2.1 Preparation of Radix Actinidiae chinensis extract
1000 g RAC were weighed, dissolved in 5000 mL of ethanol, refluxed for 10 h, extracted for 3 times, and the extracts were combined, then evaporated, dried, gradient elution, and the solvent was removed, and the powder was collected and vacuum-dried for 24 h. 100 mL of distilled water was added to 100 g of dry powder, and then the stock solution was diluted to 0 mg/ml, 3.16 mg/ml, 5.62 mg/ml, 10 mg/ml, 15.84 mg/ml, 31.6 mg/ml, 56.23 mg/ml, 100 mg/ml, followed by sterile filtration with a 0.22 µm microporous membrane, divided into packages, and stored in a 4°C refrigerator for later use.
2.2 Cell culture
786-O and A498 cell lines were cultured in RPMI 1640 medium containing 10% calf serum, penicillin, and streptomycin at 37°C and 5% CO2. When the cell growth density reached 80%, the old medium was discarded, digested with 0.25% trypsin, resuspended by adding fresh medium.
2.3 CCK8 assay
The logarithmic phase cell suspension was taken, then dosed and grouped according to ‘0, 3.16, 5.62, 10, 15.84, 31.6, 56.23, 100 mg/ml RAC’, ‘0, 1, 5, 10, 15, 30, 50 mg/ml RAC’, and ‘0, 4, 8, 12, 24, 48, 72 h’. It was inoculated in a 96-well plate, and after culturing for a corresponding time, CCK-8 solution was added and incubated. The absorbance at 450nm was measured, and the cell viability was calculated. Five replicate wells were assayed in parallel.
2.4 Colony assay
The cells in the logarithmic phase were digested, seeded at 500–1000 cells/well in a plate containing 30% FBS in complete medium, and cultured at 37°C, 5% CO2. The medium was changed every 3 days and the cell status was observed. Photographs were taken when individual cloned cells were large enough to be observed. Then it was stained with 0.1% crystal violet.
2.5 Migration and invasion assay
Matrigel was diluted with a serum-free DMEM high-glycemic culture medium, uniformly coated in the Transwell chamber, incubated overnight. After 24 h, the Transwell chamber was put in a 24-well plate, and cultured. Matrigel and bottom cells of the upper chamber were wiped off, fixed with paraformaldehyde, washed with PBS, stained with crystal violet, then photographed and counted the number of cells migrated and invaded. It was repeated three times.
2.6 Apoptosis assay
The cells in logarithmic phase were seeded in 6-well plates. The cells were collected after 24 h of treatment in the ‘0, 1, 5, 10 mg/ml RAC’, ‘0 mg/ml RAC, 10 mg/ml RAC, 3-MA, 10 mg/ml RAC + 3-MA’. Then washed twice with PBS. 500 µL of buffer was added, the supernatant was discarded by centrifugation, 100 µL of buffer was added and mixed, 5 µL of Annexin V-FITC and 10 µL of PI were added respectively, and then thoroughly mixed. The reaction was performed. Finally, 400 µL of buffer was added, and the apoptosis was detected within 1 h. It was repeated three times.
2.7 Cell cycle assay
The cell suspensions in the logarithmic phase were inoculated into plates, and the culture plates were pre-cultured for 24 h. Then the experiment was carried out according to four doses of ‘0, 1, 5 and 10 mg/ml RAC’. After 24 h of administration, the cells were detected the cell cycle by flow cytometry.
2.8 Transmission electron microscope assay
The cells were fixed in glutaraldehyde solution, and washed 4 times with 0.1 M pH7.0 PBS. It was then fixed with osmic acid and rinsed again. Cells were dehydrated with gradient concentrations of ethanol solution, then treated with 100% ethanol, and then treated with pure acetone. Subsequent gradient permeabilization was performed by using an embedding medium, and then the cells were placed in a 0.5 ml dry tube (pre-filled with about 300 ul of embedding medium), and the polymerizer was heated at 70°C overnight. The sections were then sliced by using an ultrathin microtome, stained with 100 ul of uranyl acetate 50% ethanol saturated solution for 20 min, rinsed with 100 ul of lead citrate in double distilled water and stained for 15 min, and finally photographed
2.9 AOPI assay
The AOPI staining was used to detect the effect of the combination of RAC and 3-MA on autophagy in RCC. The RCC in the logarithmic phase were placed on the coverslip, and the ‘0 mg/ml RAC, 10 mg/ml RAC, 3- MA and the 10 mg/ml RAC + 3-MA’ were divided into groups and cultured in a 37 ℃, 5% CO2 for 24 h. It was rinsed 3 times through PBS, dripped with freshly prepared 1 mg/L AOPI staining solution, incubated at 37°C, aspirated the AOPI staining solution, and rinsed 3 times with PBS. The slides were mounted with 50% glycerol and placed under an inverted fluorescence microscope to observe the acidic autophagic vesicles.
2.10 Western blot
First, the total protein in RCC was collected and the BCA kit (Solarbio, pc0020) was used to detect the total protein. The SDS-PAGE electrophoresis and transfer membrane were performed. The PVDF membrane was blocked with 5% skimmed milk powder, then it was put into the primary antibody Smad2 (AF6449, 1:1000, Affinity), p-Smad2 (AF3450, 1:1000, Affinity), Smad3 (AF6362, 1:1000, Affinity), p-Smad3 (AF3362, 1:1000, Affinity), TGF-β1 (AF1027, 1:1000, Affinity), and α-SMA (AF1032, 1:1000, Affinity) were incubated overnight. Then the membrane was rinsed and the secondary antibody GAPDH (AF7021, 1:15000, Affinity) was incubated. The ECL was used to detect protein bands, and the protein gray value was calculated by Image J.
2.11 Statistical analysis
SPSS 20.0 was used for data analysis. One-way ANOAY analysis of variance is used to measure data across multiple groups, and SNK test is used for comparison between groups. While Kruskal-Wallis H test is used for variance. All data were expressed as mean ± standard deviation. P < 0.05 suggested that the difference was statistically significant.