Thirty-two male C57BL/6 mice with the average weight of 20–25 g and aged 6-8 weeks were studied. The mice were obtained from the Pasteur Institute (Tehran, Iran). This study was approved by the Ethics Committee of Kurdistan University of Medical Sciences (IR.MUK.REC.1395.382).

In this experimental study, adult male C57BL/6 mice (Purchased from Pasteur Institute of Iran; Tehran) weighing 20–25 g on average at the age of 6–8 weeks were randomly allocated to four groups, each including eight mice. Group one (Bleo): 2 mg of bleomycin sulfate (Nippon Kayaku, Tokyo, Japan) were dissolved in 0.1 ml carboxymethyl cellulose (CMC) (Sigma Corp. St. Louis, Missouri, USA) 0.5%, and then administered via intraperitoneal injection on Days 1, 8, and 15. Group two (Bleo+Thal) received bleomycin as was mentioned for the Bleo group plus 4 mg thalidomide (Sigma Corp. St. Louis, Missouri, USA) dissolved in 0.1 mL CMC 0.5% via intraperitoneal injection five times a week for one month. Group three (Thal) received 4 mg thalidomide (Sigma, USA) dissolved in 0.1 mL CMC 0.5% via intraperitoneal injection five times a week for one month, and Group four (CMC) received 0.1 mL CMC 0.5% via intraperitoneal injection five times a week for one month. At the end of the experiment, the animals were sacrificed using an overdose of ketamine. The thorax was opened, the lungs were removed, and after being washed with PBS, the lungs were placed in formalin 10%. Then, the tissues were dehydrated at different levels, molded, and sections with 6-µm thickness were prepared. The samples were then stained using hematoxylin (Merck KGaA, Darmstadt, Germany) and eosin (Sigma-Aldrich Corp. St. Louis, Missouri, USA) (H & E) to investigate the histological changes, and stained by Masson’s trichrome (Sigma Corp. St. Louis, Missouri, USA) to evaluate the collagen deposition within the lung parenchyma under light microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany). Morphological changes of the pulmonary tissue were evaluated. Moreover, the percentages of alveolar spaces and pulmonary connective tissue were determined by point-counting methods per volume unit. In this method, for each group, 15 fields of 15 sections were examined with 40X magnification.

The expression of Cox-2 protein was evaluated by immunohistochemistry. Sections with the thickness of 6 µm were prepared by sectioning tissue blocks embedded in solid paraffin. The sections were mounted on silanized slides (Sigma, USA). The slides were then placed in an oven at 60 ºC and deparaffinized and rehydrated in three steps. After that, antigen retrieval was performed by applying citrate buffer solution for 17 minutes. Endogenous peroxidase activity was blocked by adding 3% H₂O₂ for 10 minutes. To neutralize extra proteins, 2% bovine serum albumin solution (Pars Tos, Iran) was used. The solution obtained was then incubated overnight with rabbit-specific primary antibody against Cox-2 (Abcam, Cambridge, UK) at 4 ºC. Then, the samples were rinsed, and incubated with rabbit-specific secondary antibody against Cox-2 (Abcam, England) was added. After rinsing, diaminobenzidine (DAB) (Roche, Germany) was added, and the samples were kept in darkness for 10 minutes. The nucleus staining was performed by using hematoxylin, and the samples were mounted on slides. Cox-2⁺ cells were counted using Motic software (Motic Image Plus 2.0, Richmond, Canada). The software calibration was performed using a scaled slide to measure in micrometers, and the number of Cox-2 expressing cells was determined in 15 fields of 15 sections 25.

The experimental results are presented as mean ± Standard Deviation (SD). Following the assurance of normal distribution of data, data significance and correlations was estimated using one-way analysis of variance (ANOVA) with the Newman–Keuls post hoc test in GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA), and also in all the tests, P<0.05 was considered statistically significant.

The qualitative results obtained from the histopathological evaluation of the pulmonary tissue demonstrated that the level of inflammation and fibrosis, the deposition of collagen fibers, and the expression of Cox-2 protein were different across various experimental fibrotic changes were observed. In addition, the infiltration of inflammatory cells in alveolar spaces, disorganization of the alveolar structure, impairment of alveolar walls, development of cystic changes in alveoli, and the amount of connective tissue also increased (Figure 1A). Collagen deposition in the interstitial connective tissue was observed as blue fibers in the Masson’s trichrome staining (Figure 1B). According to the results obtained from immunohistochemical studies, Cox-2-expressing cells were present between the epithelial cells of the alveoli and the connective tissue (Figure 1C).

Histological and Immunohistochemical analysis on the lung tissue in Thal group shows that the lung tissue alveoli have a normal appearance. There is no evidence of the congestion and inflammatory cells. Also, there is no evidence of interstitial tissue remodeling (Figure 2A). The results of photomicrographs from Masson’s trichrome staining showed areas with lower level collagen (Figure 2B). Cox-2 immunohistochemistry (anti- Cox-2) shows that Cox-2+ cells are rarely observed (Figure 2C).

Figure 2

However, the Bleo+Thal group showed a relative reduction in the number of inflammatory cells, thinner alveolar walls, and a better organization of the alveolar structure. Fewer changes associated with alveolar impairment were observed. Moreover, remodeling in connective tissue is less visible than group one. Also, both type 1 and type 2 pneumocyte cells can be seen (mostly type 2) in epithelial cells of the alveoli (Figure 3A). The level of collagen fiber deposition relatively reduced (Figure 3B), and immunohistochemical studies showed the expression of Cox-2 among the connective tissue (Figure 3C).

On the other hand, results obtained from tissue sections stained with H&E (Figure 4A), Masson's trichrome (Figure 4B) and immunohistochemistry (Figure 4C) in the CMC group (IV) showing no significant change in the lung tissue and collagen levels. No evidence of increased fibrosis and Cox-2 positive cells in the connective tissue epithelium were observed. Furthermore, the changes were similar to those demonstrated in group two.

Figure 4

Quantitative results related to the pulmonary alveoli and connective tissue of the mice in different groups were obtained by the morphometric method, and the percentages of contact points of alveolar spaces and pulmonary connective tissue for the four groups were determined. The mean values for the contact point of alveolar spaces in the volume unit were 39.20% ± 8.11, 52.77% ± 3.39, 59.17% ± 7.97, and 64.15% ± 5.42 for the Bleo, Bleo+Thal, Thal, and CMC groups, respectively. The results indicate an increase in the alveolar spaces in the Bleo+Thal group compared to the Bleo group. Statistical analysis showed that the groups differed significantly from each other regarding the percentage of the alveolar spaces (P<0.001). However, in this regard, comparison of the Thal and Bleo+Thal groups (P= 0.1) and the Thal and CMC groups (P=0.04) did not show statistically significant differences. The mean percentage of contact points of connective tissue in the volume unit determined for the Bleo, Bleo+Thal, Thal, and CMC groups were 59.59% ± 8.87, 47.22% ± 3.39, 40.82% ± 7.97, and 35.84% ± 5.43, respectively. This result demonstrates an increase in the amount of connective tissue in the Bleo group compared to the Bleo+Thal group. The groups differed significantly in this respect (P<0.001). Nevertheless, comparison of the Thal and Bleo+Thal groups (P= 0.1) and the Thal and CMC groups (P=0.04) did not show significant differences. Furthermore, the mean number of Cox-2⁺cells in the Bleo, Bleo+Thal, Thal, and CMC groups were 11.08 ± 0.6, 7.54 ± 0.6, 2.36 ± 0.32, and 3.14 ± 0.48, respectively. According to the results obtained from the statistical analyses, the number of Cox-2⁺ cells in the Bleo group compared to Thal group were significantly increased (P=0.006). Similarly, a significant increase in the number of Cox-2⁺ cells was detected in the Bleo group compared to the CMC group (P=0.007). The number of Cox-2⁺ cells was significantly reduced in the Bleo+Thal group when compared to the Bleo group (P=0.012). However, the Thal and CMC groups were not significantly different from each other in this regard (Figure 5).

Figure 5

In the present study, the effect of thalidomide on the lung fibrosis, induced by intraperitoneal administration of bleomycin in mice, was investigated. The qualitative and quantitative examinations on the lung tissue of different groups were performed using histomorphometric and immunohistochemical approaches. The histological results of the Bleo group showed an impairment in alveolar walls, development of cystic modifications, increased connective tissue, and deposition of higher amounts of collagen fibers in the pulmonary connective tissue vis-à-vis the Bleo+Thal group. In this study, a significant reduction of connective tissue and a significant increase in alveolar spaces in the bleomycin thalidomide group compared to the bleomycin group suggested a decrease in fibrosis following thalidomide treatment.

As shown by the immunohistochemical studies, the number of Cox-2-expressing cells in the Bleo group increased compared to the Bleo+Thal group. According to morphometric studies, the percentage of contact points of alveolar spaces in the volume unit in the Bleo+Thal group was significantly higher than that in the Bleo group (P<0.001). In addition, the percentage of contact points of connective tissue in the volume unit in the Bleo group increased significantly when compared with the Bleo+Thal group (P<0.001). Increased connective tissue and decreased alveolar spaces in the Bleo group indicate pulmonary fibrosis induced by bleomycin. Moreover, according to the results of the current study, administration of thalidomide in the Belo + Thal group led to a statistically significant increase in connective tissue and an increase in the alveolar spaces compared to the Bleo group. These results indicated the alleviation of fibrosis following administration of thalidomide. Moreover, Tabeta et al. (2007) showed that thalidomide has an inhibitory effect on bleomycin-induced pulmonary fibrosis in laboratory mice 23. They employed intraperitoneal administration of bleomycin for induction of fibrosis. This study showed that thalidomide had an inhibitory effect on the synthesis of collagen, TGF-β, IL-6, and VEGF; thus, it reduced the pulmonary fibrosis. In their histopathological evaluations using Azan stains, these researchers reported an increase in the thickness of the intra-alveolar wall, as well as infiltration of inflammatory cells and collagen deposition in the interstitial lung tissue in the Bleo group compared to the Bleo+Thal group 23. In addition, Zhao et al. (2009) demonstrated that thalidomide has a therapeutic effect on lung fibrosis. To induce lung fibrosis in mice, they administered subcutaneous bleomycin on a daily basis. In the treatment group, oral bleomycin was administered one week before bleomycin injections. According to their results, thalidomide could be effective in the treatment of lung fibrosis via its inhibitory effect on hydroxyproline (HYP) and α-SMA protein 24. Singhal et al. (1999) demonstrated that the inhibitory effect of thalidomide on the expression of IL-6 is related to cell growth and angiogenesis; hence, the drug has been administered in multiple myeloma 22. Moreover, it has been reported that thalidomide reduces the level of TGF-β, which is related to cell differentiation, and shows anti-inflammatory 11, anti-angiogenesis 12 , and immunomodulatory effects 26,27,28. It seems that thalidomide can reduce induced lung fibrosis via inhibition of angiogenesis, regulation of the immune response and proliferation, and its anti-inflammatory effects. Horton et al. (2012) demonstrated that oral thalidomide in patients with lung fibrosis could significantly alleviate patients’ cough 29. Severe cough is one of the symptoms of lung fibrosis in patients, which is resistant to conventional therapies. It is a disabling symptom, and there are as yet no effective treatments for it. It is suggested that thalidomide can be effective in treating such patients by influencing the respiratory tract sensory nerves 30,31. In the current study, the qualitative results of immunohistochemical evaluations indicated that the distribution of Cox-2 protein in the Bleo+Thal group was mainly in the epithelial regions of pulmonary alveoli and respiratory bronchioles. Moreover, Cox-2⁺ cell count using the morphometric approaches demonstrated that the number of these cells in the Bleo+Thal group was significantly lower than that in the Bleo group (P<0.001). It was shown in the current study that thalidomide protects against lung fibrosis by decreasing the number of Cox-2⁺ cells. There are few reports in this aspect, and the role and expression of Cox-2 in different types of lung fibrosis is unknown. Paivaniemi et al. demonstrated in a study that an increase in the number of Cox-2-expressing epithelial cells in the lung tissue is possibly a positive prognostic factor for inhibition of fibrosis 32. Prostaglandins are generated from the action of prostaglandin synthases, called cyclooxygenases. Cox-2 is the second isoform of Cox, which is inducible and is expressed following inflammation. The enzyme contributes to the development of pain, inflammation, and edema. The Cox-2 expression could be induced by inflammatory stimulants, cytokines, growth factors, and tumor promoters. This is while the enzyme is suppressed by steroidal and non-steroidal anti-inflammatory drugs 33. Vancheri et al. (2004) demonstrated that in lung fibrosis, fibroblasts are unable to produce Cox-2, thereby having a defect in the production of prostaglandin E2 34,35,36. Additionally, Keerthisingam et al. (2001) and Xaubet et al. (2004) reported that the levels of Cox-2 and prostaglandin E2 in cultured fibroblasts derived from IPF patients 37,38. However, it is shown that in cancer thalidomide inhibits lipopolysaccharide-mediated induction of inflammatory factors including Cox-2 and prostaglandin E2 in a dose-dependent manner. Moreover, the drug is shown to increase the degradation of Cox-2 mRNA, which can be helpful in the treatment of the disease 13. Considering the various roles Cox-2 plays in different diseases and the complexity of the molecular mechanisms that thalidomide and Cox-2 are involved in, further studies are required in this field.

In this work, a significant reduction of connective tissue and a significant increase in alveolar spaces in the bleomycin thalidomide group compared to the bleomycin group could indicate a decrease in fibrosis following thalidomide use.

Thalidomide decreases the severity of damages in the lung, increases the number of pulmonary alveoli, and decreases the amount of connective tissue following induction of fibrosis, which is probably mediated by decreasing the expression of Cox-2 protein. As already mentioned, the thalidomide mechanism of action is complex, and there are many unknown aspects of lung fibrosis. Therefore, administration of thalidomide via other routes, as well as the induction of fibrosis in other animal models, and other times from initiation of the fibrosis process would be further investigated to compare with the results of the current study.

Ardeshir Moayeri, Ayoob Rostamzadeh, Ghazal Keshavarz and Mohammad Jafar Rezaie designed the study and performed the experiments. Mansour Amraei conducted the data analysis. Mansour Amraei, and Mohammad Jafar Rezaie participated in drafting the paper; Ardeshir Moayeri and Mohammad Jafar Rezaie wrote the manuscript. All authors provided data analysis and read and approved the manuscript.

The authors report no conflicts of interest in this work.

Ardeshir Moayeri, Ayoob Rostamzadeh, Ghazal Keshavarz and Mohammad Jafar Rezaie designed the study and performed the experiments. Mansour Amraei conducted the data analysis. Mansour Amraei, and Mohammad Jafar Rezaie participated in drafting the paper; Ardeshir Moayeri and Mohammad Jafar Rezaie wrote the manuscript. All authors provided data analysis and read and approved the manuscript.

We thank the Deputy Director of Research and Technology at Kurdistan University of Medical Sciences for supporting this study and the Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, for their kind cooperation. Moreover, we would like to appreciate the kindness of Dr. Kaka for his assistance.

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