- BLM-induced animal model of pulmonary fibrosis
Thirty-five 7-8 weeks male C57BL/6J mice were purchased from Charles River(Beijing, China). All mice were housed and cared for in a pathogen-free facility at Nankai University. The mice were acclimatized in a room with constant temperature(25 ± 2℃) and relative humidity(60 ± 2%) and allowed free access to food and water. All animal experiments were approved by the Animal Care and Use Committee at Nankai University. The mice were randomly divided into 5 groups(n=7 per group): Control group, BLM group, Pirfenidone-treated group(200mg/kg), Low-Ellagic acid-treated group(10mg/kg), High-Ellagic acid-treated group(20mg/kg), Pirfenidone were purchase from Dalian Meilun Biotechnology(Dalian, China), Ellagic acid were purchased from Macklin Biochemical(Shanghai, China). The mice were orally exposed to Ellagic acid, Pirfenidone and water is once a day for 7-13 day. For BLM administration, mice were anesthetized with 10% chloral hydrate(Sangon, Shanghai, China) and then intratracheally injected with bleomycin(Nippon Kayaku Co., Ltd., Tokyo, Japan) at a dose of 2U/kg body weight for analysis of the fibrotic response. The sham-operated group received intratracheal injections of the same amount of saline. The drug administration group began to be administered daily from the seventh day of modeling to 13th day. Mice were sacrificed on day 14 and lung tissue was harvested for the following experiments to evaluate the degree of pulmonary fibrosis.
- The isolation and culture of pulmonary fibroblasts
Primary pulmonary fibroblasts(PPF) were isolated from and treated with 0.9% saline solution or BLM wild-type C57BL/6J mice, Mouse lung fibroblasts cell line(Mlg), and Mouse embryonic fibroblasts cell line(NIH-3T3), human lung fibroblasts (HLF-1) (kindly supplied by Professor Wen Ning, Nankai University) were cultured in DMEM medium (Solarbio, Beijing, China) or F12K medium (Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel) and antibiotics(100mg/ml streptomycin, and 100 U/ml penicillin G) in a 37 ℃ atmosphere of 95% humidified air and 5% CO2. Chloroquine(CQ) were purchase from Macklin (Shanghai, China), Bafilomycin a1(Baf a1) were purchased from Cayman Chemical (Wuhan, China).
- Western blot
The sample of lung tissues and cells were homogenized in RIPA lysis (Beyotime Biotechnology, Shanghai, China) buffer with PMSF and NaF (phosphatase inhibitor; need to add when extracting the phosphorylating protein), then centrifuged(10000 rpm, 10min) to obtain supernatants. The total protein concentration was measured by BCA Protein Assay kit(Beyotime Biotechnology, Shanghai, China). Secondary antibodies: goat anti-rabbit IgG-HRP(Abcam, Share, UK), goat anti-mouse IgG-HRP(Abcam, Share, UK). Relative density of each band was analyzed by Image J. The following first antibodies were used:
Antibody
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Company &Item No.
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Antibody
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Company &Item No.
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GAPDH
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Affinity, AF7021
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P62
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Proteintech, 8420-1-AP
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β-tubulin
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Affinity, T0023
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Atg16L1
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CST, 8089T
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Lamin B1
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Affinity, DF6687
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Beclin1
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CST, 3495S
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β-catenin
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CST, 8480T
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LC3A/B
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CST, 12741S
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p-β-catenin(s33/37/Thr41)
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CST, 9561T
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mTOR
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Abcam, ab32028
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p-β-catenin(Thr41/Ser45)
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CST, 9565T
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p-mTOR(S2448)
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Abcam, ab109268
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α-SMA
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Affinity, BF9212
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S6 ribosomal protein(S6RP)
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Affinity, AF7831
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Collagen I
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Affinity, AF7001
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p-S6 ribosomal protein(S235/S236)
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CST, 4858T
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Fibronectin
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Affinity, AF5335
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Caspase3
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CST, 9662S
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Akt
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SANTA CRUZ, sc-56878
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Cleaved-Caspase3
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CST, 9664T
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p-Akt(Ser473)
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SANTA CRUZ, sc-514032
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Caspase9
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CST, 9009T
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Erk1/2
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SANTA CRUZ, sc-514302
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Cleaved-Caspase9
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CST, 9504T
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p-Erk1/2(Thr202/Thy204)
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SANTA CRUZ, sc-81492
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|
|
- Quantitative real-time PCR (qRΤ-PCR)
The total RNA was extracted using TRIzol Reagent(Invitrogen, Carlsbad, CA, USA). The cDNA was obtained from total RNA through reverse transcribed. qRT-PCR was performed by using SYBR GreenER qPCR SuperMix Universal(Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocols. The relative quantification of gene expression (a-SMA, Collagen I, Fibronectin, Cyclin D1, MMP7, Wisp1) was measured relative to the endogenous reference gene β-actin using the comparative CT method in the experiment. Sequences of the specific primer sets are as follows:
α-SMA (NM_007392.2), 5-GCTGGTGATGATGCTCCCA-3 and 5-GCCCATTCCAACCATTACTCC-3;
Col1a1 (NM_007742.3), 5-CCAAGAAGACATCCCTGAAGTCA-3 and 5-TGCACGTCATCGCACACA-3;
Fn (NM_010233.1), 5-GTGTAGCACAACTTCCAATTACGAA-3 and 5-GGAATTTCCGCCTCGAGTCT-3;
Cyclin D1 (NM_007631), 5-GCGTACCCTGACACCAATCT-3 and 5-CAGGTCTCCTCCGTCTTGAG-3;
MMP7 (NM_010810), 5-CTTACCTCGGATCGTAGTGGA-3 and 5-CCCCAACTAACCCTCTTGAAGT-3;
Wisp1 (NM_018865), 5-CAGCACCACTAGAGGAAACGA-3 and 5-CTGGGCACATATCTTACAGCATT-3;
β-actin (NM_007393.3), 5-AGGCCAACCGTGAAAAGATG-3 and 5-AGAGCATAGCCCTCGTAGATGG-3.
- Hematoxylin-eosin staining (HE staining)
Left lungs were fixed in 10% formalin for 24 hours and embedded in paraffin. Then lung sections(5µm) were prepared and stained with hematoxylin-eosin staining. HE staining images were collected using an upright transmission fluorescence microscope and opened in Image-Pro Plus Version 6.0 (Media Cybernetics, Inc, USA). Quantification of pulmonary fibrosis was performed as described previously[31]. In brief, the software selection tool can select the entire lung tissue area and automatically calculate the total pixel (Pw) of the region, and then use the same method to calculate the total pixel (Pf) of the fibrosis region, fibrosis ratio = fibrosis area pixel (Pf)/total lung pixel (Pw).
- GFP-LC3 and Cherry-GFP-LC3 transfections and Immunofluorescence
Two mg of GFP-LC3B(mouse) and Cherry-GFP-LC3B(mouse) was transfected into NIH-3T3 cells using PEI according to the supplier’s protocol (Sino Biolgical Inc., Beijing, China). 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 α-SMA antibodies. Cells were washed with PBS, and FITC were used for immunofluorescence visualization. Next, tissues were incubated with anti-a-SMA, anti-Col1 antibody overnight at 4 °C and then secondary FITC or Rhodamine (TRITC)-labelled antibody for 30 min. Fluorescein (FITC) AffiniPure Goat Anti-Mouse IgG (H+L) and Rhodamine (TRITC) Affinipure Goat Anti-Rabbit IgG (H+L) were purchased from Jackson ImmunoResearch(Pennsylvania, USA). Nuclei were stained with DAPI (Solarbio, Beijing, China), Cells and Tissues were photographed with a TCS SP8 confocal microscope(Leica).
- Dual luciferase assay
TCF/LEF promoters were cloned into the pGL4.49 luciferase reporter vector, and NIH-3T3 cells were transfected with luciferase reporter plasmids using PEI. Renilla-luciferase was used as an internal control. Cells were treated 18 h after transfection with a series of EA for 8 hous. Cells were harvested, and the luciferase activity of cell lysates was determined using a luciferase assay system (Promega, USA) as described by the manufacturer. Total light emission was measured using a GloMax®-Multi Detection System (Promega, USA).
- Immunohistochemistry and Masson staining
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(Nikon, Japan). The experimental procedure of Masson refers to Masson's Trichrome Stain Kit(Solarbio, Beijing, China). The method of counting the positive area is as follows: 1)Using Image J to open a picture, click “Image” and “type”, change “RGB Color” to “RGB stack”; 2)Click “Image”, drop down and click “adjust”, change the B&W in the “Default” column to “Red”; 3)adjust the upper and lower pulleys to select the positive signal area, click “set”, click “ok”; 4) Then click “analyze”, click “set measurement”, choose “area fraction”; 5)Began to count positive results, click “Control+M” on the keyboard, the number of “the% Aera” is the result.
- Flow cytometric analysis of apoptosis
1X10⁶ NaCl-PPF or PPF-BLM cells/mL were seeded in six-well plate and left for 24 hours in incubator to resume exponential growth. The extent of apoptosis was measured through annexinV-FITC apoptosis detection kit (Beyotime, Shanghai, China) as described by the manufacture’s instruction. Cells were exposed to drug and incubated for 24h. then they collected and washed with PBS twice, gently resuspended in annexin-V binding buffer and incubated with annexinV-FITC/PI in dark for 15min. The number of apoptosis was detected by CytoFLEX S(Beckman Coulter, USA).
- Hydroxyproline assay
The collagen contents in right lungs of mice were measured with a conventional hydroxyproline method.[32], In brief, the right lungs were dried and acid hydrolyzed, then the residue was filtered and the PH value was adjusted to 6.5-8.0. The hydroxyproline analysis was performed using chloramine-T spectrophotometric absorbance as previously describe.
- Evaluation of pulmonary function
The C57BL/6J mice were administrated by BLM(2U/Kg) on day 0, and were orally exposed to Ellagic acid, Pirfenidone and water are once a day for day 7-13, and finally were sacrificed on day 14. The mice were anesthetized with 10% chloral hydrate in saline (i.p.), If you recognized that the breath of mice is more quickly, you need to add anesthetic or wait for a moment until the mice breathe slowly. Carefully cut the neck skin with a scalpel (try not to bleed, otherwise it will affect the experimental data of lung functions), and subsequently using the surgical line to fix the cannula. The mice were transferred into the plethysmography chamber and analyzed functions using Anires2005 system (Biolab, Beijing, China). This system automatically calculates and displays pulmonary function parameters, including forced vital capacity (FVC), dynamic compliance (Cydn), inspiratory resistance (Ri) and expiratory resistance (Re).
- Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6.0 software. 2-tailed Student t test was used to analyze statistical differences. Pearson correlation and linear regression were used to determine the concordance. Data were shown as means ± s.d. and significance was described as follows: *,P <0.05; **, P < 0.01; ***, P < 0.001, #,P <0.05; ##, P < 0.01; ###, P < 0.001.