Drugs and chemicals
All the drugs and chemicals used in this study are listed as follows (Table 1).
Table 1
Chemials | Ventor | Catalog No. | City, Country |
LPS | Sigma-Aldrich | L4391 | USA |
Erastin | Abmole | M2679 | Huston, USA |
M-CSF | MCE Chemical | HY-P7085 | New York City, USA |
PD150606 | MCE Chemical | HY-100529 | New York City, USA |
Rosiglitazone (ROSI) | MCE Chemical | HY-17386 | New York City, USA |
Bleomycin (BLM) | Nippon Kayaku | / | Tokyo, Japan |
Animal model
All animal procedures were approved by the Institutional Animal Care and Use committee of Zhongshan Hospital, Fudan University (2022-042) in compliance with the guidelines for the Care and Use of Laboratory Animals published by the National Academy Press (NIH Publication No.85 − 23, revised 1996). Balb/c mice (female, 4–6 weeks old, 18-22g) were purchased from Shanghai JieSiJie Laboratory Animals Co., LTD. All these mice were housed in the animal facility of Zhongshan Hospital, Shanghai, with a 12 h dark/light cycle. Bleomycin powder was dissolved in sterilized PBS at a concentration of 1.0 mg/ml, filtrated through a 0.22µm filter (Milipore, USA) and then stored at 4 ℃. Mice were subcutaneously injected with 0.1ml BLM on the lower back at exact location daily for 4 weeks. The moderate disease group injected with a concentration of 0.5 mg/ml, while severe group with 1mg/ml.
Animal treatment and groups
All the mice were randomly assigned into the following groups of 8 animals each with comparable mean body weight:①Blank control: received PBS;② BLM group: received BLM; ③ ACSL4 inhibition: Balb/c mice with daily subcutaneously injection of BLM, then treated with intraperitoneal injection of ROSI (daily, 0.5mg/kg, 0.1ml/day dissolved in sterilized PBS, lasts for 21 days); ④ Calpain inhibition: Balb/c mice with daily subcutaneously injection of BLM, then treated with intraperitoneal injection of PD150606 (daily, 3mg/kg, 0.1ml/day dissolved in DMSO, lasts for 21 days). After treatment, all mice were sacrificed to collect skin tissue, pulmonary tissue and serum.
Histological analysis
Left lung tissues and local skin tissues were inflated and immersed in fresh 4% neutral buffered paraformaldehyde for 36 h at room temperature, then embedded in paraffin.
4-µm-thick sections were stained with hematoxylin and eosin (H&E) ,and we randomly selected 5 high-power field (HPF, 100X) of each slide for semi-quantitative measurement of inflammation score as previous described [21]. Likewise, sections of skin and lung tissues were stained with Masson’s trichrome for fibrosis analysis, calculating the collagen volume fracture (CVF, %) as positive area was stained blue with Image J software (NIH, USA).
Isolation of primary bone marrow-derived macrophages (BMDM)
As the previous study indicated [22], cleaned femurs and tibia bones were cut from the control and BLM disease model mice. Then they were centrifuged to collect bone marrow cells in sterile PBS and plated in Dulbecco’s modified Eagle’s medium (DMEM; Biosharp) additional supplemented with macrophage colony-stimulating factor (M-CSF; 10ng/ml). After 5 days culture, cells were harvest for later treatment and detection.
Cell culture and establishment of stable cell lines
Raw264.7 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and were cultured in DMEM supplemented with 10% heated-inactive fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Biosharp) at 37°C with 5% CO2.
Sequences of calpain 1 and calpain 2 knockdown and over-expression are listed in supplementary materials table 1. Then these sequences were packed in lentivirus for transfection (ZuoRun Biotech, Shanghai, China). After 5 days infection, the infection efficiency was firstly determined by counting the numbers of green fluorescent protein (GFP)-expressing cells under a fluorescence microscope (DMI4000B, Leica Microsystems, Germany)
Western blotting
Tissues and cells were lysed in a mixture of RIPA lysis buffer (P0013, Beyotime, Shanghai, China) and Phenylmethanesulfonyl fluoride (PMSF; ST506, Beyotime, Shanghai, China). Then after total protein collected, the supernatant was quantified by a BCA kit (ST2222, Beyotime, Shanghai, China). Proteins from each sample were subjected to electrophoresis on 12% sodium dodecyl sulfate-poly-acrylamide gel (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes on a semi-dry electro transferring unit (Bio-Rad, USA). PVDF membranes were blocked with 3% Albumin Bovine V for 2 h and incubated with the diluted primary antibodies overnight at 4℃. All the primary antibodies used in this study are listed as follows (Table 2). After incubation, membranes were washed by phosphate buffered saline with 0.1% Tween-20 (PBST) and incubated with HRP-conjugated secondary antibody (A0208, Beyotime, Shanghai, China) for 2 h. After extensive washing, blots were detected with enhanced chemiluminescence assays.
Table 2
Primary Antibodies for Western Blotting
Primary antibody | Ventor | Catalog No. | Working dilution |
(for western blotting) |
ACSL4 | Abmart | T510198 | 1: 1000 |
FTH1 | Abmart | T56955 | 1: 1000 |
GPX4 | Abmart | T56959 | 1: 1000 |
SLC7A11 | Abmart | T57046 | 1: 1000 |
Calpain1 | CST | #2556 | 1: 1000 |
Calpain2 | CST | #2539 | 1: 1000 |
Capns1 | Abclonal | A6539 | 1: 1000 |
Collagen Ia | Abclonal | A5786 | 1: 1000 |
Collagen III | Abclonal | A3795 | 1: 1000 |
α-SMA | Abclonal | A17910 | 1: 1000 |
β-actin | Abclonal | AC026 | 1:100000 |
RNA extraction and quantitative real time PCR (qRT-PCR)
Total RNA was extracted using the RNAiso Kit (R0024, Beyotime, Shanghai, China). cDNA was synthesized using the PrimeScript RT Master Mix (RR036A, takara, Janpan), and PCR reaction mixtures were prepared using qPCR SYBR Green Master Mix (CatNo.11202ES08, Yeasen, shanghai, China). The RT-PCR reaction process was as follows: 95°C for 5 min, followed by amplification in 40 cycles of 95°C for 10 s, 20 s at 60°C and 20 s at 72°C, then melting in default using the ABI 7900 Real-Time PCR System. The primer sequences are listed as follow. β-Actin served as the internal reference, and the 2–ΔΔCt method was used to quantify the relative expression of mRNA. All the primers used in this study are listed in supplementary table 2.
Histological analysis
Extension of inflammation of lung tissue was graded into the following categories and two independent researchers involved in grading for the slices: grade 0, normal lung tissue without inflammation, scored 0; grade 1, minimal alveolitis (+), widened alveolar septa due to inflammatory cells infiltration, the lesions confirmed to less than 20% of the whole lung, scored 1.0; grade 2, moderate alveolitis (++), the lesions extended to 20–50% of the whole lung, scored 2.0; grade 3, severe alveolitis (+++), diffuse lesions in more than 50% of the whole lung, scored 3.0.
Cell viability assay
Cell viability was assessed by using the cell counting kit 8 (CCK8; M4839, Abmole) according to the manufacturer’s instructions. Cell were seeded in 96-well plates (20,000 cells per well) and treated with 10µl erastin or PBS in the incubator for 24h. Then the culture medium was replaced with 100µl fresh supplemented DMEM containing 10µl CCK8 in each well of the plate. The cells were incubated in the cell incubator for 2h and then the absorbance at 450nm was measured using the microplate reader.
Calpain activity assay
The tissue lysates were assay for calpain activity using a calpain Assay Kit (ab65308, Abcam, UK) according to the manufacturer’s instructions. After incubating at 37℃ for 1h in dark, the calpain activity was detected by the fluorescence counter.
Hydroxyproline (HYP) measurement
Weighing 100 mg skin and lung tissue for HYP content measurement is followed the manufacturer’s instructions of a HYP Test Kit (A030-2, Jiancheng, Nanjing, China). All sections were examined independently by two investigators in a blinded manner.
Iron measurements
The fresh blood was collected by heart puncture and immediately centrifuged for the serum. The serum iron level was detected by the Iron Assay Kit (A039-1-1, Jiancheng, Nanjing, China) according to the manufacturer’s instructions.
Malondialdehyde (MDA) analysis and glutathione (GSH)- oxidized glutathione (GSSG) analysis
The MDA concentration of tissue and cell lysates were assessed using the MDA Assay Kit (S0131, Beyotime, Shanghai, China) according to the manufacturer’s instructions.
Total GSH and GSSG levels of tissue ane cell lysates were measured using the GSH-GSSG Assay Kit (S0053, Beyotime, Shanghai, China) according to the manufacturer’s instructions. A standard curve was established by the addition of GSH to PBS and net GSH concentration was determined as subtract GSSG concentration from total GSH content.
Immunohistochemical (IHC) analyses
4-µm-thick sections were deparaffinized, dehydrated and the endogenous peroxidase blocked for 15 min. Then after restoring antigen, the sections were incubated with diluted primary antibodies of ACSL4 (1:200), F4/80 (1:200) and secondary antibody EnVision+/HRP before visualizing by a DAB kit and evaluating under light microscopy (Nikon, Japan). The number of positive cells (brown) was calculate with Q500IW image analysis system (Leica, Germany) and Image-Pro Plus 6.0 software.
Statistical analysis
Data were represented as mean ± SD or mean. One-way ANOVA followed by Tukey’s multiple comparison test or Student’s t-test were applied for calculating statistical differences. The data processing and graphs were performed by using GraphPad Prism software (version: 9.0.0.121) and Image J software (version: 1.8.0). P-value < 0.05 was considered to be statistically significant.