2.1 Extraction and preparation of EOFAZ
The essential oil was extracted from the fruit of Alpinia Zerumbet, which was collected in Zhenfeng County, Guizhou province, China. The fruit was identified by Professor Qingde Long who worked for the department of Pharmacognosy in Guizhou Medical University .The volatile oil was obtained by Soxhlet extractor, and the extraction rate was 1.3%. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the chemical constituents of volatile oil from whole fruit of medicinal materials[23,24]. The EOFAZ was exactly extracted from the analytical balance and dissolved with dimethyl sulfoxide(DMSO). The concentration of stock liquor was 1×107 μg·L-1 and the microporous filter membrane was sterilized and placed at 4℃. Different concentrations of solutions were prepared by medium in the experiment.
2.2 Reagents
Transforming growth factor-β1 (TGF-β1) was purchased from Peprotech (NJ,USA). Human umbilical vein endothelial cells(HUVECs) and Endothelial cell culture medium (ECM) were purchased from Sciencell (CA,USA). Transforming growth factor-β1 inhibitor (LY2109761) was purchased from Selleck (TX,USA). KLF4 small interference RNA was purchased from Genepharma (Shanghai,China). KLF4 adenovirus transfection reagent(HBAD-EGFP, HBAD-h-KLF4) was purchased from Hanheng Biotechnology Co.,Ltd (Shanghai,China). Lipofectamine 2000 reagent was purchased from Invitrogen (CA,USA). The Transwell chamber and Immobilon western chemiluminescence reagent were purchased from Millipore (MA,USA). Matrigel was purchased from BD Biosciences (NY,USA). TransZol Up Plus RNA Kit was purchased from Quanshijin Biotechnology Co., Ltd. (Beijing, China). Immunoprecipitation kit was purchased from Sangon Biotech (Shanghai,China). Protein quantitative kit was purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Cell Lysates(RIPA) was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Antibody directed against vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA), zinc finger transcription factor Snail, Krüpple-like factor 4 (KLF4), Notch-1, Notch-3, Histone H3, Acetyl-Histone H3 and p300 were from Cell Signaling technology (MA,USA). Antibody directed against histone deacetylase 2 (HDAC2) was from Proteintech (Hubei, China). Antibody directed against β-actinwas from Affinity Biosciences (Shenzhen,China). Antibody directed against GAPDH and horseradish peroxidase (HRP)-conjugated secondary antibody (1: 7000 dilution)were from Bioworld (Nanjing, China).
2.3 Transient transfection
siKLF4 sequence is as follows: 5’-GGACUUUAUUCUCUCCAAUTT-3’ , 5’-AUUGGAGAGAAUAAAGUCCTT-3’. According to the manufacturer's instructions, when the cell fusion rate was 60%, HUVECs were transfected with Lipofectamine 2000. After 6 hours, they were replaced with 5%ECM and cultured for 24 hours. The adenovirus infection index (virus number: cell number) was 30:1. HBAD-h-KLF4 4 μL was added to the 2 mL system and changed to normal medium after 6 h, then cultured the cells for 24 hours. The knockout and overexpression efficiency of KLF4 were detected by Western blotting. The cells successfully transfected were used in subsequent experiments.
2.4 Cell Culture and Treatment
HUVECs cultured in Endothelial Cell Medium containing 5% fetal bovine serum and incubated in a humidifier at 37 °C and 5% CO2. HUVECs at passages 4-5 were used in this study. Cells were assigned to the following experimental groups: ⅰ Control group(5%ECM), TGF-β1 group(10ng·mL-1), Inhibitor of TGF-β1 group(LY2109761, 2μmol·L-1 and TGF-β1, 10ng·mL-1), siKLF4+ TGF-β1 group(siKLF4 and TGF-β1, 10ng·mL-1). ⅱ Control group(5%ECM), TGF-β1 group(10ng·mL-1), siKLF4 group, siKLF4+ TGF-β1 group(siKLF4 and TGF-β1, 10ng·mL-1), EOFAZ high dose(EOFAZ-H, 4μg·L-1 and TGF-β1, 10ng·mL-1), EOFAZ low dose(EOFAZ-L, 1μg·L-1 and TGF-β1, 10ng·mL-1). ⅲ ①Control group(5%ECM), TGF-β1 group(10ng·mL-1), EOFAZ group(EOFAZ, 4μg·L-1 and TGF-β1, 10ng·mL-1). ②Control group(5%ECM), Ad-NC group(HBAD-EGFP, 5%ECM), Ad-KLF4 group(HBAD-h-Null-KLF4, 5%ECM). ③Control group(5%ECM), TGF-β1 group(10ng·mL-1), AdKLF4+TGF-β1 group(AdKLF4 and TGF-β1, 10ng·mL-1), siKLF4+ TGF-β1 group(siKLF4 and TGF-β1, 10ng·mL-1), EOFAZ group(EOFAZ, 4μg·L-1 and TGF-β1, 10ng·mL-1). Cell transfection was carried out according to the method in section 2.3. After pre-incubation with EOFAZ for 2 h, TGF-β1 was added and incubated for 72 h to induce EndMT.
2.5 Wound-healing assay and Transwell migration assay
For wounding-healing assay, 1×104 ·mL-1 cells were grown to confluence in 24-well plates. The cell monolayer was scratched using a 10-μl pipette tip and the drugs prepared by serum-free medium were added.Representative photographs were taken after 12hunder inverted microscope. Five marked points were randomly selected to determine the data and calculate the mobility. Mobility = (average value of 0h scratch distance-average value of 12h scratch distance) / average value of 0h scratch distance. For Transwell migration assay, 2×104 cells were plated into the upper chamber of Transwell inserts precoated in 24-well plates. The lower chamber was filled with ECM supplemented with 5% FBS. After incubation for 6h at 37 °C, cells that moved to the bottom surface of the chamber were stained with 0.1% crystal violet and counted under an inverted microscope.
2.6 Angiogenesis experiment
The Metrigel and serum-free ECM were diluted in a proportion of 1:1, evenly spread in 24-well plates and coagulated in a incubator for 30 minutes, then 2×105·mL-1 cells were inoculated in 24-well plates. Representative photos were taken and counted after 4-6 hours.
2.7 Immunofluorescence staining
The cells were inoculated in a 24-well plate with a density of 1×104 mL-1, and the cells were fixed with 4% paraformaldehyde for 15 min after treatment as described above. The cells were incubated with anti-VE-cadherin, anti-α-SMA antibody (1:50 dilution; CST) or anti-Histone H3, anti-Acetyl-Histone H3(1:200 dilution;CST) followed by Alexa Fluor 488/cy3-conjugated secondary antibody. Nuclei were stained with DAPI (4,6-diamidino-2-phenylindole;1:1000 dilution; Sigma-Aldrich). Stained cells were visualized using a fluorescence microscope and the fluorescence intensity was detected by enzyme labeling instrument.
2.8 Western blotting
Cells were lysed in RIPA lysis buffer containing protease inhibitors and then loaded on SDS-polyacrylamide gels for electrophoresis before transfer to PVDF membrane. After blocking non-specific binding sites, the membranes were probed with the first antibody mentioned above at 4°C. Blots were incubated with horseradish peroxidase-conjugated secondary antibody, and detected by chemiluminescence. The immunoreactive bands were produced by the Syngene Gel Imaging system (Bio-Rad, California, USA) and quantified using ImageLab 4.0 software. All antibodies and dilution ratio were listed in Table 1.
Table 1 Specifc antibodies for Western blot
Antibody
|
Company
|
Catalogue number
|
Dilution
|
VE-cadherin
|
CST
|
2500
|
1:1000
|
α-SMA
|
CST
|
19245
|
1:1000
|
Snail
|
CST
|
3879
|
1:1000
|
KLF4
|
CST
|
4038
|
1:1000
|
Notch-1
|
CST
|
3608
|
1:1000
|
Notch-3
|
CST
|
5276
|
1:1000
|
Histone H3
|
CST
|
4499
|
1:1000
|
Acetyl-Histone H3
|
CST
|
9649
|
1:1000
|
p300
|
CST
|
86377
|
1:1000
|
HDAC2
|
Proteintech
|
12922-3-AP
|
1:1000
|
β-actin
|
Affinity Biosciences
|
T0022
|
1:7000
|
GAPDH
|
Bioworld
|
MB001
|
1:10000
|
2.9 Quantitative real-time PCR analysis(qRT-PCR)
Total RNA was extracted using TransZol Up Plus RNA Kit and reverse transcribed to cDNA followed by Real-time PCR reaction. Thermal profile conditions were as follows: preincubation at 94 °C for 30s followed by 39 cycles of amplification at 94 °C for 5s and 56 °C for 30s. VE-cadherin, α-SMA, KLF4, Snail, Notch-1 and Notch-3 transcript levels were calculated by the 2−ΔΔCT method. All primers above were synthesized by Sangon (Shanghai, China). Primer sequence is show in Table 2.
Table 2 Specifc primers for quantitative RT-PCR
Gene
|
Forward
|
Reverse
|
VE-cadherin
|
CAAGGACACTGGCGAAA
|
ACGCATTGAACAACCGA
|
α-SMA
|
CGTGGCTATTCCTTCGTT
|
ACGTTCATTTCCGATGGT
|
CollagenⅠ
|
GCCTCAACATCCCCTACA
|
CAGCCCACGAAGAACAGA
|
Snail
|
ACATCCGAAGCCACACG
|
TGGGGACAGGAGAAGGG
|
KLF4
|
AGGAGCCCAGCCAGAAA
|
TCCAGTCACAGACCCCATC
|
Notch-1
|
AGGCTCTGCCGACATCA
|
AGGAAGGGGTGCTCTGG
|
Notch-3
|
GCTGTTCCCCTTGACTGG
|
CTTTGTGGGGCTGCTGT
|
β-actin
|
GACATGCCGCCTGGAGAAAC
|
AGCCCAGGATGCCCTTTAGT
|
2.10 Immunoprecipitation assay
When the cells were harvested at the end of observation, an appropriate amount of IP cleavage buffer (including protease inhibitor) was added, then cracked on ice for 30 minutes after ultrasound, and the supernatant was absorbed after centrifugation. A small amount of lysate was taken for Western blot analysis, and the remaining lysate was added 1ug antibody and 18ul protein A/G-beads to the cell lysate and incubated overnight at 4 ℃. After immunoprecipitation reaction, centrifugation for 5 minutes, protein A/G-beads was washed with lysate for 6-7 times. Finally, adding appropriate volume of loading buffer and heating at 95℃for 5 minutes, SDS-PAGE gel electrophoresis was carried out.
2.11 Statistical analysis
Statistical significance was calculated by Student’s t-test. The variance homogeneity test and One-Way ANOVA analysis were used for the comparison among groups. A value of p<0.05 was considered significant. Datas are expressed as mean ± S.D. Each experiment was performed in triplicate.