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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 3  |  Page : 1081-1087

The effect of atorvastatin on bleomycin-induced pulmonary fibrosis in rats


1 Department of Clinical Pharmacology, Faulty of Medicine, Mansoura University, Mansoura, Egypt
2 Department of Pathology, Faulty of Medicine, Mansoura University, Mansoura, Egypt
3 Department of Medical Biochemistry, Faulty of Medicine, Mansoura University, Mansoura, Egypt

Date of Submission15-Sep-2015
Date of Acceptance15-Dec-2015
Date of Web Publication31-Dec-2018

Correspondence Address:
Eman A Ali
Department of Clinical Pharmacoloy, Faculty of Medicine, Menoufiya University, Shebin Elkom, Menufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_379_15

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  Abstract 


Objective
This work aims to investigate the effect of atorvastatin on bleomycin-induced pulmonary fibrosis in rats.
Background
Pulmonary fibrosis is a lung disease refractory to treatment with poor prognosis. It is characterized by progressive irreversible destruction of lung architecture resulting from scar formation and organ dysfunction. Atorvastatin ameliorates pulmonary fibrosis in rats via having antioxidant and antifibrotic effects.
Materials and methods
The study was conducted for 3 weeks using 30 male Wister albino rats divided into three groups of 10 rats each as follows. Group 1: injected with saline intratracheal single dose and received saline orally. Group 2: injected with a single intratracheal dose of bleomycin (1 mg/kg) and received saline orally. Group 3: injected with bleomycin intratracheal and received atorvastatin (10 mg/kg/day) orally. The following procedures were done: (a) baseline body weight, final body weight, lung weight, and lung coefficient. (b) Biochemical measurements: lung malondialdehyde (MDA), lung reduced glutathione (GSH), and serum transforming growth factor-β1 (TGF-β1). (c) Histopathological examination of hematoxylin and eosin and Masson's trichrome stained sections followed by α-smooth muscle actin (α-SMA) immunostaining.
Results
Toxic group (group 2) showed a significant decrease in body weight and lung GSH associated with increase in lung weight, lung coefficient, serum TGF-β1, lung MDA level, histopathological fibrosis score, and H-sore of α-SMA compared with the normal group (group 1). In contrast, atorvastatin-treated group (group 3) showed a significant increase in body weight and lung GSH with reduction in lung weight, lung coefficient, serum TGF-β1, lung MDA level, histopathological fibrosis score, and H-sore of α-SMA compared with (group 2), but still significant to normal group levels.
Conclusion
It is concluded that atorvastatin attenuated bleomycin-induced pulmonary fibrosis in rats by reduction of oxidative stress markers, suppression of TGF-β1, and improving fibrosis score and H-score of α-SMA.

Keywords: atorvastatin, bleomycin, pulmonary fibrosis


How to cite this article:
Ali EA, Abd El Salam EM, Daba MHY, Yassin AA, El kady NM, Badr EA. The effect of atorvastatin on bleomycin-induced pulmonary fibrosis in rats. Menoufia Med J 2018;31:1081-7

How to cite this URL:
Ali EA, Abd El Salam EM, Daba MHY, Yassin AA, El kady NM, Badr EA. The effect of atorvastatin on bleomycin-induced pulmonary fibrosis in rats. Menoufia Med J [serial online] 2018 [cited 2019 Jun 16];31:1081-7. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/1081/248743




  Introduction Top


Pulmonary fibrosis is a progressive interstitial lung diseases characterized by excessive deposition of extracellular matrix, fibroblast proliferation, and disruption of lung architecture leading to the appearance of symptoms of respiratory dysfunction such as dyspnea, cough, pulmonary hypertension ending in respiratory failure and death[1],[2]. Pulmonary fibrosis may arise due to viral infection, rheumatoid arthritis, scleroderma, radiation, inorganic substances (silica, asbestos) and may be due to drug induced (bleomycin and amiodarone), but the most common and most severe etiology is the idiopathic form of pulmonary fibrosis[3].

Oxidative stress plays an important role in the pathology of many organs including the lung. The pathogenesis of pulmonary fibrosis is still unclear, but may be partly attributed to the release of toxic reactive oxygen species (ROS) and nitrogen species(RNS)aswellasthat overrides the compensatory capacity of the lung antioxidant system.[4],[5]. ROS enhances TGF-β-induced fibrosis through the activation of latent TGF-β and increasing its expression and secretion of TGF-β in many types of cells like macrophages. TGF-β, in turn, activates the production of more ROS producing a vicious circle denoting the role of antioxidants in therapy[6].

TGF-β1 is considered the main fibrogenic mediator in various organs. Increased production of TGF-β1 is followed by an increased transformation of fibroblast into myofibroblasts, increased synthesis of collagen, proteoglycans, and fibronectin[7].

Bleomycin model is the most common animal model for pulmonary fibrosis. Bleomycin is an antibiotic used as an anticancer agent[8].

Patients subjected to bleomycin treatment suffer from pulmonary toxicity, which can be fatal in about 10% of cases. This toxicity is related to the deficiency of bleomycin metabolizing enzyme; bleomycin hydrolase, which already has its lowest levels in the lung and the skin, genetic predisposition, and release of reactive oxygen species with subsequent liberation of inflammatory cytokines leading to bleomycin-induced pneumonitis (BIP) and eventually, fibrosis[9].

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase inhibitor used as a treatment for dyslipidemia and prevention of cardiovascular disease[10]. Besides its lipid lowering effect, it showed to have pleiotropic effects including anti-inflammatory, antioxidant, and immunomodulatory effects[11],[12]. It is speculated to have an inhibitory effect on the proliferation of myofibroblasts through the inhibition of TGF-β1 and α-smooth muscle actin (α-SMA) expression in myofibroblasts putting this drug as a candidate topic worthy to study in the treatment of pulmonary fibrosis[13],[14].

This work aims to study the effect of atorvastatin on bleomycin-induced pulmonary fibrosis in rats.


  Materials and Methods Top


Thirty male albino rats of local strains of average weight 200 g were brought and acclimatized for 1 week before the start of the experiment in a well-ventilated room at room temperature with free access to water and diet. The study was approved by the ethics committee of the Faculty of Medicine, Menoufia University.

The baseline body weight is measured at the start of the experiment. Rats were divided into the following three groups of 10 rats each:

  1. Group 1 (normal group): rats were anesthetized using intraperitoneal ketamine (50 mg/kg) (EIPICO Co. Heliopolis, Cairo, Egypt) and a mid-line incision was made in the skin and subcutaneous tissue overlying the proximal portion of the trachea. A single intratracheal injection of saline 0.9% was given and received saline 0.9% orally for 3 weeks
  2. Group 2 (toxic (bleomycin) group): a single intratracheal injection of bleomycin (Sigma Aldrich Co., SAINT LOUIS, Missouri, USA) at a dose of 1 mg/kg dissolved in saline 0.9% was injected as described in the normal group and received saline orally for 3 weeks[15]
  3. Group 3: rats injected with bleomycin (as in group 2) and received atorvastatin (Pfizer Co., New York, USA) orally dissolved in saline in a dose of 10 mg/kg/day for 3 weeks[16].


At the end of the study the following was done:

  1. Rats were weighed for final body weight
  2. Collection of blood samples and determination of TGF-β1: blood samples (2 ml) were obtained from the retro-orbital plexus using heparinized capillary tubes under anesthesia using diethyl ether[17]. The samples were incubated at room temperature until clotting occurs. Thirty minutes later, the samples were centrifuged at 2000g for 15 min. Then the serum samples were separated and stored at −80°C for the determination of TGF-β by using enzyme-linked immunoassay kits (San Diego, California, USA) and the results are shown as ng/l
  3. Determination of lung coefficient: after collection of blood samples, the rats were decapitated and the lung was extracted and weighed and the lung coefficient was determined (lung weight in g/basal body weight in g)[18]
  4. Determination of lung malondialdehyde (MDA) and lung reduced glutathione (GSH) level: after extraction, the right lung was perfused in PBS solution, pH 7.4 containing 0.16 mg/ml heparin to remove any red blood cells and clots and then homogenized in cold buffer (50 mM potassium phosphate, pH 7.5. 1 mM EDTA) using a glass tissue homogenizer. The homogenate was centrifuged at 4000 rpm for 20 min and the supernatant was kept at −80°C until used for MDA (Biodiagnostic: Dokki, Giza, Egypt) and reduced glutathione (GSH) (Biodiagnostic) detection. Lung MDA levels in the supernatant were determined by monitoring thiobarbituric acid reactive substance formation using the spectrophotometric method described by Ohkawa et al.[19] and the results were expressed as nmol/g tissue. GSH levels in the supernatant were determined by the spectrophotometrical method according to Beulter et al.[20]. The results are expressed as mmol/g tissue
  5. Histopathological examination: after scarification, left lung was kept in 10% neutral-buffered formalin and sent to the pathology department for histopathological examination of hematoxylin and eosin (H and E) and Masson's trichrome (MT) stained sections for the detection of collagen deposition. Qualitative histological scoring of fibrosis was divided into absent, mild, moderate, and severe
  6. Immunohistochemical staining of α-SMA expression: 4 μm thick sections were cut from paraffin-embedded blocks, and then deparaffinized and dehydrated in xylene and graded series of alcohol, respectively. The sections were boiled in 10 mmol/l citrate buffer (pH 6.0) for 20 min for antigen retrieval and then were left to cool at room temperature. An overnight incubation at room temperature with anti-α-SMA. Clone1A4 (mouse monoclonal antibody) (Dako, Glostrup, Denmark) with dilution of 1:50 followed by using the detection kit (Dako) to determine α-SMA expression using an appropriate substrate/chromogen (diaminobenzidine reagent) followed by Mayer's hematoxylin as a counterstain. Subjective quantitative histoscore (H-score) was used to evaluate SMA expression as negative (0), weak (1), moderate (2), and strong (3). This number was multiplied by the percent (%) of stained cells. The score ranged from 0 to 300[21].


Statistical analysis

The results were collected, tabulated, and statistically analyzed using the SPSS program for windows (version 16; SPSS Inc., Chicago, Illinois, USA). Results were expressed as Mean ± SD. Statistical evaluation was carried out using Mann–Whitney U test for quantitative data, χ2-test for qualitative data, and Spearman's test for correlation. Results were considered significant at P value less than 0.05.


  Results Top


Body weight, lung weight, and lung coefficient

Baseline body weight was nonsignificant for all the groups (P > 0.05) [Table 1]. Bleomycin group (toxic group) showed significant reduction in the final body weight compared with the normal group (P < 0.001). Lung weight and lung coefficient were significantly increased compared with the normal group (P < 0.001). A 3-week treatment with atorvastatin resulted in significant increase in the final body weight compared with the toxic group (P < 0.01); On the other hand, lung weight and lung coefficient were significantly decreased in relation to toxic group (P < 0.001, 0.01, respectively), but the treatment did not normalize the atorvastatin group: P less than 0.05 for final body weight, P less than 0.001 for lung weight, and P less than 0.001 for lung coefficient [Table 1].
Table 1: Comparison between the studied groups as regards basal body weight and the effect of atorvastatin on final body weight, lung weight, lung coefficient after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats

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Biochemical parameters

Serum transforming growth factor-β1 level

The toxic group exhibited significantly increased serum TGF-β1 level compared with the normal group (P < 0.001). Treatment with atorvastatin resulted in significant reduction of TGF-β1 level compared with the toxic group (P < 0.001) [Table 2] and [Figure 1].
Table 2: The effect of atorvastatin on serum transforming growth factor-β1, lung malondialdehyde, and lung reduced glutatgione levels after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats

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Figure 1: The effect of atorvastatin on serum transforming growth factor-β level after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats. ***P < 0.001 versus toxic (bleomycin) group, ##P < 0.01 versus normal group. ###significant for normal group.

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Lung malondialdehyde level

Lung MDA level was markedly increased in the toxic group compared with the normal group (P < 0.001). The atorvastatin group showed a notable reduction of MDA level compared with the toxic group (P < 0.001) [Table 2] and [Figure 2].
Figure 2: The effect of atorvastatin on lung malondialdehyde level after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats. ***P < 0.001 versus toxic (bleomycin) group; ###P < 0.001 versus normal group.

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Lung reduced glutathione level

The toxic group showed a considerable reduction in lung reduced glutathione (GSH) level compared with the normal group (P < 0.001), while atorvastatin treatment led to a significant elevation of reduced glutathione compared with the toxic group (P < 0.001) [Table 2]. Despite treatment, the biochemical parameters still were significant regarding the normal group: P less than 0.01 for serum TGF-β1, P less than 0.001 for lung MDA, and P less than 0.01 for GSH levels [Table 2].

Correlation between serum TGF-β1 and lung coefficient: lung coefficient showed a significant strong positive relationship with serum TGF-β1 (r = +0.86, P < 0.001) [Table 5] and [Figure 3].
Table 5: Correlation between serum transforming growth factor-β and both lung coefficient and histoscore (H-score) after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats

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Figure 3: Correlation between serum transforming growth factor-β level and lung coefficient after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats.

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Pathological parameters

Histopathological study

H and E ([Table 4] and [Figure c]&[Figure e]-plate 4) and Masson's trichrome staining of lung sections were examined using light microscopy. Staining revealed that lung section from the normal group showed normal bronchioles and alveoli with no fibrosis. The toxic group showed excessive collagen deposition ([Figure b] – Plate 4) and increased fibrosis score compared with the normal group (P < 0.001). Treatment with atorvastatin reduced collagen deposition ([Figure d] – Plate 4) and significantly decreased fibrosis score compared with the toxic group (P < 0.001). However, the treated group showed a higher fibrosis score than the normal group (P < 0.001) [Table 3].
Table 4: Histoscore (H-score) of α-smooth muscle actin expression after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats

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Table 3: Fibrosis score after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats

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Imunnohistochemical staining of anti-smooth muscle actin

Lung sections of the normal group showed negligible α-SMA immunostaining with a mean H-score of 0 ± 0. In contrast, the toxic group showed a significant increase in α-SMA immunostaining ([Figure c] – Plate 4) with increased H-score mean to 113 ± 34.7 compared with the normal group (P < 0.001). Atorvastatin-treated group showed a significant reduction in α-SMA immunostaining ([Figure e] – Plate 4) and decreased H-score to 40 ± 18.9 compared with the toxic group, although, the treated group showed a higher fibrosis score than the normal group (P < 0.001) [Table 4] and [Figure 4].
Figure 4: (a) Normal group: normal bronchioles (thick arrow) and alveoli without interstitial fibrosis (H and E × 200). (b) toxic (bleomycin) group: peribronchial fibrosis (thick arrow) (MT × 200). (c) Toxic (bleomycin) group: peribronchial (thick arrow) and interstitial fibrosis (thin arrow) (α-SMA × 200). (d) Atorvastatin-treated group: mild peribronchial (thick arrow) (MT (×100). (e) Atorvastatin-treated group: stained with SMA revealed minimal peribronchial fibrosis (thick arrow) (α-SMA × 100). H and E, hematoxylin and eosin; MT, Masson's trichrome; α-SMA, α-smooth muscle actin.

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Correlation between serum transforming growth factor-β1 and H-score

There was a significant strong positive relationship between serum TGF-β1 and H-score (r = +0.89, P < 0.001) [Table 5] and [Figure 5].
Figure 5: Correlation between serum transforming growth factor-β and histoscore (H-score) after 3 weeks of treatment of bleomycin-induced pulmonary fibrosis in rats.

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  Discussion Top


Pulmonary fibrosis is a lung disease resistant to treatment with high mortality rate[22].

Induction of pulmonary fibrosis using bleomycin is the most widely used animal model for such disease. It has a high reproducibility and it produces a fibrotic picture similar to human disease[23].

In bleomycin-induced pulmonary fibrosis by intratracheal instillation, inflammatory phase begins with the release of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1, interleukin-6, interferon-γ) which lasts for about a week after bleomycin instillation followed by fibrotic phase and production of TGF-β1 which reaches its peak 2 weeks after fibrosis induction by bleomycin[24].

In the current study, a single intratracheal injection of bleomycin has led to a significant decrease in final body weight and in reduced glutathione level in the lung tissue was detected compared with the normal group. This was associated with a significant increase in lung weight, lung coefficient, lung MDA level, and serum TGF-β1 level compared with the normal group. This is further ascertained by the histopathological picture of H and E and Masson's trichrome showing a significant increase in fibrosis score, in addition to, a significant increase in H-score of fibrosis of α-SMA immunohistochemical stain compared with the normal group.

Regarding oxidative stress, bleomycin-induced pulmonary fibrosis occurs as a result of occurrence of DNA strand breaks and the generation of free radicals causing cell injury followed by inflammation and development of fibrosis[8].

TGF-β is considered a rate-limiting cytokine involved in the beginning and end of tissue repair. Its prolonged elevation leads to fibrosis[25].

The Reinert et al.[9] study demonstrated that bleomycin-induced pulmonary toxicity is attributed to liberation of reactive oxygen radicals resulting in direct pulmonary toxicity and leading to inflammatory reactions and release of mediators such as leukotrienes, PG, and cytokines like the platelet-derived growth factor (PDGF), TGF-β, IL-1, and macrophage inflammatory protein 1 from macrophages leading to fibrosis in bleomycin animal models.

In the present study, after 3 weeks of treatment with atorvastatin, a significant increase in final body weight and in lung GSH level in lung tissue was detected compared with the toxic group. This was associated with a significant decrease in lung weight, lung coefficient, lung MDA level, and serum TGF-β1 level compared with the toxic group. This is further ascertained by the histopathological picture of H and E and Masson's trichrome showing a significant decrease in fibrosis score, in addition to a significant decrease in H-score of α-SMA immunohistochemical stain compared with the toxic group. However, the treatment was significant compared with the normal group.

These results were in agreement with a study done by Zhu et al.[16] who showed that atorvastatin treatment significantly lowered lung weight and lung coefficient compared with the bleomycin treated rats; moreover, atorvastatin improved histopathological picture and this could be explained due to their antioxidant effect which prevented the accumulation of hydroxyproline in lung tissues supported by significantly decreased lung MDA level as well as NO level (as markers of oxidative stress) after atorvastatin treatment. In addition, atorvastatin may downregulate CTGF protein expression modulating the collagen synthesis in the bleomycin model.

In contrast, Xu et al.[26] reported that statins can induce lung inflammation and fibrosis in mice due to the activation of NLRP3-inflammosome.

As regards TGF-β1 suppressive effect, a supporting study showed that atorvastatin inhibited the proliferation and differentiation of rat lung fibroblasts culture obtained from the Wister rat model of TGF-β1-induced pulmonary fibrosis which in turn led to the attenuation of lung fibrosis, alteration of myofibroblasts shape and to decrease α-SMA expression suggesting its antifibrotic effect[27]. The antioxidant and antifibrotic effects of atorvastatin were further clarified by studies done on liver and kidney[28],[29].

Atorvastatin improved the histopathological picture and fibrosis score, this was in accordance with Khodayar et al.[30] in a paraquat-induced lung fibrosis; administration of atorvastatin 1 week before and 3 weeks after paraquat administration prevented pulmonary fibrosis through decreasing tissue hydroxyproline and serum levels of MDA induced by paraquat. Moreover, histopathological semiquantitative Ashcroft scoring of fibrosis and the amount of lung indices showed the preventive role of atorvastatin in rat pulmonary fibrosis induced by paraquat.


  Conclusion Top


It is concluded that atorvastatin attenuated bleomycin-induced pulmonary fibrosis in rats through having an antioxidant effect and suppression of transforming growth factor beta, the main mediator of the fibrotic process; hence, improving the demographic parameters and histological picture of the treated animals, although it did not normalize treated rats. So, further studies should be conducted using higher doses of the drug and/or longer duration of treatment along with studying other mechanisms of suppressing fibrosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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