Drugs
Sal D (Batch No. P22N9F75815) was provided by Shanghai yuanye Bio-Technology Co, Ltd (Shanghai, China). Sal B (Batch No.18062901), Fa (Batch No.17092501), La (Batch No.18042602), Ra (Batch No.18030901), Ca (Batch No.171228041), R1(Batch No.18052908), Rb1 (Batch No.18082204), Rg1(Batch No.18071601), Rd (Batch No.18011503) and Re (Batch No.18062501) were provided by Chengdu Feipude Biotechnology Co, Ltd (Chengdu, China). Sal B, Sal D, Fa, La, Ra, Ca, R1, Rb1, Rg1, Rd and Re were dissolved in sterile water. They were diluted with DMEM to the final concentration.
Pericytes culture
MBVP were obtained from BeNa Culture Collection (Beijing, China, BNCC 342014) cultured in MBVP growth media (89%DMEM+ 10% FBS+1% penicillin and streptomycin (Gibco, New York, USA)). The cells were placed in a 37℃incubator containing 5% CO2. When the cells grew to 80% - 90%, they were passed for subsequent experiments.
Oxygen–glucose deprivation, drug treatments
MBVP were washed with PBS (Solarbio, Beijing, Chain) and cultured in glucose-free DMEM (Gibco, New York, USA). Then placed MBVP in 37°C anoxic chamber (Stemcell, Vancouver, Canada) which was filled with 95 % N2 and 5 % CO2 for 6 h. After hypoxia, medium of the OGD/R group was changed to DMEM (Gibco, New York, USA). Medium of the treatment group was changed to the DMEM (Gibco, New York, USA) containing drugs of Sal B, Sal D, Fa, La, Ra, Ca, R1, Rb1, Rg1, Rd and Re. And then cultured at 37℃ incubator containing 5% CO2 for 18 h. The control group was cultured in DMEM for 24 h in 37℃incubator with 5% CO2.
Detection of cell viability
Cells were cultured in 96-plates and exposed to OGD 4 h followed by reoxygenation 20 h (OGD4h /R20h). Then, cell viability was measured using Cell Counting Kit-8 (CCK-8, CK04, Japan). DMEM with 10% CCK-8 solution was added to the cells which were washed once with HBSS. Cells were placed in 37 °C incubator for 30-60 mins. The absorbance was measured at 450 nm using a microplate analyzer (Infinite F50, Switzerland). Lactic dehydrogenase (LDH) release was measured using LKolate Dehydrogenase Assay Kit after OGD 4 h followed by reoxygenation 20 h (CK12, Japan). bEnd.3 cells supernatant were collected and centrifuged (4℃, 1000 rpm, 5min). 50µl of each sample and 50µl of reaction mixture was pipetted into a 96-well plate. The absorbance of the plates was measured at 490 nm with a microplate analyzer (Infinite F50, Switzerland).
Detection of SOD and ROS
The MBVP were seeded in 96-well plates, OGD 6 h/R 18 h were performed after the cells fused. Then, Superoxide Dismutase (SOD) and Reactive Oxygen Species (ROS) of MBVP in each group were detected according to the manufacturer’s instructions of SOD Activity Kit (Nanjing Jiancheng Bioengineering Institute, Nangjing, Chain) and ROS Assay Kit (Beyotime Institute of Biotechnoligy, Shanghai, Chain).
Flow Cytometry
MBVP were dissociated with trypsin after OGD/R. The cells were collected after centrifuged at 500g for 5 min, and washed with pre-cooling PBS for 2 times. After re-suspended by binding buffer, the MBVP were labeled with V-FITC and PI was for 10 min. The cells were suspended by binding buffer and detected by Flow Cytometry. The assay was performed according to manufacturer’s instructions of Annexin V-FITC/PI Cell Apoptosis Detection Kit (40302ES60, Yeasen Biotechnology, Shanghai, China).
Scratch assay
MBVP were seeded in 6-well plates, OGD 6 h/R 18 h were performed after the cells fused. Then, 200 μL pipette tips were used to make the scratch gap. Photos were taken at the following 12 h, 24 h and 48 h. Quantitative analysis was conducted with Image J software,as previously described (Fu et al. 2019). Wound healing rate(WH)= [the wound area at time ( 12h,24h,48h-) its initial area( 0h)]/ its initial area(0 h) × 100%.
Western blotting
After OGD 6 h reperfusion for 18 h, the proteins were extracted from the 6-well plate using RIPA lysis buffer and were quantified according to the instructions of the BCA kit (23227, Thermo Fisher Scientific). The treated sample was added to a 4%-12% SDS-PAGE gel to separate the proteins by size. The separated proteins transferred to PVDF membranes were scaled with 5% skim milk. The antibodies JNK (ab124956, Abcam, Cambridge, USA), p-JNK (ab47337, Abcam), Caspase-3 (ab13847, Abcam), PI3K(ab151549, Abcam), p-mTOR (ab84400, Abcam), mTOR (ab32028, Abcam), PDGFR-β (ab32570, Abcam), VEGF (ab32152, Abcam), Cleaved-Caspase-3 (9661S, CST, Bostom, USA), AKT (4691S, CST), p-AKT(4060S, CST), p-P38 (4511S, CST), P38(8690S, CST), p-ERK(4370, CST), ERK(12950, CST), Bcl-2 (3498S, CST), β-actin (4967S, CST), Bax (14796S, CST), Ang-1 (BS2829, Bioword, USA) were diluted in PBS at a ratio of 1:1000. PDVF membranes were incubated overnight in antibodies at 4 ° C. Membranes were washed with TBST, and incubate with goat anti-rabbit or oat anti-mouse (zsgb-bio, Beijing, China) horseradish peroxidase for 1h. The blots were observed with Amersham imager 600 (GE Healthcare, Chicago, US), the gray value was analyzed by Image J software.
Real-Time PCR
After OGD 6 h reperfusion for 18 h, the total RNA was extracted from the 6-well plate using the TRIzol® reagent (Invitrogen/Life Technologies, Carlsbad, CA). The cDNA reverse transcription kit (Applied Biosystems, Foster City, USA) was used to generate cDNA. The mRNA was carried out using SYBR Green PCR Mix Kit (CWBIO, Jiangsu, Chain) with 7500 sequence detection system (Applied Biosystems, Foster City, USA). The mRNA level was normalized to β-actin level, old change calculated according to the threshold cycle (Ct) =2(− ΔΔCt) of the treated cells with respect to target amplification. Specific primers which were designed by Nanjing Jiancheng Institute of Biology (shanghai, China) are listed in Table 1.
Table 1 Primer sequences
Genes
|
Primer/Probe sequences(5’to3’)
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β-actin
|
F 5’-GTAAAGACCTCTATGCCAACA-3’
R 5’-GGACTCATCGTACTCCTGCT-3’
|
Ang-1
|
F 5’- CACAGGGACAGCAGGCAAACAG -3’
R 5’- CACAGGCATCGAACCACCAACC -3’
|
VEGF
|
F 5’- CACAGGGACAGCAGGCAAACAG -3’
R 5’- CACAGGCATCGAACCACCAACC -3’
|
PDGFR-β
|
F 5’- CACCTTCTTGCAGCGACACTCC -3’
R 5’- TCCATGTAGCCACCGTCACTCTC -3’
|
Statistical Analysis
Between different groups were performed by one-way ANOVA analysis with Tukey’s multiple comparison test using SPSS 18.0 statistical software. Data are presented as mean±SEM from at least three independent experiments. P<0.05 was considered as significant.