Cell line and culture
CCC-ESF-1 normal human embryonic dermal fibroblasts were obtained from the Cell Resource Center, Peking Union Medical College and were used between passages 10 and 14. Cells were maintained in DMEM supplemented with 10% fetal bovine (Gibco) in a humidified atmosphere of 5% CO2 at 37 °C.
Drug
Baicalein (Cat# 111595-201607) was obtained from National Institutes for Food and Drug Control (Beijing, China) and prepared in stock at the dose of 20 mM in dimethyl sulfoxide (DMSO) and the working concentrations were prepared by diluting the stock in medium. TGF-β1 and PDGF were obtained from Peprotech (Rochy Hill, NJ, USA). The inhibitor of TGF β receptor type I (LY364947) and the selective PDGFRα/β inhibitor (crenolanib) were obtained from MedChemExpress.
Measurement of collagen accumulation and secretion
CCC-ESF-1 cells were seeded in collagen I-coated plates for in vitro models of fibrosis and cultured 48 h to 80-100% confluence. After a washing with PBS, the medium was then changed to a serum-free DMEM supplemented with 1% insulin-transferin-selenium (ITS), which was the basic medium used throughout this study, for 24 h and then cells were incubated in the medium with or without TGF-β1(5ng/mL) or PDGF(40ng/mL) for additional 48h in the presence or absence of vehicle, different concentrations of baicalein. Some inhibitors (1 μM LY364947, 2 μM crenolanib) were used as positive controls. The deposits of collagen were assessed by Picro-Sirius red (PSR) staining and quantitative analysis as previously reported[12]. Briefly, cells were fixed with methanol overnight at -20°C and incubated in 0.1% PSR staining solution for 4 h. Then cells were washed twice with 0.1% acetic acid and PSR was eluted in 0.1 M sodium hydroxide. The optical density (OD) value was measured at 540nm. Collagen secretion was detected by measuring soluble collagen cell culture supernatant using the Sircol Soluble Collagen Assay (Biocolor, Country Antrim, UK) according to the manufacturer’s instructions.
Cytotoxicity assay
Cytotoxicity was assessed using lactate dehydrogenase release (LDH) assay. 120 μL supernatant from each well was collected and detected for LDH release according to the manufacturer’s instructions (Beyotime, China). Cytotoxicity was determined by the optical density (OD) value of LDH release by cells.
ELISA assays of collagen type I and α-SMA
CCC-ESF-1 cells were lysed with three freeze-and-thaw cycles between -80℃ and 37℃ in PBS and centrifuged at 12000g for 15min at 4℃. Collagen type I and α-SMA in the supernatants were measured using ELISA kits according to the manufacturer’s manual (CUSABIO, China). Results were expressed as the ratio of collagen I or α-SMA per total protein.
Reverse transcriptase quantitative polymerase chain reaction (qPCR) analysis
Total RNA in cells was isolated by the RNeasy Mini kit (Qiagen). cDNA was prepared from 2 μg of total RNA using the RevertAid First strand cDNA Synthesis Kit (Thermo Scientific™) and then subjected to qPCR to quantify the gene expression using SYBR Green SuperReal Premix Plus (Tiangen Co. China). The primer pairs used for analyses were listed in Table 1. GAPDH was used to normalize for the amounts of loaded cDNA. Relative expression of the target genes was calculated using 2−ΔΔCt method.
Table 1 Primer sequence
Genes
|
Primer Sequence
|
Length
|
human COL1A1
|
Forward: 5’-TCAAGAGAAGGCTCACGATGG-3’
Reverse: 5’- TCACGGTCACGAACCACATT -3’
|
69
|
human COL1A2
|
Forward: 5’- GGTCAGCACCACCGATGTC -3’
Reverse: 5’- CACGCCTGCCCTTCCTT -3’;
|
51
|
human COL3A1
|
Forward: 5’- GCTGGCTACTTCTCGCTCTG -3’
Reverse: 5’- TCCGCATAGGACTGACCAAG -3’
|
97
|
human CTGF
|
Forward: 5’- CAGCATGGACGTTCGTCTG -3’
Reverse: 5’- AACCACGGTTTGGTCCTTGG-3’;
|
115
|
human fibronectin
|
Forward: 5’- TTCTAAGATTTGGTTTGGGATCAAT -3’
Reverse: 5’- TCTTGGTTGGCTGCATATGC -3’
|
51
|
human TGF-β1
|
Forward: 5’- CAATTCCTGGCGATACCTCAG -3’
Reverse: 5’- GCACAACTCCGGTGACATCAA -3’
|
86
|
human α-SMA
|
Forward: 5’- GACAATGGCTCTGGGCTCTGTAA -3’
Reverse: 5’- CTGTGCTTCGTCACCCACGTA -3’
|
147
|
human GAPDH
|
Forward: 5’- GACCACTTTGTCAAGCTCATTTCC -3’
Reverse: 5’- GTGAGGGTCTCTCTCTTCCTCTTGT -3’
|
151
|
Mouse model of scleroderma
Six to seven -week old female C57BL/6J mice were obtained from Beijing Vital Laboratory Animal Technology (Beijing, China) and maintained in specific pathogen free lab, with food and water ad libitum. All animal procedures were in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines with the approval of the Institutional Animal Ethical and Welfare Committee (Approval Number: protocol #20162032).
The bleomycin-induced skin fibrosis model was initially described by Yamamoto et al[27]. Bleomycin (BLM, Nippon Kayaku, Tokyo, Japan) was dissolved in 0.9% NaCL at a concentration of 1 mg/mL and sterilized by filtration. Mice were randomly divided in 5 groups with at least 9 mice in each group. 100μL of BLM or 0.9% NaCL were injected subcutaneously into the shaved back well defined areas (1cm2) of the mice daily and orally gavaged with vehicle control (0.5% carboxymethylcellulose sodium), D-penicillamine (125mg/kg) or baicalein (25,50 and 100 mg/kg) once a day for 4 weeks.
Quantification of fibrosis in mice
The injected skin areas were fixed in 4% paraformaldehyde in PBS, embedded in paraffin and stained with Masson’s Trichrome staining. Dermal thickness was determined by measuring the largest distance between the epidermal–dermal junction and the dermal-subcutaneous fat junction. The collagen content in lesional skin was analyzed by hydroxyproline assay according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and was expressed as μg hydroxyproline per mg of tissue wet weight.
ELISAs for serum autoantibodies and cytokines
Serum levels of anti-Scl70, anti-dsDNA, anti-centromeres and anti-PM-Scl were measured using ELISA kits (Euroimmun Medizinische Labordiagnosika AG, Germany) according to the manufacturer’s instructions with minor modifications. A 1:5 serum dilution was used for the determination of all autoantibodies. The secondary antibody was a goat polyclonal anti-mouse IgG H&L conjugated to horseradish peroxidase (HRP) (Abcam, UK). The optical density (OD) value was measured at 450/650nm. Serum levels of IL-1β, IL-2, IL-4, IL-6, IL-17A, TNF-α, MCP-1 and MIP-1β were detected using MILLIPLEX MAP kits (Merck Millipore, Germany) according to the manufacturer’s instructions.
Flow cytometric analysis
Single-cell suspensions were obtained from mouse spleen in PBS. Erythrocytes were lysed with RBC lysis buffer. Spleen cells were labeled with mAb specific for cell surface markers B220 (PE-Cy7, RA3-6B2) from BD Pharmingen and CD27 (PE, LG.3A10) from Miltenyi Biotec. All samples were assessed by CytoFLEX flow cytometry system (Beckman Coulter Inc., CA, USA). At least 30000 lymphocyte-gated cells were routinely collected for each sample. B cells were identified as B220+ lymphocytes. Memory B cells were identified as CD27+B220+ lymphocytes.
Statistical Analysis
Data were expressed as mean ± SD or mean ± SEM. All statistical analyses were performed using GraphPad Prism 8.0.2 Software. Statistical significant differences of multiple groups were determined by ordinary ANOVA followed by Dunnett’s test for post-hoc comparisons. In each case, P value<0.05 was considered statistically significant.