2.1.1 Cell Line
HepG2.2.15 cells were purchased from Guangzhou Genio Biotechnology Co., Ltd.; HepG2 cells for human liver cancer were gifted from the Tumor Research Institute of Xiangya Medical College, Central South University.
2.1.2 Drugs and main reagents
Polyphyllin I, curcumin, sorafenib, Erastin, Ferrostatin-1 (MCE company, batch numbers are: HY-N0047, HY-N0005, HY-10201, HY-15763, HY-100579); DMEM-high Sugar, fetal bovine serum, trypsin (0.25%), penicillin-streptomycin solution (GIBCO, USA, batch numbers: 11965092, A3160802, 25200056, 15240062); cell proliferation/toxicity detection kit (CCK-8 kit ) (USA APExBIO, batch number: K1018); dimethyl sulfoxide DMSO (cell culture grade) (Beijing Soleibao Company, batch number: D8371); FeRhonox-1 iron ion (II) live cell imaging probe (Japan Goryo Company) , Lot number: GC901); lactate dehydrogenase (LDH) detection kit (Beijing Biolab Technology Co., Ltd., lot number: GL2055); JC-1 solution, reactive oxygen detection kit, DAPI staining solution (Shanghai Biyuntian Bio Technology Co., Ltd., batch number: C2005, S0033S, C1005) RIPA lysate (Shanghai Biyuntian Biotechnology Co., Ltd., batch number: P0013C); Phosphate Buffer solution (PBS) (Wuhan Punuosai Life Technology Co., Ltd., Lot number: PB180327); BCA protein quantification kit (Beijing Baierdi Biotechnology Co., Ltd., lot number: DEM109-500T); RRM2 antibody, SRC antibody, ACACA antibody (British Abcam, lot numbers: ab154964, ab47405, ab269273) .
370 series Steri-Cycle high temperature sterilization constant temperature CO2 incubator, 1300 Series A2 ultra-clean workbench, 3001 microplate reader (American Thermo Company); flow cytometer-CyFlow Cube6 (Guangzhou Jiyuan Biological Company); inverted microscope CKX41 (Olympus, Japan); DFM-60D inverted fluorescence microscope (Shanghai Caikang Optical Instrument Co., Ltd.); 1645052 high-current electrophoresis instrument, transfer tank, electrophoresis tank, ChemiDoc XRS gel imaging system (Bole, USA ).
2.2.1 Drug preparation
Polyphyllin I: Dissolve 5 mg of Polyphyllin I in 1ml of DMSO, and add 57ml of complete medium to obtain a mother liquor with a concentration of 100μmol/L when it is completely dissolved.
Curcumin: Dissolve 1 mg of curcumin in 1 ml of DMSO, and add 26 ml of complete medium to obtain a mother liquor with a concentration of 100 μmol/L when it is completely dissolved.
Sorafenib: Dissolve 1mg Sorafenib in 1ml DMSO, and add 106ml complete medium to obtain a mother liquor with a concentration of 100μmol/L when it is completely dissolved.
Erastin: Dissolve 1mg Erastin in 1ml DMSO, and add 17ml complete medium to obtain a mother liquor with a concentration of 100μmol/L when it is completely dissolved.
Store in a refrigerator at -80°C, and dilute with culture medium to the required concentration when used.
2.2.2 Cell culture and passage
HepG2 and HepG2.2.15 cells were both inoculated in complete medium (90% DMEM + 10% FBS + 1% double antibody) and incubated in a constant temperature 37°C 5% CO2 sterile incubator. The cell density reaches 80% for passage, digestion with 0.25% trypsin, complete medium is added to terminate the digestion, the cell suspension is pipetted and transferred to a centrifuge tube for centrifugation (1000 rpm, 3 min), and the supernatant is discarded and re-added to complete Divide the culture medium into new culture flasks to continue the culture. Take the cells whose growth cycle is in the logarithmic growth phase for further experiments.
2.2.3 CCK-8 method to detect cell viability
Take 0.25% trypsin to digest HepG2 and HepG2.2.15 cells, collect the cells after centrifugation for cell counting, and inoculate 100μL cell suspension per well (about 8x103 cells) in a 96-well plate. Incubate for 24 hours, add 10μLCCK-8 detection reagent to each well and continue to incubate for 2 hours. Use a microplate reader to detect the absorbance (A) at 450nm in each well. Graph PadPrism8.0 analyzes and calculates the half-inhibition rate concentration (1C50) of the drug. The experiment was repeated three times. In the next experiment, the concentration of the half inhibitory rate was used as the concentration to intervene the cells. In the experiment, the concentration of Ferrostatin-1 was 1μmol/L.
2.2.4 Cell grouping and administration
Take the HepG2 and HepG.2.15 cells in the logarithmic growth phase, wash 3 times with PBS buffer, digest with 0.25% trypsin, add complete medium to terminate the digestion, and place them in a 15ml centrifuge tube for cell centrifugation (1000rpm, 3min) , Discard the supernatant, pipette with complete medium to form a cell suspension, inoculate 3×105 cells per well in a 6-well plate, and incubate in an incubator for 24 hours; divide the cells into a blank group and PPI group, Cur group, sorafenib group, Erastin group, and Ferrostatin-1 group, they were observed and photographed under an optical microscope after 24 hours of drug intervention.
2.2.4 FeRhoNox-1 staining to observe the intracellular Fe2+ status
The HepG2 and HepG2.2.15 cell suspensions were inoculated in a 6-well plate with cell slides and placed in an incubator for 24 hours to adhere to the wall. Each drug group continued to intervene for 24 hours. Take out FeRhoNox-1 from the refrigerator, put it at room temperature for 30 min, centrifuge for 1 min, configure FeRhoNox-1 with DMSO solution as a storage solution with a final concentration of 1 mM, store at -20°C in the dark, and take PBS solution to store the solution when used Dilute to a final concentration of 5μM and preheat to 37°C. After 24 hours of drug intervention in the cells, aspirate the culture medium, wash with PBS buffer solution 3 times, add pre-heated FeRhoNox-1 staining solution, incubate in an incubator for 50 minutes, discard the staining solution, wash 3 times with PBS, and place Observe and take pictures under a fluorescence microscope.
2.2.5 Detection of LDH release rate
Take well-growing HepG2 and HepG2.2.15 cells in log phase, trypsin to digest the suspension, count the cells, inoculate them in a 12-well plate at a cell density of 5×104 cells/mL, 2 mL per well, each drug group Operate according to the instructions of the LDH kit after 24 hours of intervention. Measure the wavelength at 490 nm in the microplate reader. Cell LDH release rate (%)= (LDH activity in the medium/total LDH activity)×100%. The experiment was repeated three times.
2.2.6 Determination of intracellular ROS levels
The fluorescent probe DCFH-DA is used to detect the ROS level. By detecting the DCF fluorescence intensity in SKOV3 cells, the ROS level is evaluated. The DCF fluorescence intensity is directly proportional to the intracellular ROS level. The pre-treatment method of cells is the same as that under 2.2. After 24 hours of intervention in each drug group, the cells are collected and suspended in diluted DCFH-DA (final concentration of 10 μm·L-1), incubated at 37 ℃ for 20 min, flow cytometry The instrument detects the intensity of DCF fluorescence (the fluorescence spectrum of DCF is very similar to FITC, so use FITC parameter settings to detect DCF). The experiment was repeated three times.
2.2.7 JC-1 staining to observe the mitochondrial membrane potential
Take well-growing HepG2 and HepG2.2.15 cells in the logarithmic phase, trypsin to digest the suspension, count the cells, inoculate them in a 12-well plate at a cell density of 5×104 cells/mL, 2 mL per well, each drug group After intervention for 24h, add the prepared JC-1 solution with a final mass concentration of 10μg/mL, incubate at 37°C for 30min in the dark, wash twice with PBS, add 200μL of new medium, and detect by flow cytometry. The experiment was repeated three times.
2.2.8 Western blot detection of protein expression of RRM2, SRC and ACACA
Each drug group was intervened for 24 hours, and the complete medium group was used as a blank control group to culture for 24 hours, lysed with RIPA lysis buffer (adding protease inhibitors), lysed on ice for 30 minutes, centrifuged to collect the supernatant, which is the total protein solution. The BCA kit detects the concentration of the collected protein. Add 5* reduced protein loading buffer to the protein solution at a ratio of 4:1, denature in a boiling water bath for 15 minutes, load 50μg protein per well, separate the protein by SDS-PAGE gel electrophoresis, transfer to PVDF membrane, and 5% degreasing The milk powder was sealed at room temperature for 2 h. Add RRM2 (1:1000), SRC (1:1000), ACACA (1:1000) according to the antibody instructions, and incubate in a shaker overnight at 4°C. Add TBST, place it on a decoloring shaker and wash the membrane three times, each time for 5 minutes, incubate for 1 hour at room temperature of the secondary antibody, add TBST again and wash the membrane three times, each time for 5 minutes. According to the ECL kit instructions, perform exposure and observe the protein expression, and use image J software to analyze the gray value. The experiment was repeated three times.
2.2.9 Statistical methods
Statistical analysis was performed using GraphPad Prism7 software. The measurement data is expressed as`x±s , and the T test is used for statistical analysis when comparing the results of the two groups, and the test level is α = 0.05.