2.1 Reagents
Ethanol was purchased from Zhen Xin Co. Ltd. (Shanghai, China). Isoflurane was provided by Baxter Healthcare Co. (Deerfield, IL, USA). TRIzoI reagent kits were obtained from Omega Bio-Tek (Doraville, USA). The RNA-Solv reagent and HiBindTM PCR DNA extraction kit were obtained from Omega Bio-Tek Inc. (Norcross, GA, USA). Primary antibodies against Col2a1 (ab34712), ERG (ab214341), and PTHLH (ab239527) were purchased from Abcam (Cambridge, UK); anti-Col10a1 (bs-0554R-FITC) obtained from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Real-time qPCR (RT-PCR) kits were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). All oligonucleotide primers specific to rat genes were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
2.2 Animals and treatment
Specific pathogen-free (SPF) Wistar rats (females weighing 210–250 g, and males weighing 270–320 g) were purchased from the Experimental Center of Hubei Medical Scientific Academy (No. 2015–2018, certification number: 42000600032526, Wuhan, China), and animal handling and experimental procedures were carried out with approval from the Institute of Health Sciences Institutional Animal Care and Use Committee. All rats were fed adaptively for 1 week, and 2 females were mated with 1 male every night. The day when sperm was found on a vaginal smear was considered GD 0. Then, the pregnant rats were randomly separated into two groups: the control group and the PEE group (n = 24 in each group). The PEE group was administered 4 g/kg·d ethanol by gavage from GD9 to GD20, while the control group was administered the same volume of distilled water. Eight randomly selected pregnant rats from each group were anesthetized with isoflurane and decapitated on GD14, GD17, and GD20. At these three time points, one fetal rat was randomly selected from each litter and fixed with 95% alcohol for subsequent skeleton analysis. The knee joints of the remaining rats in the two groups were removed under an anatomical microscope, the right limbs were fixed in a 4% paraformaldehyde (PFA) solution, and the left limbs were stored in a -80 ℃ freezer for gene detection. The scheme of animal experimental procedures was as follows.
2.3 Whole-mount skeletal staining
Whole-mount skeletal staining using Alcian blue and Alizarin red was performed as described previously(Singh et al. 2018). Rat embryos of GD17 and GD20 were collected, rinsed in PBS and fixed in 95% ethanol for three days, followed by overnight fixation in 100% acetone. Then, the tissues were stained for three days in a 1:1:1:17 volume mixture of glacial acetic acid:0.3% Alcian blue 8GX (Sigma-Aldrich) in 95% ethanol:0.1% Alizarin red in 70% ethanol:70% ethanol. After staining, the tissues were washed using 1% KOH and imaged under a Canon EOS 70D camera (Canon, Tokyo, Japan).
2.4 Histological measurement
Rat embryonic limbs on GD14, GD17, and GD20 were dissected, fixed in 4% PFA at 4°C for three days, and embedded in paraffin, and 4 µm sections were cut along the parasagittal plane using a microtome. For histological analysis, Alcian blue and safranin O were applied to detect the glycosaminoglycan (GAG) content. Histological images were captured using a Nikon NIS Elements BR light microscope (Nikon, Tokyo, Japan).
Immunohistochemical staining and tissue immunofluorescence staining were performed following the manufacturer’s protocol. Briefly, after dewaxing, EDTA containing antigen retrieval buffer (pH 8.0) was used for antigen retrieval. BSA was used to block the previously added primary antibody, and the primary antibody dilution ratios were as follows: anti-Col2a1 (1:200 dilution), anti-Col10a1 (1:200 dilution), anti-PTHrP (1:250 dilution), and anti-ERG (1:250 dilution). Immunohistochemistry was conducted using a DAB staining kit (GeneTech Company, Ltd., Shanghai, China). For tissue immunofluorescence, the primary antibody was detected with a fluorescent Cy3-conjugated goat anti-rabbit IgG (H + L) (1:50 dilution) secondary antibody, after which the tissue sections were stained with 4′, 6-diamidino-2-phenylindole (DAPI) and sealed with an anti-fluorescence quenching agent. All images were captured and then analyzed with a Nikon NIS Elements BR light microscope (Nikon, Tokyo, Japan). The staining intensity was calculated by measuring the mean integrated optical density (MOD) in 10 different fields for each sample.
2.5 Bioinformatics analysis
The miRNAs targeting ERG were predicted according to three bioinformatics programs, miRDB (http://mirdb.org/), TargetScan (http://www.targetscan.org/vert_71/), and microRNA.org (http://www.microrna.org). The overlapping miRNAs are shown in the Venn diagram.
2.6 Chondrogenic differentiation of BMSCs in alginate bead culture
The extraction of BMSCs and their culture in alginate beads were conducted as described in a previous study(De Ceuninck et al. 2004; Deng et al. 2012). Monolayer cultures were trypsinized, washed, and centrifuged. The isolated BMSCs were suspended at a concentration of 1×107 cells/ml in 1.25% alginate (Sigma-Aldrich, USA) in 0.15 M NaCl. The cell suspension was drawn into a syringe and slowly added to a 102 mM calcium chloride solution dropwise with a needle. Beads with approximately 5×105 cells/bead were cultured in chondrogenic medium, containing high-glucose DMEM supplemented with 1% insulin, transferrin, and selenous acid (ITS) (Sigma-Aldrich, USA), 100 nM dexamethasone (Sigma-Aldrich, USA), 50 µg/ml ascorbic acid-2‐phosphate (Sigma-Aldrich, USA), 40 µg/ml l‐proline (Sigma-Aldrich, USA), and 10 ng/ml transforming growth factor-β1 (TGF-β1) (PeproTech, USA). After chondrogenic differentiation for 3 weeks, alginate bead sections were stained with safranin-O to assess the number of cells and to locate GAG deposits. During the period of chondrogenic differentiation, the culture medium with or without ethanol at concentrations of 0, 30, 60, and 120 mM was replaced every other day.
2.7 Primary chondrocyte culture
Chondrocytes were isolated from GD17 rats and plated at a density of 2 × 105 cells per well in 6-well plates in medium (DMEM/F12 medium supplemented with 10% fetal bovine serum, 100 mg/ml streptomycin, and 100 U/ml penicillin). Primary chondrocytes at 80% confluence were used for further experiments. During the culture period, the cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
2.8 Transfection
ERG and PTHLH overexpression plasmids were produced by Shanghai GenePharma Co., Ltd. (Shanghai, China). The miR-200b-3p inhibitor and mimic were synthesized by RiboBio (Guangzhou, China). Subconfluent GD17 rat primary chondrocytes in 6-well plates were transfected in triplicate with 2.5 µg of ERG and PTHLH overexpression plasmids using Lipofectamine™ 3000 and P3000™ transfection reagent (Invitrogen, USA) according to the manufacturer’s protocol. For miRNA inhibitor and mimic transfection, cells were transfected with 100 nM miR-200b-3p inhibitor or 50 nM miR-200b-3p mimic using Lipofectamine™ 3000 transfection reagent (Invitrogen, USA) according to the manufacturer’s protocol. After 8 hours, the transfection efficiency was determined using a fluorescence microscope. After 24 and 48 hours, the cells were collected for further analysis of gene and protein levels.
2.9 Cellular immunofluorescence staining
After treatment, the cells cultured on coated plates were washed three times with PBS, fixed in 4% formaldehyde for 15 min, blocked with 3% BSA for 1 hour, and permeabilizes with 0.1% Triton X-100/PBS for 10 min. The cells were then incubated overnight at 4°C with primary antibodies in 0.5% BSA, including rabbit anti-Col2a1 (1:200 dilution), rabbit anti-Col10a1 (1: 50 dilution), mouse anti-ERG (1: 100 dilution), and mouse anti-PTHrP (1: 100 dilution). The cells were washed with PBS and incubated with 1:200 diluted Cy3-conjugated goat anti-rabbit and Fluor 594-conjugated goat anti-mouse secondary antibodies for 1 hour at room temperature. The nuclei were stained with DAPI at a 1:500 dilution for 5 min. The slides were washed twice with PBS, and fluorescence images were captured using a confocal microscope (Smartproof 5, Carl Zeiss, Oberkochen, Germany). The staining intensity was determined by measuring the IOD in 10 different fields for each sample.
2.10 Western blotting
Briefly, resuspended cells were rinsed with ice-cold PBS and then lysed for 30 min at 4°C in RIPA lysis buffer containing phosphatase inhibitor cocktail, followed by analysis with the BCA Assay Kit for protein quantification. A total of 20 µg of protein was loaded into each lane, separated by SDS-PAGE, and blotted onto PVDF membranes (Millipore, MA, USA). The membranes were blocked in 5% nonfat milk for 1 hour and incubated overnight at 4°C with the primary antibody. The dilution concentrations of the primary antibodies were as follows: Col2a1 (1:1000), Col10a1 (1:800), PTHrP (1:3000), and ERG (1: 3000). Then, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG, 1:5000 and goat anti-mouse IgG, 1:5000) for 1.5 hours and visualized using ECL HRP substrate (PerkinElmer Inc., Boston, MA, USA). The antibody binding signals were detected using a ChemiDoc Image Analyzer (Bio-Rad, Hercules, CA, USA). The relative protein level was standardized to the GAPDH protein level. The protein band intensities were analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA) from 3 independent bands.
2.11 Total RNA extraction and RT-PCR
Total RNA was isolated from cartilage tissues and primary chondrocytes using TRIzol reagent following the manufacturer’s protocol. The RT-PCR procedure was described by Li et al.(Qing-Xian et al. 2020). RNA was assayed for GAPDH, Col2a1, aggrecan, tenascin-C, Col10a1, PTHLH, and ERG espression. All primers were designed with Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, USA). The primer sequences for the rat genes are shown in Table 1. GAPDH served as the reference gene to normalize the expression of other genes. The relative expression levels of target genes were calculated by the 2−ΔΔCt method.