Cell culture and treatment. THP-1 (Human acute monocytic leukemia cell line) cells and L02 cells were purchased from ATCC. The cells were all cultured in RPMI 1640 containing 10% heat-inactivated FBS and 1% penicillin-streptomycin in a 5% CO2 atmosphere at 37°C. THP-1 cells were treated with 160 ng/ml phorbol 12-myristate 13-acetate (PMA) alone and then incubated for 48 h in RPMI medium to obtain macrophage-like M0 cells. They were then cocultured with L02-SCR or L02-Sh cells in a 6-well Transwell plate.
Animals. FPN1flox/flox conditional gene knockout mice were constructed by Nancy Andrews of the USA. Two LoxP sites were inserted into exon 6/7 of the SLC40A1 gene, and the mice were maintained on a 129/SvEvTac background. SLC40A1-LoxP transgenic mice (129S-Slc40a1tm2Nca/J) were purchased from Jackson Laboratories (USA), backcrossed on a C57BL/6 background and bred in-house. When they were bred with liver cell-specific promoter (Alb-Cre) mice, all of the offspring obtained exhibited hepatocyte-specific knockout. Mice with downregulation of the FPN1 gene were used as the model mice for this experiment. Unless indicated, age-matched animals were used at 8–12 weeks of age. Homozygote (FPN1−/−), heterozygote (FPN1−/+) and wild type (FPN1+/+) mice were obtained by mating, and the genotypes were determined by agarose gel electrophoresis. All animals in this study were raised in SPF-level animal rooms and fed a standard diet. The housing of the animals and the experiment procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health) and were approved by the Ethical Committee for Care and Use of Laboratory Animals of
Guangzhou Medical University. This study was carried out in compliance with the ARRIVE guidelines 2.0.
Real-time Quantitative PCR. Total RNA was isolated from THP-1 cells, M0 macrophages, and M0 macrophages cocultured with L02-SCR or L02-Sh using TRIzol according to the manufacturer’s instructions. One microgram of RNA was reverse transcribed into cDNA using a First Strand cDNA Synthesis Kit. cDNA was quantified using the Applied Biosystems Step-One Real-Time PCR system with a SYBR Green Real-time PCR Master Mix kit. The following primer sequences that were used are shown in Table 1 (It is showed in supporting information). GAPDH served as the housekeeping gene.
Table 1
List of the sequence of gene primers.
Gene name | Forward(5’-3’) | Reverse(5’-3’) |
IL-1β(mice) | CCAGGATGAGGACATGAGCA | CGGAGCCTGTAGTGCAGTTG |
IL-4(mice) | CGGCACAGAGCTATTGATGG | TCCGTGGATATGGCTCCTG |
IL-6(mice) | AGTTGCCTTCTTGGGACTGA | CCTCCGACTTGTGAAGTGGT |
IL-10(mice) | TAGAGCTGCGGACTGCCTTC | TTCCGATAAGGCTTGGCAAC |
IL-12α(mice) | CTGGCGTCTACACTGCTGCT | CGTGATTGACACATGCTGGA |
IL-12β(mice) | GCTGGTGTCTCCACTCATGG | TCTTCAGGCGTGTCACAGGT |
TNF-α(mice) | TATGGCTCAGGGTCCAACTC | CCCATTTGAGTCCTTGATGG |
CSF-1(mice) | CGACTTCCCGTAAAGGCATA | AGCAGAGGGCACTTAAGCAA |
TGF-α(mice) | CATTGATCTGCCCAGGTCTT | ACTTCTGCCTGGAGCTGTGT |
TGF-β(mice) | TGCCCTCTACAACCAACACA | GTTGGACAACTGCTCCACCT |
IFN-γ(mice) | CCTTTGGACCCTCTGACTTG | AAACAGCCATGAGGAAGAGC |
HGF(mice) | CTCCCGAGAACTTCAAATGC | GCAGTAGCCAACTCGGATGT |
VEGF(mice) | CCCTTCGTCCTCTCCTTACC | AAGCCACTCACACACACAGC |
FPN1(mice) | GCA GGC TCT GTT CTG GTC CT | GAT GAT TCC GCA GAG GAT GA |
IL-1β(human) | ACGATGCACCTGTACGATCA | TCTTTCAACACGCAGGACAG |
IL-4((human) | GCCTTCAGCACATCTTCACACCTC | ATCGCTTCTCTGCACCTGTTCTTG |
IL-6(human) | GGTGTTGCCTGCTGCCTTCC | GTTCTGAAGAGGTGAGTGGCTGTC |
IL-10(human) | TGCCTTCAGCAGAGTGAAGA | GTCTTGGTTCTCAGCTTGGG |
IL-12α(human) | GAGTTCAAGACCAGCCTGACCAAC | ACCTCCACCTCCGAGTTCAAGC |
IL-12β(human) | GAGCAGGCATCAGCACCATCTG | ACACCATCAGCAGCATCACCTTG |
TNF-α(human) | AGCTGGTGGTGCCATCAGAGG | TGGTAGGAGACGGCGATGCG |
CSF-1(human) | CAGAAGGAGGACCAGCAAGTGAAG | GCCAGCAAGACCAGGATGACAC |
TGF-α(human) | GCCTCTGCCGATCTTGAACATCTC | TGCCTACACCTACCTGCTTACCTG |
TGF-β(human) | CACGTGGAGCTGTACCAGAA | GAACCCGTTGATGTCCACTT |
IFN-γ(human) | TGTTACTGCCAGGACCCATA | CTTCCTTGATGGTCTCCACA |
HGF(human) | AATGGCACGATCTTGGCTCACTG | AGGAGTGGTGGTGGCAGGTG |
VEGF(human) | TAGCTGCCTGCCTGGTGACTG | CAGAAGGACCACAGGACACAACAC |
FPN1(human) | TGA ATG CCA CAA TAC GAA GG | CCA AGT TCC ATC CCG AAA TA |
β-Actin | GCC ACT GCC GCA TCC TCT TC | AGC CTC AGG GCA TCG GAA CC |
14146 | GGC ATT CCC AAC ACT TTA GC | |
14147 | CCC ATA GGT TAA ACT GCT TCA A | |
20239 | TGCAAACATCACATGCACAC | |
20240 | TTGGCCCCTTACCATAACTG | |
olMR5374 | GAAGCAGAAGCTTAGGAAGATGG | |
ELISA. The levels of IL-4, IL-10, TGF-β, TNF-α, and IFN-γ (Cell signaling Company, USA) in the serum and cell culture supernatant were measured by ELISA following the manufacturer’s instructions. All ELISA reagent kits were purchased from ExCellBio. All samples were measured in triplicate. The concentration of certain cytokines in the serum and cell culture was quantified by standard curve. The fold change in the expression of cytokines was calculated compared to the control group.
Agarose Gel Electrophoresis. Agarose Gel Electrophoresis was performed as described previously34. DNA was extracted using a GeneJET Genomic DNA Purification kit (Thermo, USA). Extracted DNA samples were amplified by PCR of the FPN1 gene by restriction fragment length polymorphism-PCR. The PCR mixture contained 12 µL of OneTaq Quick-Load 2X Master Mix with standard buffer, 4 µL of each primer (40 µM), and 8 µL of DNA sample (< 10 ng), resulting in a total volume of 28 µL. The reaction was carried out under the following conditions: initial denaturation at 95 ℃ for 15 min followed by 34 cycles of denaturation at 95 ℃ for 1 min, annealing at 56 ℃ for 1 min and elongation at 72 ℃ for 1 min with a final extension at 72 ℃ for 10 min. Gel electrophoresis was performed on 1.5 ~ 2% agarose gels supplemented with 11 mM MgCl2 and SYBER™ safe DNA gel stain in TAE buffer (0.5×) at pH 8. The samples of interest were mixed with 20% loading buffer (6×) and then loaded into agarose gel wells. The gel was run at 70 V for 2.5–3 h and visualized using a UVP scanner.
Western Blot. The cells were washed with ice-cold PBS and then lysed in ice-cold RIPA lysis buffer containing 1 mmol/L PMSF. The protein concentrations were calculated using BCA assay kits. A total of 40 µg of total cellular protein was subjected to 8% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% defatted milk powder at room temperature for 1.5 h, incubated with primary antibodies (FPN1, CD11b, CD206, iNOS, and GAPDH were come from Cell Signaling Company, USA) at 4°C overnight and then incubated with HRP-conjugated secondary antibodies at room temperature for 1.5 h. Following each step, the membranes were washed five times with TBS-T for 5 min. Finally, the blots were developed using the enhanced chemiluminescence system.
Immunohistochemical analysis. Immunohistochemical analysis was played according to the procedure reported by Hau-Wen Li35. Deparaffinization of the tissue sections was executed using a graded ethanol series, and then 0.3% hydrogen peroxide was used to block endogenous peroxidase activity. Antigen retrieval was achieved by placing the sections in 10 mmol/L citrate buffer (pH 6.0) and heating in an autoclave for 5 min. The sections were then rinsed with phosphate-buffered saline (PBS, pH 7.2) and incubated with 10% nonimmunized goat serum for 30 min at room temperature to inhibit nonspecific binding. The tissue sections were then incubated at 4°C overnight with antibodies. After 24 h, the slides were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit secondary antibodies for 30 min at 25°C. Then, staining with 3,3-diaminobenzidine (DAB) was carried out for 90 seconds at room temperature; hematoxylin staining was performed as a control.
Histology. Tissue from mice was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (thickness = 5 µm). Masson staining was conducted by a Masson Stain Kit (Solarbio, Beijing, China). Sirius red staining was performed to visualize collagen fibers using a Sirius Red Stain Kit (Solarbio, Beijing, China). Each section was assessed under light microscopic fields.
Flow cytometry. F4/80+, CD16/32+, and CD206+ cells were quantified by antibody labeling and flow cytometry. Briefly, cells were dispersed with 0.25% EDTA-trypsin and washed with PBS. For each group, 106 cells were resuspended in 500 µl PBS and labeled with 5 µl of antibody. The cells were incubated at room temperature for 30 min and resuspended 2–3 times during incubation. The data were acquired on an LSRII and analyzed by FlowJo v8.8.6.
Antibodies and reagents. The antibody to FPN1( #L3266) was obtained from Santa Cruz Biotechnology (USA). The antibody to GAPDH (#5174) was obtained from cell signaling Company (USA). The antibody to Collagen-1 (#ab34710), Collagen-4 (#ab6586), Laminin (#ab11575), MMP9 (#ab38898), a-SMA (#ab5694), VEGF(#ab46154), TGF-B(#ab92486), INOS(#ab15323) were obtained from abcam (USA). The antibody to F4/80 (#123107) and CD206 (#141706) were obtained from Biolegend. The antibody to F4/80(#12-4801-82), CD68(#11-0689-42) and CD16/32 (#4303632)were obtained from Invitrogen. Sirus Red Staining Kit (#G1471) was obtained from Solarbio (China) and Masson Staining Kit (#D026-1-3) was obtained from NanJing JianCheng (China)
Iron assay. The relative iron concentration in cell lysates was assessed using an Iron Assay Kit (#ab83366, Abcam) according to the manufacturer’s instructions.
Statistical analysis. Statistical analysis was performed using GraphPad Prism software version 6.0. All data are expressed as the mean ± standard deviation (SD). Comparisons between two groups were performed using Student’s t test, and three or more groups were evaluated for significance using one-way ANOVA combined with Bonferroni's post hoc test. A P value < 0.05 or 0.001 considered statistically significant.