Preparation of Taohong Siwu Decoction (TSW):
Taohong Siwu Decoction was prepared from six different medicinal plants, including: 20 g of peach red, 10 g of safflower, 20 g of angelica, 20 g of raw land, 20 g of red oak, 10 g of Chuanxiong. The medicinal materials are provided by the Chinese Pharmacy of the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine. Preparation method of traditional Chinese medicine: Weigh the traditional Chinese medicine decoction pieces according to the prescription dose, add the decoction pieces to the medicine tank, add 10 times of double distilled water of raw medicine, soak for 30 min, fry the fire with boiling fire until it is boiled, and simmer for 30 min, filter out the liquid medicine. Add 5 times of double-distilled water to boil, and decoct for 30 minutes. Mix 2 times of the drug solution, concentrate to 2 g of raw drug per 1 ml, and store in a refrigerator at 4 °C.
Animals and breeding environment
Ten male Sprague-Dawley rats weighing 200–260 g were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., and the experimental animal production license number is SCXK (Xiang) 2013-0004. The rats were raised at the Experimental Animal Center of Hunan University of Traditional Chinese Medicine. They were housed in a standard animal room with temperature of 21 ° C -25 ° C, humidity at 50% ± 5, 12 hours of day and night light and dark alternate time. The cages are cleaned once a week and the litter are replaced as needed. Animals are free to water and food. Rat food was provided by the Experimental Animal Center of Hunan University of Traditional Chinese Medicine. Rats were fed ad libitum for 1 week, and 1 week later, TSW was administered to the stomach for 1 week. All the animal experiments and protocols were approved by the Local ethics committee.
Preparation of serum containing TSW
Ten male Sprague-Dawley rats weighing (200–250) g were administered TSW according to the following formula: Rat dose (g/kg) = 6.25 × [adult dose (g) / adult body mass (60 kg)] × 3. Rats were administered at a dose of about 25 g/kg/d intragastrically twice each day. The rats in the blank group were given the same volume of normal saline for 10 days. After the last 2 hours of gavage, they were anesthetized with 10% chloral hydrate.
The blood was collected from the abdominal aorta. Briefly, the animals were anesthetized, placed on the surgical frame on the back, and the abdominal cavity was opened to expose the abdominal aorta clearly. Gently pry open the fat around the blood vessels with a small forceps, and then wipe the excess fat covering the blood vessels with a cotton ball until the blood vessels are clearly seen. The abdominal aorta is above the spine, next to the inferior vena cava, the inferior vena cava is thick, the color is dark, and the abdominal aorta is light in color. After finding it, clamp a 1.5 cm abdominal aorta with 2 arterial clips, and gently lift the artery wall with a small forceps at the distal end. Then use the blood collection tube for abdominal aortic puncture to see the blood return, and the other end is inserted into the vacuum tube. When you open the proximal blood vessel clamp, blood will flow out. 5 ml of blood was collected and left at 4 °C overnight, centrifuged. Then the serum was collected, sterilized by filtration using a 0.22 µM filter, and was inactivated at 56 ° C, and stored at -80 ° C until use.
Isolation, Identification and Culture of Rat Aortic Endothelial Cells
For the aortic endothelial cells preparation, use dissection scissors to open the abdomen from the midline, and expose the abdominal aorta. Open the chest cavity and expose the heart and lungs. Cut the abdominal aorta at the middle with dissection scissors to release the blood. Fill a 1 mL syringe (with 25 G needle) with 1 mL of PBS containing 1,000 U/mL of heparin. Inject PBS containing 1,000 U/mL of heparin to the left ventricle and perfuse the aorta. Push the heart and the lung with forceps at the great arteries to the right side of the mice to expose the thoracic aorta. Quickly remove the thoracic aorta using micro-dissection forceps and put it in ice-cold 1x PBS (sterile), then transport the container into a laminar airflow hood. Insert a 1 mL syringe fitted with 25 G needle into one end of the aorta, and gently flush the aorta with ice-cold PBS to remove the blood. Use micro-dissection forceps to remove as much of the attached adipose tissue and small lateral vessels as possible. Immediately transfer the aorta to endothelial growth medium. Cut the aorta into 1 mm rings using a sterile scalpel blade. Harvest about 8–10 rings per aorta. Open each aortic ring using a pair of micro-dissection scissors. The rat aorta was selected, separated, placed in a petri dish, and the morphology of the cells was observed with a fluorescent inverted microscope, and the cells were stained with an immune factor-related antigen.
The MTT method was used to detect the effect of serum containing serum of Taohong Siwu Decoction on the cell viability of vascular endothelial cells. Briefly, endothelial cells were plated into 96-well plate with 3000 cells/well and cultured for 6 h. Then cells were added with 10% KB blank, 2.5% TSW serum, 5% TSW serum, and serum 10% TSW serum and cultured for overnight. Then cells were added with 100 µl of MTT solution (Cat#: M2128, Sigma, USA) and incubated for another 4 hours. After incubation, the solution was aspirated and the cells were added with DMSO, 100 µl/well, and the absorbance was measured by a microplate reader at a wavelength of 490.
The Transwell chamber migration experiment was used to detect the effect of the serum containing TSW and HIF-1α inhibitor on the migration of vascular endothelial cells. The cells were seeded into 24-well plates with 5 × 103 cells/well. The drug-containing serum or TSW serum together with HIF-1α inhibitor (CAY10585, cat#: ab144422, Abcam, USA) were used to treat the cells as described above. After overnight treatment, the cells at the bottom chamber were fixed with paraformaldehyde, and then stained with the 0.1% crystal violet. Then migrated cells were observed and quantified by inverted microscope. At least 5 different fields with more than 200 cells were counted and the images were acquired.
Tube formation assay
96-well plate were coated with gel 50 µl/well Matrigel. Then the isolated endothelial cells were plated into 96-well plates with 1.5 × 104 cell/well. Cells were treated with TSW-containing serum in the presence and absence of HIF-1α inhibitor as described before. 24 hours after incubation, the tube formation was checked, and images were acquired by C5 microplate reader. Images from at least 5 different fields were acquired the IPP software was used to calculate the length of the lumen.
The isolated endothelial cells were plated into 24-well plates and then treated with TSW-containing serum in the presence and absence of HIF-1α inhibitor as described before. Different groups of cells were scratched with a 20-µL pipette to create the wound. Cells were washed with PBS buffer to remove the debris. Images were acquired at 0, 24 and 48 hours after treatment to determine the wound healing at 3 random locations. The wound area in each group was quantified.
Quantitative real-time PCR (qRT-PCR)
Total RNAs were extracted from treated cells and then reverse transcribed into cDNA with reverse transcriptase (Cat#: K1622,Thermo USA). Then the relative expression levels of HIF-1α, pVHL and VEGF were detected by qRT-CPR by using T100™ Thermal Cycler. GAPDH was used as endogenous control and the relative expression of target genes was calculated by using the 2−ΔΔCt method. The primes used for qRT-PCR were listed in the Table 1.
Total proteins were prepared by digested the cells treated with different treatment with lysis buffer (P0013, Beyotime Bio, China). Then, total of 30 µg proteins from each group were separated by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). After the membranes were blocked with 5% fat-free milk in PBS buffer, the membranes were incubated with anti-HIF-1α (Abcam, USA), VEGF (Abcam, USA), VHL (Abcam, USA) and β-actin (Proteintech, USA) antibodies for overnight at 4 °C. Then, membranes were washed four times with PBST buffer with 5 minutes for each time. The secondary antibodies were incubated with membranes for 1 hour at room temperature. After the membranes washed with PBST buffer, the protein expression was detected using enhanced chemiluminescence (Advans Group). The quantification of protein expression was evaluated by ImageJ (National Institutes of Health).
All the data were presented as mean ± SEM from at least three independent experiments. All the results were analyzed by using GraphPad Prism 8 software (GraphPad Prism Software Inc., San Diego, USA). The difference between two groups was analyzed by using t test. P < 0.05 was considered statistically significant.