. The therapeutic approach is complex, and there is a need to investigate the action of new drugs, not only to alleviate the symptoms but also to treat the morphological lesions caused by the different clinical phenotypes. Our group recently published a study in which we used a well-described model of cigarette smoking-induced emphysema mainly due to the imbalance between oxidants and antioxidants. After the development of disease, two statins (atorvastatin and simvastatin) were found to stimulate lung repair [20]. In the present study, we used a characteristic model of protease – antiprotease imbalance through the intranasal administration of elastase, which generated severe emphysema with lung morphological alterations of greater amplitude than the previously used cigarette smoking model. Similar to the previous study, we treated the animals with atorvastatin after establishment of disease on the 32nd day for the following 32 days. We observed that lesions of the pulmonary structures, such as destruction of the alveolar septa with consequent enlargement of the alveoli, persisted even in the absence of the elastolytic stimulus. This phenomenon was verified by the measurement of Lm, Vv of alveoli, Vv of collagen fibers, and Vv of elastic fibers in the PPE 64d groups. The aim of this study was to evaluate whether atorvastatin, a statin that showed the best anti-inflammatory effects in a previous study, was able to revert the severe morphological changes caused by elastase and whether this reversal was dose-dependent. The A5 mg and A20 mg groups showed a significant improvement in the Lm and Vv of VSV-G Peptide and elastic and collagen fibers. The A1 mg group showed no change in the Lm and Vv of elastic fibers. Takahashi et al. [18] performed a similar experiment in which mice received elastase intratracheally and were treated with simvastatin. The animals were treated with varying doses of simvastatin (4, 20 and 100 μg ip) 21 days after elastase administration, and the authors observed a significant reduction in Lm at all doses compared with the control group. The 100 μg group presented the greatest improvement. Takahashi et al. attributed this reduction in Lm mainly to the proliferation of epithelial cells observed by immunohistochemistry. In the acute protocol, the mice received 20 μg of simvastatin, and the concentrations of hydroxyproline and desmosine (markers of collagen and elastin rupture, respectively) were measured in the bronchoalveolar lavage (BAL). The authors concluded that there was a reduction in hydroxyproline concentration on the 3rd day after administration of elastase, but not for desmosine. In our study, we also observed a reduction in Lm at the highest dose of treatment as well as at a later stage (32 days after elastase administration). Although we analyzed the collagen and elastic fibers in lung tissue, we observed an improvement in Vv of collagen fibers at all doses, except for the Vv of elastic fibers at doses of 5 mg and 20 mg. Based our results, we suggest that, due to the extensive degree of fiber impairment, larger doses may be required to promote the synthesis of new elastic fibers. The number of leukocytes in the bronchoalveolar lavage and the total number of macrophages and neutrophils in the alveolar space of the PPE 64d group was higher than that in the control group. Jiang et al. [29] investigated the role of the serotonin (5-HT or 5-HTT) transporter and the effect of simvastatin regulation in a rat model of cigarette smoke-induced pulmonary hypertension. They observed that cigarette smoke increased the influx of leukocytes in BAL, which was reduced by simvastatin treatment. In normoclasticemic patients with coronary artery disease, Walter et al. [30] investigated the effects of atorvastatin treatment on the expression of cellular adhesion molecules in leukocytes. They concluded that treatment with atorvastatin reduced chronic inflammation by decreasing the expression of adhesion molecules in monocytes and neutrophils. Statins are known to interfere with cell binding by reducing leukocyte adhesion to endothelial cells [31]. This function of statins explains their ability to weaken the upregulation of P-selectin typically seen in activated endothelial cells and to interfere with the adhesion of monocytes and lymphocytes to endothelium via suppression of intercellular adhesion molecule I ((ICAM-I)) and antigen 1 associated with lymphocyte function-associated antigen 1 (LFA-1) counts. Oliveira et al. [32] studied the effect of a model of emphysema induced by multiple tracheal instillations (4 instillations beginning at day every 7 days) of PPE to examine changes in the inflammatory profile in the pulmonary septum after each instillation. They observed a considerable increase in the macrophage and neutrophil populations after the last instillation (day 21). We used a similar protocol; however, it comprised short times between instillations. We also observed an increase in total macrophages and neutrophils in the alveolar space in the PPE 64d group compared with the control group, although we did not characterized this increase in time intervals. In our study, we observed a significant reduction of elastic and collagen fibers both in the PPE 32d and PPE 64d groups, which may, in part, explain the perpetuation of the inflammatory process since elastin and collagen fragments act as chemoattractants recruiting more macrophages and neutrophils, thus creating a cycle [1,3]. The atorvastatin-treated groups showed a reduced total leukocyte count in the bronchoalveolar lavage fluid, with more significant results obtained for the A20 mg group than the A1 mg and A5 mg groups. However, only the 20 mg dosage reduced macrophage counts in the alveoli. Regarding neutrophils, atorvastatin doses of 5 mg and 20 mg were more effective than 1 mg. Kourliouros et al. [33] performed a study using high doses of atorvastatin (80 mg/kg) in preoperative cardiac surgery patients to prevent postoperative inflammatory complications such as systemic inflammatory response syndrome. The authors concluded that maximizing the statin dose in preoperative patients was beneficial by relying on decreased cardiac and renal damage as measurable evidence. They suggested that this preoperative protection is dose-dependent and is attributed, among other effects, to the change in transendothelial migration of neutrophils and in the reduction of MMP-9 production in patients undergoing surgery.