Scrutiny of the Effect of Dexamethasone on Cytokines and Expression of Selected Genes Involved in Fibrosis: A Study in a Mouse Model of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic form of interstitial lung disease, characterized by an inflammatory infiltrate, deposition of extracellular matrix (ECM) components like collagen, and change in the architectural lung parenchyma. IPF has a poor prognosis and is lethal, with a median survival of 2 to 3 years. The etiology of the disease is obscure; however, several studies indicated that the disease is initiated by inflammation and the release of profibrotic growth factor, namely transforming growth factor β (TGF-β). The definitive treatment for patients has not yet been approved. Although. However, evidence suggests that inflammation plays an essential role in the development and pathogenesis of IPF as a stimulus. However, inflammation is sometimes referred to as a secondary event in fibrosis, partly due to the failure of anti-inflammatory drugs in clinical trials. Therefore, more studies are needed to evaluate the effectiveness of anti-inflammatory therapies. In primary injury or inflammation, TGF-β, a critical cytokine and regulator of fibrosis, promotes inflammation and increases the activity and proliferation of fibroblasts at the site of inflammation, differentiation into myofibroblasts, and production of extracellular matrix, leading to severe pulmonary fibrosis. As a downstream mediator, the connective tissue growth factor (CTGF) is induced by TGF-β and promotes its fibrotic effects, enhancing lung fibrosis through fibroblast proliferation and collagen deposition. Evidence has shown that CTGF expression is increased in fibroblasts of IPF patients. In addition, Endothelin-1 (ET-1) is another downstream mediator of TGF-β fibrogenic responses in fibroblasts, which produces an extracellular matrix and differentiates fibroblasts into myofibroblasts. Also, the expression of tumor necrosis factor-alpha (TNF-α), which in addition to its inflammatory properties, also has fibrogenic properties, is significantly increased in the lungs of IPF patients. This expression is associated with increased fibroblasts and the deposition of extracellular matrix proteins in the interstitial region of the lung. In general, these findings indicatthat these factors are reasonable targets for designing treatment strategies or evaluating the effect of proposed drugs in the treatment of IPF.Broad-acting anti-inflammatory molecules, including glucocorticoids like Dexamethasone, were considered potential therapy. Glucocorticoids are anti-inflammatory agents that can reduce pro-inflammatory molecules by suppressing cellular and humoral immunity. Dexamethasone is one of the most potent glucocorticoid drugs; however, despite the various investigations on the inhibition of pulmonary fibrosis, its anti-fibrosis effects are still debated, and there are contradictory findings in this regard. Also, the underlying mechanism of its impact on IPF is not clear. Therefore, in this study, the therapeutic effect of Dexamethasone on pulmonary fibrosis was scrutinized, based on the impact of various players of this scenario, namely the expression of TGF-β, TNF-α, CTGF, ET-1and hydroxyproline.

6 to 8 week-old male mice were used in this study. All animal care conditions, including temperature, light, and humidity, were observed. Mice aged 8-10 weeks were randomly divided into the following groups, and ten mice were placed in each group.1.  Control group treated with normal saline (Control) 2.  Bleomycin inoculated group on day one (BLM)3.  Bleomycin inoculated on day one and dexamethasone-treated for 14 days (BLM+DEXA)The experiments were performed according to the approved guideline from the Faculty Ethics Committee (IR.IUMS.FMD.REC1396.9511127007). Lung fibrosis was induced intratracheal in anesthetized mice using 50 µL bleomycin (5mg/kg) by a single dose. The intervention with Dexamethasone (1mg/Kg/day) was done by 14 days intraperitoneal injection under sterile conditions. The mice were euthanized on day 21 under deep anesthesia, and their lungs were extracted. For evaluation, architectural changes occurred in the lungs based on bleomycin administration and dexamethasone intervention; the left lungs were fixed in formalin. The right lungs were frozen in liquid nitrogen for RNA extraction, gene expression analysis, ELISA, and hydroxyproline assay and were stored at -80 ºC. After 24 hours of immersion in formalin, paraffin blocks were prepared from the left lungs, and tissue incisions were made and transferred to the slide. After paraffin removal and leaching of slides, pathological examinations were performed using Masson Trichrome staining. The amount of hydroxyproline was measured using a hydroxyproline kit. Quantitative measurement of mRNA from CTGF and ET-1 genes in the lungs of mice was performed using real-time PCR. Data analysis of different groups was performed using Prism software and one-way ANOVA and Tukey multiple comparison test.In order to evaluate the anti-fibrotic effect of Dexamethasone in IPF, the bleomycin-induced mouse model was treated with Dexamethasone. After fibrosis was induced by intratracheal BLM administration, histopathological evaluation and hydroxyproline assay, ELISA for measurement of TGF-β and TNF-α, and RT-PCR were performed to evaluate the expression CTGF and ET-1 genes.

Histological examination indicated the deposition of collagen after administration of one dose of Bleomycin; an enzymatic analysis of hydroxyproline showed that administration of a single dose of BLM intratracheally leads to extensive fibrosis in the lungs of C57BL/6 mice on day 21. In comparison, 14 days of intraperitoneal treatment with Dexamethasone reduced the severity of fibrosis. Histological examination of Mason trichrome-stained tissue sections from lungs of control mice (receiving saline intratracheally) showed normal lung structure including no extracellular matrix deposition (based on the absence of blue color in the interstitium) and the alveolar space was distinct. While the lung tissue sections of BLM mice showed histopathological changes, including increased fibrotic areas and increased collagen deposition (based on increased blue color), and decreased alveolar spaces compared to the control group. However, lung tissue sections from dexamethasone-treated mice (BLM+DEXA group) showed only mild fibrosis. Dexamethasone treatment reduced the amount of hydroxyproline compared to the Bleomycin treated group; however, this reduction was not statistically significant (P> 0.05). Dexamethasone treatment significantly reduced TGF-β levels in the lungs of the BLM + DEXA group compared with the BLM group (P <0.001). Further, treatment of received Bleomycin (BLM) received mice with Dexamethasone (BLM + DEXA) reduced the amount of TNF-α when compared with Bleomycin received only group nevertheless, this reduction was not statistically significant to mice but statistically significant (P <0.05). The levels of TNF-α and TGF-β in the lungs of Bleomycin treatment mice (BLM) were higher when compared with Normal saline-treated mice (Control) (P <0.001). Dexamethasone treatment can induce its effect by inhibiting a prominent signaling pathway, namely TGF-β. Further, Dexamethasone treatment of Bleomycine received mice significantly reduced the expression of CTGF gene (P <0.001). CTGF expression occurs before the TGF- β cytokine is expressed during the phenomenon of fibrosis. It seems that CTGF is responsible for extracellular accumulation in the fibrosis pathway. In addition, 14 days of treatment of Bleomycin received mice with Dexamethason reduced ET-1 genes in the lungs of the BLM+DEXA group compared with the BLM group (P <0.001). ET1 gene is responsible for the differentiation of fibroblasts to myofibroblasts.It is known that Dexamethasone can ameliorate fibrosis; however, it is not known how this steroid can induce its effect or whether its impact is based on its role in the inhibition of inflammation

Our findings showed that intratracheal inoculation of Bleomycin in mice resulted in extensive accumulation of extracellular matrix in the lungs of C57BL/6 mice. Our study demonstrated that administration of Dexamethasone attenuated Bleomycin-induced pulmonary fibrosis in C57BL/6 mice by reducing hydroxyproline, production of two critical cytokines, the TGF-β and TNF-α, also by reduction of CTGF and ET-1 gene expression. Still, further investigations are required to understand how this compound can reduce fibrosis.