AUDA Attenuates Bleomycin-induced Pulmonary Fibrosis In Mice Via Inhibiting Smad/P38 Signaling Pathway

Objective:Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia of unknown cause with a severe prognosis, with a survival of 3 to 5 years since diagnosis. IPF is characterized by progressive scarring of the pulmonary parechyma, leads to progressive loss of lung function, with dyspnea and hypoxemia, and, ultimately, respiratory failure and death. Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, have vasodilatory, anti-inflammatory and anti-fibrosis properties. EETs are mainly metabolized by soluble epoxide hydrolase (sEH) to their less active metabolites Dihydroxy-20-carbon triple acid (DHETs). Inhibition of sEH might increase the level of EETs and its biological activity in vivo. This study will explore the effect of sEH inhibitor on Bleomycin (BLM)-induced pulmonary fibrosis in mice and its mechanisms. Bleomycin is used to treat different types of neoplasms, its most severe adverse effect is lung toxicity. Bleomycin-induced pulmonary fibrosis is a recognized animal model in the word. So in this experiment, we investigated the role of sEH inhibitor AUDA in attenuating the BLM-induced pulmonary fibrosis in mice. Epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transformation (FMT) are considered to be important cause of fibrosis, and transforming growth factor-β1 (TGF-β1) is an important factor induced EMT and FMT. So in this experiment, we investigated the role of sEH inhibitor AUDA in inhibiting the TGF-β1 induced EMT and FMT in vitro.Methods:In vivo, ICR mice were randomly divided into four experiment groups, control group, BLM group and BLM+AUDA (3 or 10 mg/kg) group. Mice were treated with a single dose of BLM (3 mg/kg) via intratracheal injection to induce pulmonary fibrosis, and drugs were administered one week later. At the end of the fourth week, the baseline enhanced pause (Penh, a airway resistance parameter) in unrestrained and conscious mice was assessed by whole body barometric plethysmography. Bronchoalveolar lavage fluid (BALF) was prepared for determinations of inflammatory cell accumulation. Lung tissue homogenates were used for measurement of qRT-PCR, enzyme-linked immunosorbent assay (ELISA), Western blot, hydroxyproline activity as well as histological examination. In vitro, MTT assay was used to assay cell viability to evaluate the effects of AUDA on the proliferation of epithelial cells and fibroblasts. TGF-β1 was used to stimulate 16HBE cells to establish an EMT model, we investigated the role of AUDA in regulating the TGF-β1 induced EMT and its underlying mechanism. The role of AUDA on regulating fibroblast-myofibroblast transformation was also investigated.Results:In vivo, oral administration with AUDA attenuated bleomycin-induced increase in Penh and reduce in body weight, significantly inhibited inflammatory cells, including leukocytes, lymphocytes and neutrophils accumulation in BALF. AUDA reduced the mRNA and protein expression of TGF-β1, IL-1β and MMP-9 in lung tissue. Moreover, AUDA attenuated BLM-induced alveolar structure destruction and pulmonary parenchyma, reduced the deposition of collagen fibers and the level of hydroxyproline in lung tissue. Besides, AUDA downregulated the level of α-SMA and upregulated the level of E-cadherin via Smad/p38 signaling pathway, attenuated lung fibrosis. In vitro, AUDA significantly inhibit the proliferation of epithelial cells and fibroblasts. Treatment with TGF-β1 for 48 h, expression a-SMA in 16HBE cells was up-regulated and expression of E-cadherin was down-regulated. These effects were completely blocked by pre-treatment with AUDA via smad2, smad3 and p38 signaling pathways. Additionally, AUDA could also reduce the level of a-SMA in HFL-1 cells, inhibit fibroblast-myofibroblast transformation.Conclusions:AUDA significantly reduce BLM-induced inflammation, collagen deposition and pulmonary parechyma in the lung of mice. AUDA also inhibit the proliferation of epithelial cells and fibroblasts, regulate the TGF-β1 induced EMT and FMT.