Pirfenidone Alleviates Inflammation and Fibrosis of Acute Respiratory Distress Syndrome by Modulating the Transforming Growth Factor-β/Smad Signaling Pathway

Abstract

Acute respiratory distress syndrome (ARDS) occurs as an acute onset condition, and patients present with diffuse alveolar damage, refractory hypoxemia, and non-cardiac pulmonary edema. ARDS progresses through an initial exudative phase, an inflammatory phase, and a final fibrotic phase. Pirfenidone, a powerful anti-fibrotic agent, is known as an agent that inhibits the progression of fibrosis in idiopathic pulmonary fibrosis. In this study, we studied the treatment efficiency of pirfenidone on lipopolysaccharide (LPS) and bleomycin-induced ARDS using rats. The ARDS rat model was created by the intratracheal administration of 3 mg/kg LPS of and 3 mg/kg of bleomycin dissolved in 0.2 mL of normal saline. The pirfenidone treatment group was administered 100 or 200 mg/kg of pirfenidone dissolved in 0.5 mL distilled water orally 10 times every 2 days for 20 days. The administration of LPS and bleomycin intratracheally increased lung injury scores and significantly produced pro-inflammatory cytokines. ARDS induction increased the expressions of transforming growth factor (TGF)-β1/Smad-2 signaling factors. Additionally, matrix metalloproteinase (MMP)-9/tissue inhibitor of metalloproteinase (TIMP)-1 imbalance occurred, resulting in enhanced fibrosis-related factors. Treatment with pirfenidone strongly suppressed the expressions of TGF-β1/Smad-2 signaling factors and improved the imbalance of MMP-9/TIMP-1 compared to the untreated group. These effects led to a decrease in fibrosis factors and pro-inflammatory cytokines, promoting the recovery of damaged lung tissue. These results of this study showed that pirfenidone administration suppressed inflammation and fibrosis in the ARDS animal model. Therefore, pirfenidone can be considered a new early treatment for ARDS.

Keywords: acute respiratory distress syndrome; bleomycin; inflammation; lipopolysaccharide; pirfenidone; pulmonary fibrosis.

Figure 1

Cell counting of bronchoalveolar lavage fluid. * indicates p < 0.05 compared to the control group. # indicates p < 0.05 compared to the ARDS-induced group. CON, control group; ARDS, acute respiratory distress syndrome-induced group; ARDS-P100, acute respiratory distress syndrome-induced and 100 mg/kg pirfenidone-treated group; ARDS-P200, acute respiratory distress syndrome-induced and 200 mg/kg pirfenidone-treated group.

Figure 2

Histological analysis, lung injury score, and pulmonary fibrotic level. (A) Hematoxylin and eosin (H&E) staining and picrosirius red staining. Scale bar is 200 µm. (B) Lung injury score. (C) Fibrotic area. * indicates p < 0.05 compared to the control group. # indicates p < 0.05 compared to the ARDS-induced group. CON, control group; ARDS, acute respiratory distress syndrome-induced group; ARDS-P100, acute respiratory distress syndrome-induced and 100 mg/kg pirfenidone-treated group; ARDS-P200, acute respiratory distress syndrome-induced and 200 mg/kg pirfenidone-treated group.

Figure 3

Pro-inflammatory cytokine concentrations. (A) The level of tumor necrosis factor (TNF)-α in the lung tissue. (B) The level of interleukin (IL)-6 in the lung tissue. (C) The level of IL-1β in the lung tissue. (D) The level of IL-8 in the lung tissue. * indicates p < 0.05 compared to the control group. # indicates p < 0.05 compared to the ARDS-induced group. CON, control group; ARDS, acute respiratory distress syndrome-induced group; ARDS-P100, acute respiratory distress syndrome-induced and 100 mg/kg pirfenidone-treated group; ARDS-P200, acute respiratory distress syndrome-induced and 200 mg/kg pirfenidone-treated group.

Figure 4

Expressions of transforming growth factor (TGF)-β, SMAD family member 2 (Smad2), and Smad3. (A) The relative TGF-β expression in the lung tissue. (B) The relative ratio of phosphorylated Smad2 (p-Smad2) to Smad2 in the lung tissue. (C) The relative ratio of phosphorylated Smad3 (p-Smad3) to Smad3 in the lung tissue. * indicates p < 0.05 compared to the control group. # indicates p < 0.05 compared to the ARDS-induced group. CON, control group; ARDS, acute respiratory distress syndrome-induced group; ARDS-P100, acute respiratory distress syndrome-induced and 100 mg/kg pirfenidone-treated group; ARDS-P200, acute respiratory distress syndrome-induced and 200 mg/kg pirfenidone-treated group.

Figure 5

Expressions of matrix metalloproteinase (MMP)-9 and tissue inhibitor metalloproteinase (TIMP)-1. (A) The relative MMP-9 expression in the lung tissue. (B) The relative TIMP-1 expression in the lung tissue. * indicates p < 0.05 compared to the control group. # indicates p < 0.05 compared to the ARDS-induced group. CON, control group; ARDS, acute respiratory distress syndrome-induced group; ARDS-P100, acute respiratory distress syndrome-induced and 100 mg/kg pirfenidone-treated group; ARDS-P200, acute respiratory distress syndrome-induced and 200 mg/kg pirfenidone-treated group.

Figure 6

Expression of fibronectin and concentrations of hydroxyproline and connective tissue growth factor (CTGF). (A) The relative fibronectin expression in the lung tissue. (B) The level of hydroxyproline in the lung tissue. (C) The level of CTGF in the lung tissue. * indicates p < 0.05 compared to the control group. # indicates p < 0.05 compared to the ARDS-induced group. CON, control group; ARDS, acute respiratory distress syndrome-induced group; ARDS-P100, acute respiratory distress syndrome-induced and 100 mg/kg pirfenidone-treated group; ARDS-P200, acute respiratory distress syndrome-induced and 200 mg/kg pirfenidone-treated group.