Parthenolide attenuated bleomycin-induced pulmonary fibrosis via the NF-κB/Snail signaling pathway

PTL (> 99%) was provided by Shangdeyaoyuan Co. (Tianjin, China). DEX sodium phosphate (> 98.5%) was purchased from Meilun Biological Technology Co. (Dalian, China), and BLM sulfate (> 91%) was obtained from Meilun Biological Technology Co. (Dalian, China). The NF-κB, Snail, β-actin, GAPDH, E-cadherin, vimentin, MMP1, α-SMA and Col-1 antibodies were purchased from Affinity Biosciences Co. (Beijing, China). The mouse TNF-α, mouse IL-4, mouse TGF-β1, and mouse interferon gamma ELISA Kits were purchased from Meilian Biological Technology Co. (Shanghai, China). Chlorine ammonia T (> 97.08%) and p-dimethylaminobenzaldehyde (> 97.08%) were obtained from (> 99.71%). Reverse-4-hydroxy-l-proline (> 99.4%) was purchased from Bailingwei Technology Co. (Beijing, China). Perchloric acid (> 70%) was obtained from Jingchun Biological Technology Co. (Shanghai, China).

The human pulmonary epithelial A549 cell line was obtained from KeyGen Biotech (Nanjing, China). The human fetal lung fibroblast cell line HFL1 was kindly supplied by Professor Wen Ning (Nankai University). The cells were cultured in a medium supplemented with 10% heat-inactivated (56 °C, 30 min) fetal calf serum (HyClone, USA) and maintained at 37 °C with 5% CO2 in a humidified atmosphere.

Primary pulmonary fibroblasts isolated from NaCl/BLM-treated mice were cultured in DMEM supplemented with 10% FBS and antibiotics in 5% CO2 at 37 °C in a humidified atmosphere as described previously [14]. Cells at passages 3–4 were used for cell viability and wound healing assays. Primary AECs were isolated from C57BL/6 J mice as previously described [15]. Newly isolated AECs were used for immunofluorescence and Western blotting assays.

Cell viability was determined using the MTT assay. Cells (5 × 10³ cells/mL) were seeded in 96-well culture plates and incubated overnight. Cells were treated with various concentrations of PTL for 24 h. Cell viability was measured after the addition of MTT (20 μL) at 37 °C for 4 h. Dimethyl sulfoxide (150 μL) was added to dissolve the formazan crystals. Optical density was measured at 570 nm using a microplate reader (Multiskan FC, Thermo Scientific, Waltham, MA, USA).

Cells for the wound healing assay were grown on a 35-mm dish to 100% confluency and scraped to form a 100-μm wound using sterile pipette tips. The cells were cultured in the presence or absence of PTL in serum-free media for 24 h. Images of the cells were obtained at 24 h using a light microscope (Nikon, Japan).

Primary epithelial cells were fixed in 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.2% Triton X-100 in PBS, blocked with 5% BSA and incubated with E-cadherin and vimentin antibodies. Cells were washed with PBS, and donkey anti-rabbit Fluor 555 or donkey anti-mouse Fluor 488 secondary antibodies (CWBIO, China) were used for immunofluorescence visualization. The nucleus was labeled with DAPI (Solarbio, China), and cells were photographed with a TCS SP5 confocal (Leica) microscope.

AP1, STAT3, NF-κB, snail, slug and MYC promoters were cloned into the pGL6-TA luciferase reporter vector, and A549 cells were transfected with luciferase reporter plasmids using Lipofectamine (Invitrogen). Renilla-luciferase was used as an internal control. Cells were treated 1 d after transfection with 5 μΜ (L) or 10 μM (H) PTL for 24 h. Cells were harvested, and the luciferase activity of cell lysates was determined using a luciferase assay system (Promega) as described by the manufacturer. Total light emission was measured using a Luminoskan Ascent Reader System (Thermo, Massachusetts, USA).

Specific pathogen-free ICR mice (males) (body weights 18–22 g) were purchased from the Laboratory Animal Center, Academy of Military Medical Sciences of People’s Liberation Army (Beijing, China) and housed in groups of six under a regular 12-h light/dark cycle. Mice were acclimated to laboratory conditions for one week prior to testing at a constant temperature.

Sixty mice were divided into six groups with 10 animals per group according to body weight: control group, BLM group, BLM + DEX group (0.45 mg/kg), BLM + PTL-H group (50 mg/kg), BLM + PTL-M group (25 mg/kg), and BLM + PTL-L group (12.5 mg/kg). PF was established in mice via a single intratracheal administration of BLM at 5 mg/kg body weight. Different doses of PTL were intragastrically administered daily for four weeks beginning 7 days after BLM injury, and DEX was used as the positive control. Control and model groups received an equal volume of vehicle (0.9% NaCl) using the same schedule and route of administration.

Mouse body weights were recorded daily. Mice were sacrificed on the 36th day using excess chloral hydrate hydrochloride anesthesia. Blood was obtained for ELISA analyses, and whole lungs were removed and weighed. The right lungs were fixed in 10% formalin, dehydrated, and embedded in paraffin. The left lungs were used to determine hydroxyproline. The pulmonary coefficient was calculated using the following equation: lung weight/body weight × 100%.

Mice were anesthetized with 10% chloral hydrate in NaCl (i.p.) and transferred to a plethysmographic chamber for pulmonary function analyses using the Anires2005 system (Beijing Biolab, Beijing, China). This system automatically calculates and displays pulmonary function parameters, including dynamic compliance and inspiratory and expiratory resistance.

Paraffin sections were prepared at a 4-μm thickness, stained with H&E and Masson’s trichrome using the manufacturers’ standard procedures, and observed under a photomicroscope (Olympus, Tokyo, Japan) for microscopic examination of morphological changes and fibrosis evaluation (collagen fibers).

The tissue sections were pretreated in a microwave, blocked and incubated using a series of antibodies, and stained with DAB and hematoxylin. The results were captured using a microscope (Olympus, Japan). The intensity and percentage of positive cells were measured. Multiplication (staining index) of intensity and percentage scores was used to determine the results.

Mice were anesthetized, and a microhematocrit tube was introduced to the canthus of the orbit. The microhematocrit tube was slightly advanced and rotated to allow blood flow into the lithium-heparin tube. Plasma was separated from the cellular fraction via centrifugation at 3500 rpm for 10 min at 4 °C and stored at − 80 °C.

The tracheas of mice were cannulated and lavaged three times with 1-ml sterile PBS at room temperature for BALF collection. Samples were centrifuged at 1000 rpm for 5 min, and cell pellets were recovered in 1-ml sterile PBS. Cells were counted using a hemocytometer. Smears of BALF cells were stained with hematoxylin and eosin and viewed under light microscopy to measure the inflammatory cell differential.

Plasma TGF-β1, TNF-α and IL-4 levels were assayed using ELISA Kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). Assays were performed according to the manufacturer’s instructions.

We determined the effect of PTL treatment (24 h) on the cell viability of primary pulmonary fibroblasts (PPF) isolated from NaCl/BLM-treated mice and HFL1 cell lines using MTT assays (Fig. 1a, d and g). The half-maximal inhibitory concentrations (IC₅₀) of PTL after 24-h treatment in the PPF-NaCl, PPF-BLM and HFL1 cells were 10.68 μM, 26.01 μM and 13.66 μM, respectively. These results indicate that PTL reduced the cell viability of lung fibroblasts in a dose-dependent manner.

The effect of PTL on the cellular migration of lung fibroblasts was determined using a wound-healing assay. Confluent cells were scraped with a sterile pipette tip, and the remaining cells were allowed to migrate to the resulting gap in the absence or presence of PTL. Serum-starved PPF or HFL1 cells in the control group exhibited a narrow cell wound gap in the wound area 24 h after wounding. Cells treated with PTL (2.5, 5, 10, or 20 μM) exhibited relative delays in wound closure (Fig. 1b-c, e-f and h-i).

We researched the influence of PTL on the TGF-β1-induced EMT process in lung epithelial cells to further investigate its effects on the pathological mechanisms of PF. Figure 2a shows that low doses of TGF-β, an inducer of EMT, resulted in extended pseudopodia and changes in the microfilament structure of A549 cells. The PTL-H + TGF-β and PTL-L + TGF-β groups reversed the features of EMT to different degrees. The morphology changes and EMT marker expression in TGF-β/PTL-treated primary AECs exhibited the same results (Fig. 2b). We further evaluated several EMT-related transcription factors, such as NF-κB, Snail, AP-1, c-Myc, Slug, and Stat-3. We analyzed the activity of these transcription factors in A549 cells using a dual-luciferase assay. PTL reduced the expression and activity of NF-κB and Snail, but did not influence the expression of AP-1, c-Myc, Slug, and Stat-3 (Fig. 2c and d).

We verified the effect of PTL treatment on the expression of EMT-related transcription factors NF-κB and Snail in A549 cells and primary AECs. Western blot analysis results revealed that NF-κB and Snail levels increased significantly in the A549 cells and primary AECs after exposure to TGF-β. NF-κB and Snail levels decreased slightly in the TGF-β + PTL-L group and decreased severely in the TGF-β + PTL-H group (Fig. 2e-h). The expression levels of Snail and NF-κB decreased in a dose-dependent manner.

Mice were intragastrically administered with or without PTL after BLM injection to investigate the effects of PTL on BLM-induced lung fibrosis. A protective effect of PTL was observed on weight loss. Body weight loss was significantly attenuated in mice treated with PTL compared to the model group (Fig. 3a). Treatment of bleomycin-injured mice with high/middle PTL doses significantly reduced collagen content compared to mice treated with DEX (Fig. 3b). The protein levels of collagen (Col1) and α-SMA exhibited similar results (Fig. 3c). The lung coefficients were higher in the lung tissues of the model group than the other groups, and the lung coefficient of the mice treated with PTL was considerably reduced compared to the model group (Fig. 3d). The attenuated fibrosis in PTL-treated mice was further supported by improved pulmonary function, which was observed as a decreased inspiratory resistance, expiratory resistance and increased dynamic compliance compared to BLM-treated mice (Fig. 3e-g). Collectively, these in vivo data indicate that PTL attenuated bleomycin-induced pulmonary fibrosis in mice.

Persistent inflammation exists in an early stage, and it drives fibrotic progression in bleomycin-induced fibrosis. However, the effect of inflammation in fibrogenesis is arguable. Inflammatory cell numbers in BALF and inflammatory cytokines in plasma were evaluated to determine whether PTL altered inflammatory responses. Notably, cell differentiation analysis of BALF revealed that the increase in macrophages, lymphocytes and neutrophils was attenuated in the high/middle PTL dose groups compared to the model group (Fig. 4a-c). TGF-β, TNF-α and IL-4 levels in plasma were detected using ELISA. Levels of the inflammatory cytokines TGF-β, TNF-α and IL-4 were increased in the plasma of the model group compared to the other groups. The levels of these inflammatory cytokines decreased significantly in mice treated with PTL compared to untreated mice, and this effect was dose-dependent. Our assay results demonstrated that PTL significantly reduced the inflammatory response in bleomycin-injured mice in a dose-dependent manner.

Lung tissues were pink and soft under the normal conditions, and the lungs of mice in the bleomycin-treated model group were hard, shrunken in size, and dark in color. Treatment with PTL remarkably improved the lung condition (Fig. 5a). Histopathological changes in mouse lung tissues were evaluated using H&E staining. The lung tissue specimens of the BLM-induced mice treated with PTL exhibited marked improvements in inflammation and fibrosis (Fig. 5b and c). The BLM-injured lungs exhibited fibroplasia, inflammatory cell infiltration, thickened alveolar walls, destroyed and disordered alveoli, and stenosed or partially collapsed alveolar spaces (Fig. 5b and d). The lungs of the animals treated with PTL revealed significantly reduced infiltration of inflammatory cells, edema, thrombosis, and structure destruction compared to the model group. Masson’s trichrome staining was used to examine the collagen deposition and distribution and assess fibrosis in lung tissues. Significant collagen deposition, particularly around the bronchus, was observed in BLM-injured lung tissues compared to their respective controls (Fig. 5c and e). The PTL-treated groups exhibited a significant decrease in collagen deposition compared to the model group, and PTL-H was better than PTL-L.

The present study analyzed several EMT-related proteins to determine whether PTL affected the conversion of AECs to fibroblasts (FBs). PTL increased epithelial cell markers, such as E-cad, and reduced the vimentin and α-SMA (marker of mesenchymal cells) expression (Fig. 6). These results suggest that PTL increased epithelial cell markers and reduced mesenchymal cell markers. The effect of PTL was better than DEX. PTL also inhibits Col-1 and MMP1, which are PF cytokines. Immunohistochemical staining of Col-1 and MMP1 revealed that PTL increased MMP1 levels and decreased Col-1 levels in lung tissues in a dose-dependent manner (Fig. 6).

The expression levels of NF-κB and Snail were also assessed using immunohistochemical staining. The results demonstrated that NF-κB and Snail levels decreased significantly compared to the model group in a dose-dependent manner (Fig. 7). PTL inhibited EMT via the NF-κB/Snail pathway in lung tissues. These results are consistent with the cell experimental results.

The STRING database was used to examine several types of interactions between the control and PTL treatment groups. Gene Ontology (GO) analysis was performed to analyze the function of differentially expressed mRNAs using the GO categories. GO categories are derived from Gene Ontology (http://​www.​geneontology.​org), and the categories include three integrated networks of defined terms that describe gene properties (molecular function, cellular component and biological process). PTL influenced many functions, including inflammatory responses and proliferation (Fig. 8a). We also analyzed the molecular function, cell structure, and biological processes of the expression profiling chip on the GO website after PTL treatment and compared the results with the control groups (Fig. 8b). These biological processes are closely related to pulmonary fibrosis.