Background
Idiopathic pulmonary fibrosis (IPF) is a disease characterized by diffused alveolar inflammation and extensive interstitial fibrosis, which is related with various cells, inflammatory factors, fibrosis related cytokines, and transforming growth factor-β1 (TGF-β1) signal transduction [
1,
2]. It leads to high mortality, and the average survival time after diagnosis is about 2–5 years [
3]. TGF-β1 is a key factor for the initiation of pulmonary fibrosis that promotes proliferation and transformation of lung fibroblasts and deposition of extracellular matrix [
4]. Nonetheless, the pathogenesis of IPF is not entirely understood, and hence, an efficient treatment is rather limited [
5,
6]. Therefore, it is vital to investigate the pathogenesis and therapeutic mechanisms of IPF.
Pirfenidone (PFD) is approved drug for IPF treatment with anti-fibrotic, antioxidant, and anti-inflammatory effects [
7]. It inhibits the differentiation and proliferation of fibroblasts as well as the synthesis of collagen and generation of inflammatory cytokines [
8,
9]. The clinical study indicated that PFD effectively improved the pulmonary function and quality of life for patients with IPF [
10]. Moreover, it was found that PFD could also clear oxygen free radicals and exert anti-fibrosis effect [
11]. In addition, it stimulated the generation of reduced glutathione (a major antioxidant substance), increase oxidative stress capacity, and exert antioxidant effects [
12]. Despite that the exploration of the antioxidant mechanism of PFD is not intensive.
Oxidative stress and oxidation/antioxidation imbalance play a major role in the pathogenesis and progression of IPF [
13,
14]. Nuclear factor-erythroid-related factor 2 (Nrf2) belongs to cap ‘n’ collar (CNC) transcription family that regulates the expression of a wide array of antioxidant genes. BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap‘n’collar protein) homology 1, Bach1 has been regarded as a competitive inhibitor for Nrf2 by intervening and competitively inhibiting the interaction of Nrf2 and antioxidant response element (ARE). This negatively regulates the downstream antioxidant genes including heme oxygenase-1 (Ho-1) and glutathione peroxidase 1 (Gpx1) [
15‐
17]. It was found that Nrf2 and its downstream antioxidant factors are involved in the pathogenesis of IPF [
18]. Nrf2 agonist attenuated pulmonary fibrosis induced by bleomycin (BLM) via the oxide level in lung tissue [
19]. Our previous study demonstrated that Bach1 knockout inhibited the progression of BLM-induced pulmonary fibrosis by regulating the expressions of Nrf2 and its downstream anti-oxidant factors including Ho-1 and Gpx1, thus providing novel insights into the role of Bach1/Nrf2 in the regulation of oxidative stress involved in the pathogenesis of IPF [
20].
Based on its antioxidative property of Nrf2/Bach1 in oxidative stress regulation, PFD was used to intervene mouse lung fibroblasts (MLF) induced by TGF-β1 and mice with pulmonary fibrosis induced by BLM in order to observe the influences of the drug on expressions of Nrf2/Bach1 and the downstream antioxidant factors, Ho-1 and Gpx1. Furthermore, the effects of PFD on fibrosis-related cytokines interleukin-6 (IL-6), myofibroblast markers type 1 collagen α1 (COL1A1), common indicators of oxidative stress such as reactive oxygen species (ROS) and malondialdehyde (MDA) in mouse and lung pathological alterations were also investigated. The present study explores the underlying antioxidative mechanism and therapeutic efficacy of PFD on IPF.
Methods
Experimental materials
C57BL/6 mice were acquired from Vital River Laboratories (Beijing, China) and primary mouse lung fibroblasts (MLF), MIC-CELL-0040 from PriCells (Hubei, China). RNA extraction kit was purchased from Tiangen Biotech (Beijing, China), reverse transcription kit and fluorescent quantitation PCR kit from Takara (Tokyo, Japan). Nrf2, Bach1, HO-1, GPx1 rabbit anti-mouse primary antibodies were purchased from Abcam (Cambridge, MA, USA). Protein Extraction Kit was obtained from Sigma-Aldrich (St. Louis, MO, USA) and ROS kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Enzyme linked immunosorbent assay (ELISA) Kit was obtained from R&D (R&D Systems, Minneapolis, MN, USA) and Cloud-Clone Corp (USCN Life Science Inc, Wuhan, China). Primers were synthesized by Sangon (Shanghai, China). Hematoxylin staining kit and Masson trichrome staining kit were purchased from Solarbio (Beijing, China). TGF-β1 recombinant protein was obtained from R&D, and PFD was obtained from Shionogi & Co., Ltd (Osaka, Japan).
Animals and administration
7–8 week old male C57BL/6 mice (body weight 18–22 g) were maintained under specific pathogen-free conditions and acclimated for 1 week at 22 ± 3 °C and humidity (45–60%) in a 12:12 h light before the experiments. The mice were permitted ad libitum access to food and water. Fifteen mice were randomly divided into 5 equal groups: control group (50 μL 0.9% saline, administered intratracheally once), BLM 14d group (14d after 5.0 mg/kg BLM dissolved in saline to 50 μL, intratracheally injected once, at the time of establishment of lung fibrosis), BLM 42d group (at the final time point of administration), only PFD group (300 mg/kg PFD, orally administrated daily for 4 weeks) and BLM + PFD group (300 mg/kg PFD treatment for 4 weeks after 14d of BLM administration). We assessed the model of pulmonary fibrosis after 14d and 42d of BLM administration. At the designated endpoint, serum and BALF of mice were collected. Then the mice were sacrificed, and lung tissues excised to identify pulmonary inflammation and fibrosis by pathological morphology. The lung lavage was obtained by washing the lung three times with saline (0.9%) through a tracheal cannula. Post-execution, both the lung tissues were harvested, and a part of the pulmonary lobe was preserved in liquid nitrogen after being treated by diethylpyrocarbonate (DEPC); others were fixed in 4% formaldehyde. The animal experiments were approved by the Ethics Committee of Baotou Medical College (201511306).
Cell culture and intervention
Primary mouse lung fibroblasts were cultured in DMEM medium containing 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C in a 5% CO2-humidified atmosphere. At 70–80% confluency, the cells were seeded at 1 × 105/mL in a 6-well plate. After serum starvation for 24 h, they were treated by 5 ng/mL TGF-β1 for 24 h followed by stimulation with 100, 200 and 500 μg/mL PFD, was added to the supernatant for an additional 48 h. In all in vitro experiments, gene expression (transcript and protein) was assessed at the same time point, 72 h after TGF-β1 treatment. At the final point of TGF-β1 and various concentrations of PFD administration, the cells and supernates were harvested respectively. Three biological replicates were performed, and experiments repeated three time for each.
RT–PCR
Total RNA from the fibroblasts and mouse lung tissues was extracted by RNA extraction kit, and cDNA was synthesized by reverse transcription according to the manufacturer’s instructions. The relative expression level of mRNA was detected by the PCR kit. The primer sequences designed by Primer Premier 5.0 are shown in the following Additional file
1: Table S1. Reverse transcription was carried out in a total volume of 20 μL at 65 °C for 5 min, 42 °C for 60 min, and 70 °C for 15 min. The PCR conditions (ABI Prism 7500, Applied Biosystems) were as follows: 95 °C, 30 s; 60 °C, 1 min; 72 °C, 30 s for 40 cycles. The mouse GAPDH was used as internal control, and 2
–ΔΔCT was used to calculate the relative expression level of the target genes. The experiments were performed at least 3 times (Table
1).
Table 1
PCR primers sequence
Nrf2 | Forward (5′- 3′) | GTGGTTTAGGGCAGAAGG |
Reverse (5′- 3′) | TCTTTCTTACTCTGCCTCTA |
Bach1 | Forward (5′- 3′) | ACAGGGCTACTCGCAAA |
Reverse (5′- 3′) | GTCATCTCCCAGGCTAATC |
HO-1 | Forward (5′- 3′) | GACAGAAGAGGCTAAGACCGC |
Reverse (5′- 3′) | TGACGAAGTGACGCCATCT |
GPx1 | Forward (5′- 3′) | GCACATCTACCACGCAGTCA |
Reverse (5′- 3′) | AGAGTCTCAAGAACATCGCCT |
GAPDH | Forward (5′- 3′) | AAGACCCAGAAATGAAC |
Reverse (5′- 3′) | TCTACACGATAACAACCA |
Western blot
Mouse lung tissues and lung fibroblasts were lysed for protein extraction according to the manufacturer’s instructions. The protein concentration was evaluated by BCA method. The total protein was transferred onto a nitrocellulose membrane after resolving on 10% SDS-PAGE and blocked for 1 h with 5% skim milk, Subsequently, the membrane was probed with primary antibodies, such as rabbit anti-mouse Nrf2 (1:500), Bach1 (1:300), Ho-1 (1:500), Gpx1 (1:1000), and β-actin (1:500), respectively, at 4 °C overnight. Then, secondary antibody labeled with horseradish peroxidase (1:10000) was added for 1 h at room temperature and protein bands were visualized by using double infrared laser scanning imaging system (Licor, USA).
ELISA
IL-6 and COL1A1 levels in supernate of lung fibroblasts, serum, and BALF as well as MDA expression in serum and BALF were performed by ELISA kit according to the manufacturer’s instructions.
Chemiluminescence
Single cell suspensions were obtained from lung tissues of mice by enzymatic dissociation and the number of cells counted no less than 1 × 106. ROS were measured using the 2′,7′- dichlorofluorescin diacetate (DCFH-DA) assay. The cells were incubated with 10 μM DCFH-DA for 30–60 min at 37 °C and then were washed with PBS. Fluorescence density was measured by FACScan instrument (Becton Dickinson, San Jose, CA, USA) at the excitation wavelength of 485 nm and the emission wavelength of 530 nm.
Pathological morphology
Lung tissues were fixed with 4% formalin for 12–16 h, then dehydrated and embedded in paraffin. The sample blocks were sliced into 5 μm and stained by HE to observe the inflammatory infiltration and integrity of the alveolar structure. Masson trichrome was used to assess the amount of fibrosis in the lung. The degree of pulmonary alveolitis was scored according to Szapiel et al. [
21], and pulmonary fibrosis by Ashcroft score [
22].
Statistics
Statistical analysis was performed by SPSS 17.0, and the data were expressed as mean ± SD. Comparison among groups was analyzed by one-way analysis of variance (ANOVA), and P < 0.05 was statistically significant.
Discussion
PFD is known to inhibit proliferation, differentiation, and the collagen synthesis of fibroblasts. It also inhibits the generation and activity of fibrosis-related cytokines TGF-β1, fibronectin, and connective tissue growth factor, thus exerting anti-inflammatory and anti-fibrosis effects that have been regarded to treat IPF [
8,
23‐
25]. A recent in vivo study indicated that PFD suppressed chemotaxis and accumulation of fibroblasts in the lungs of a mouse with bleomycin-induced pulmonary fibrosis [
9]. Importantly, the previous study reported that PFD alleviated the oxidization through its ability to suppress NADPH-dependent lipid peroxidation in liver microsome. This suggested that the anti-fibrosis activity of PFD is involved in oxidative stress [
26]. Other studies also found that PFD exerted an anti-fibrosis effect by alleviating lipid peroxidation and clearing the active intracellular oxygen [
11], thus demonstrating that anti-fibrosis of PFD could be associated with the oxidative stress.
Nrf2 is a central regulator for oxidation reaction, enhancing the oxidative stress in tissues and cells by stimulating the generation of downstream antioxidant substances. It indicated that stimulating Nrf2 alleviated the lung damage and suspended the progression of ILD [
16,
19,
27]. Moreover, Bach1 is a competitive inhibitor for Nrf2, which also plays an essential role in the regulation of oxidative stress [
15]. The defense mechanism against oxidative stress-induced damage is mediated by the transcription and protein expression of ARE and Nrf2 dependent antioxidant factors, including Ho-1, Gpx1, and others [
28]. Previous evidence has shown that slicing Nrf2 down-regulated the Ho-1 expression and further promoted the transformation from fibroblasts to myofibroblasts [
29]. Furthermore, it was also found that the activity and expression of Gpx1 and Ho-1 were significantly decreased in damaged lung of a mouse with bleomycin-induced pulmonary fibrosis [
18,
30], suggesting that the decline of Nrf2 dependent antioxidants promoted the formation of fibrosis. Intriguingly, Bach1 competitively inhibits the interaction between Nrf2 and ARE, leading to the suppression of ARE-dependent gene [
15]. However, the influence of Bach1 on the transcription and expression of ARE-dependent genes is yet controversial [
31‐
33]. Additionally, whether PFD exerts antioxidation and anti-fibrosis effects by regulating Nrf2/Bach1 equilibrium and expression of Nrf2-dependent antioxidants, remain unclear.
The amount of extracellular matrix generated by fibroblasts and the transformation into myofibroblasts is one of the principal characteristics of IPF [
34]. TGF-β1 is one of the key factors for generation of pulmonary fibrosis that promote proliferation and transformation of lung fibroblasts and deposition of extracellular matrix [
4]. BLM improves the production of oxygen radical, damage of alveolar epithelium, and proliferation of fibroblasts, further leading to pulmonary fibrosis [
35,
36]. In the present study, we found that the mRNA and protein expressions of Nrf2 and down-stream antioxidants such as Ho-1 and Gpx1 were significantly decreased in the MLF induced by TGF-β1 and lung tissue of mouse induced by BLM. However, the mRNA and protein expressions of Bach1 were significantly increased, demonstrating that the equilibrium state of Nrf2/Bach1 and Nrf2-dependent antioxidant factor were involved in the pathogenesis of IPF. Our previous study supported that and Bach1 knockout by siRNA enhanced the expression of Nrf2 dependent antioxidants in the TGF-β1 induced MLF and in mice of BLM induced pulmonary fibrosis [
20]. Take together, the overexpression of Bach1 may be associated with the pathogenesis of IPF by affecting the antioxidant/oxidant balance. Nevertheless, following varied PFD administration, the mRNA and protein expression levels of Nrf2, Ho-1, and Gpx1 in MLF were significantly increased, but in Bach1 were significantly decreased, as compared with the TGF-β1 stimulated group. We also found that the promoting effect on Ho-1 was significant with 200 μg/mL PFD. In addition, present study showed that the mRNA and protein expression levels of Nrf2, Ho-1, and Gpx1 in mouse pulmonary fibrosis induced by BLM were significantly increased, but in Bach1 were significantly decreased with PFD treatment for 4 weeks. Our results further suggested that PFD regulated the equilibrium of Nrf2/Bach1 and expression of a Nrf2-dependent antioxidant factor, further mitigating the inhibitory effect of antioxidant level in TGF-β1 induced MLF and in mice with BLM-induced pulmonary fibrosis, thus facilitating the antioxidant effect.
Several fibrosis-related factors are involved in the pathogenesis and progression of IPF, including TGF-β1, tumor necrosis factor-α (TNF-α), IL-6, and COL1A1 by promoting the proliferation of fibroblasts and transformation to myofibroblasts, leading to huge deposition of extracellular matrix [
2]. Here, our results demonstrated that the expressions of fibrosis-related cytokines IL-6 and myofibroblast markers COL1A1 were significantly increased in the supernatant of TGF-β1 induced MLF, serum, and BALF in mice with BLM-induced pulmonary fibrosis, especially in BLM 42d group. However, after PFD treatment, the levels of COL1A1and IL-6 were significantly decreased, indicating that PFD inhibited the production of a pro-fibrosis factor and the development of lung fibrosis. In addition, to determine whether PFD inhibits BLM-induced pulmonary fibrosis by scavenging free radicals, we measured the ROS generation in the lung tissues and MDA level in serum and BALF to evaluate the changes in BLM-induced oxidative damage. Excessive ROS induced by oxidative stress contributes to pulmonary fibrosis by accelerating epithelial-mesenchymal transition (EMT), infiltrating of inflammatory cell and collagen accumulation [
37]. MDA, a reactive carbon compound, is regarded as one of key indicator of oxidative stress [
38]. Consistent with previous studies [
39,
40], our results showed that ROS generation and MDA expression there was a significant increase in BLM administered mice and more obvious in 42d than 14d. Moreover, PFD treatment showing the significant decrease in ROS and MDA levels in BLM-induced mice imply that PFD is beneficial in regulating the equilibrium of oxidant-antioxidant.
In order to further explore the antioxidant and anti-fibrosis effect of PFD, we elucidated the lung histopathological changes in mice of lung fibrosis induced by BLM with or without PFD. Following 2 weeks of BLM administration in present experiment, the alveolus collapsed or disappeared, the septum width was significantly increased, and extensive inflammatory infiltration, that is in accordance with the pathological alterations during pulmonary fibrosis. Similarly, the results of Masson showed that there was a larger blue staining area (fibrillation) and destruction of the alveolar structure after BLM administration, complying to the above [
41]. The mouse modeling with IPF was thus successfully established and thus was chosen as the intervention time of PFD. After BLM d42, inflammatory infiltration was similar as d14 while the degree of fibrosis and structural damage had no obvious difference because of self-resolving of BLM- induced lung fibrosis and inflammation repair prior to fibrosis reversal. Previous study suggested that bleomycin model was a self-resolving model of lung fibrosis and fibrosis was resolved over time after a peak between d14 and d28 [
42]. But in the present study, we found that a self-resolving of bleomycin- lung fibrosis from d28 to d42 expressed as alleviated inflammation, and reversible repaired of fibrosis and structural damage may take longer. It is possible that our model of pulmonary fibrosis differs from previous studies in the repair time of fibrosis because of the well-known heterogeneity of fibrosis development in that mouse model.
However, after PFD therapy for 4 weeks, the inflammatory cells infiltration and the damaged structure of lung tissue were alleviated as compared to the BLM group (d42), suggesting that PFD relieves the pathological progression of IPF.
Based on the antioxidant and anti-fibrosis effects, PFD was used to treat MLF induced by TGF-β and mouse with pulmonary fibrosis. This demonstrated the influence of PFD on Nrf2/Bach1 equilibrium and expression of Nrf2-dependent antioxidants in TGF-β induced MLF and mice with BLM-induced pulmonary fibrosis. The present evidence showed that PFD promoted the expression of antioxidant factors by reversing the inhibitory effect of antioxidant ability induced by TGF-β1 and BLM in vitro and in vivo. Moreover, such antioxidant effect was realized by regulating the equilibrium of Nrf2/Bach1 and its down-stream antioxidants. Also, PFD inhibits the up-regulation of fibrosis-related factors such as COL1A1and IL-6 induced by TGF-β1 and BLM along with indicators of oxidative stress such as ROS and MDA level induced by BLM, alleviates the inflammatory infiltration and pathological damage of pulmonary fibrosis, while only PFD treatment for wild-type mice do not display these effects. It provides the experimental evidence for the underlying antioxidant mechanism of pirfenidone treatment as well as the novel approach for pathogenesis and therapeutic targets of IPF.