Main chemicals and reagents
RPMI-1640 medium (C11885500BT) and 100 U/mL penicillin and 100 U/mL strepto-Mycin were acquired from Gibco (Grand Island, NY, USA).Vitamin E (V8011) and MTT (M8180) were purchased from Solarbio (Beijin, China). ROS、SOD and CAT kits were acquired from Beyotime Biotechnology (Shanghai, China). Mouse Melanin ELISA kit was purchased from mlbio (Shanghai, China). Cytoplasmic-Nuclear protein extraction kit was acquired from Boster (Wuhan, China). Bicinchoninic acid (BCA) protein quantification kit and PI3K inhibitor LY294002 were obtained from Beyotime Biotechnology (Shanghai, China). FITC Annexin V Apoptosis Detection Kit I (556547) was acquired from BD Biosciences (San Jose, USA). PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (RR047A) and TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (RR820A) were purchased from Takara (Osaka City, Osaka Prefecture, Japan).
Phospho-Akt (p-Akt, ser473, 4060T), Akt (4691T) and Nrf2 (12721S) were purchased from Cell Signaling Technology (Danvers, MA, USA). HO-1 (ab68477) and Lamin B1 (ab229025) were acquired from Abcam (Cambridge, UK). β‑actin (AC026) and FITC goat anti-rabbit IgG (H + L) (AS011) were obtained from Abclonal (Wuhan, China). Goat anti-rabbit IgG-HRP (abs20002) was purchased from absin (Shanghai, China).
Preparation of herbs extracts
Qinglongyi (batch number: 030490102) was purchased from Sanyitang Traditional Chinese Medicine Decoction Pieces Co., Ltd. (Anhui, China), and Buguzhi (batch number: 200501) was obtained from Zhonghe Traditional Chinese Medicine Decoction Pieces Co., Ltd. (Jinan, China). The voucher specimens (Qinglongyi: NO. 20201101; Buguzhi: NO. 20201102) were deposited in the Chinese medicine herbarium of Shandong University of Traditional Chinese Medicine (Jinan, China).
Q and B were separately smashed to a powder. Q (30 g) was extracted with 75% ethanol at reflux for 2 h two times. The ethanol extract was concentrated with a rotary evaporator at 40 ℃ to produce a brown gum. The extraction method of B and QB (mass ratio 1:1, 1:2 and 2:1 ) was the same as that of Q [8, 19–21]. The extract yields (w/w, dried extract/crude herb) of Q, B, QB (mass ratio 1:1, 1:2 and 2:1 ) were 10.00%, 12.87%, 14.42%, 9.06%, 11.43%, respectively. The extract was suspended in RPMI-1640 medium to prepare the drug solution of 2mg/ml. Then, the drug solution was filtered by 0.22 µm microporous membrane to remove bacteria, sub-packaged and stored at -20 ℃. Before the experiment, the drug stock solutions were diluted to the desired concentration separately.
Quality control
To ensure the quality of herbs extracts, psoralen, isopsoralen and total flavonoids (calculated by quercetin), which were identified as a chemical marker for quality monitoring, were analyzed by high performance liquid chromatography (HPLC) and ultraviolet-visible spectroscopy (UV-vis), according to the Chinese Pharmacopoeia (2020 version) and Standard of traditional Chinese Medicine in Liaoning Province (Volume 2). The Standard of traditional Chinese Medicine in Liaoning Province (Volume 2) stipulates that the total flavonoids in terms of quercetin (C15H10O7) and dried products should not be less than 0.7% in Qinglongyi, and the Chinese Pharmacopoeia (2020 version) stipulates that the total amount of psoralen (C11H6O3) and isopsoralen (C11H6O3) should not be less than 0.70% in terms of dried products in Buguzhi.
Cell culture
B16F10 mouse melanoma cells (CL-0319) were purchased from Procell (Wuhan, China), and cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin. The cultivation conditions were set at 37 ℃, 95% air and 5% CO2. When cell growth density reached 80–90%, gently removed the original medium. Then added 1 mL trypsin (0.25%) to the culture dish, digested the cells for 1 minute, waited for the cells to become round, added 1mL serum medium to stop digestion. Next, the cells were transferred with a pipette to prepare the cell suspension, and then inhaled into two 1.5 mL centrifuge tubes respectively, and centrifuged at 1000 rpm for 5 minutes. After discarding the supernatant, 1mL culture medium was added to resuspensionand, the cells were finally transferred to two new culture dishes for continuous culture.
Establishment of experimental models
B16F10 cells (1x104 cells/well) were seeded into 96‑well plates and were treated with H2O2 solutions at final concentrations of 50, 100, 200, 400 and 600 µM for different times (2 h, 4 h, 6 h, 8 h). MTT was used to detect cell survival rate. An appropriate concentration and time of H2O2 was determined to construct a model of oxidative stress injury in B16F10 cells. In this work, 200 µM H2O2 for 4 h was chosen to establish the oxidative stress injury model.
Selection of herbs extracts action concentrations
The experimental procedures for screening drug action concentrations were as follows: Firstly, the drug stock solution was diluted to 0.02, 0.04, 0.08, 0.16, 0.32 and 0.64 mg/mL in fresh RPMI1640 medium. Secondly, B16F10 cells (1x104 cells/well) were inoculated into 96-well plates overnight, and then treated with different concentrations of Q, B, QB (0.02, 0.04, 0.08, 0.16, 0.32 and 0.64 mg/mL) for 24 h. Finally, MTT was used to determine cell survival rate, and then selected the drug concentration which had no significant effect on cell survival rate for follow-up experiments. In this study, 0.02mg/mL, 0.04mg/mL and 0.08mg/mL were selected as action concentrations of Q, B and QB for follow-up experiments.
Evaluation of antioxidation properties of herbs extracts in vitro
Cell grouping
Cells were assigned to 17 groups: normal control group, H2O2 group, Q, B, and QB groups. Cells in normal control group were cultured in RPMI-1640 medium. To establish the oxidative injury model, the other 16 groups of cells were treated with a final concentration of 200 µM H2O2 solution for 4h. To explore whether Q, B, and QB pretreatment alleviates oxidative injury, B16F10 cells in the Q, B, and QB groups were treated with Q, B, and QB (0.02mg/mL, 0.04mg/mL and 0.08mg/mL) for 24 h before H2O2.
Detection of cell survival rate
The MTT method was used to detect the cell survival rate. The cells in logarithmic growth phase were routinely digested, centrifuged and counted. The number of cells was adjusted to 1×10− 5 cells / mL and seeded in 96-well plates with 100 µL per well. Then the culture plates were transferred to 37 ℃ and 5% CO2 incubator overnight, and added corresponding drugs according to the grouping, zeroing group only added culture medium. During the period of incubation, 20 µL MTT (5 mg/mL) was added to each well and the cells were further incubated for 4 h under the culture conditions. Then, each well added 150 µL DMSO, and the optical density (OD) at 490 nm was measured using a microplate reader (Bio Tek Cytation 5). Calculating the cell survival rate, cell survival rate (%) = (Experimental group OD-zeroing group OD)/ (normal control group OD-zeroing group OD)× 100%.
ROS detection
The flow cytometry was used to detect ROS level. The cells in logarithmic growth phase were routinely digested, centrifuged and counted. The number of cells was adjusted to 1×10− 5 cells/mL and seeded in 6-well plates with 2 mL per well, then the culture plates were transferred to 37 ℃ and 5% CO2 incubator overnight. When the cell growth density reached 60%ཞ70% and added corresponding drugs according to the grouping for 24 h. The culture medium was discarded and the cells were incubated with fresh medium containing 10 mmol/L DCFH-DA for 20 min at 37 ℃, then washed with PBS (pH 7.4) three times. The fluorescence of DCF was measured using a flow cytometer (CytoFLFXS).
Superoxide dismutase (SOD) and catalase (CAT) assays
Collecting the processed cells and after lysis of cells, the protein content was detected by BCA kit, and the activities of superoxide dismutase (SOD) and catalase (CAT) were determined by superoxide SOD and CAT kits, according to the manufacturer’s instructions.
Detection of melanin
Collecting the processed cells and after lysis of cells, the melanin content was measured with a ELISA kit, in accordance with the manufacturer’s instructions.
Mechanism studies of QB based on PI3K/Akt/Nrf2 signaling pathway
Cell grouping
The cells were divided into 6 groups as follows: normal control group, H2O2 group, QB group, LY294002 (PI3K inhibitor) group, QB + LY294002 group and VE group (Vitamin E was chosen as the positive control drug). Cells in normal control group were cultured in RPMI-1640 medium. To establish the oxidative injury model, the other 5 groups of cells were treated with a final concentration of 200 µM H2O2 for 4h. To explore whether QB pretreatment alleviates oxidative injury, cells in QB group and QB + LY294002 group were treated with QB (0.08mg/mL) for 24 h before H2O2. To investigate whether QB can protect melanocytes from oxidative stress injury through PI3K/Akt/Nrf2 signal pathway, cells in LY294002 group and QB + LY294002 group were preincubated with LY294002 (20 µmol/L) for 2 h before QB.
Detection of ROS
The detection steps were the same as ROS detection.
Apoptosis assay
The cells in logarithmic growth phase were routinely digested, centrifuged and counted. The number of cells was adjusted to 1×10− 5 cells/mL and seeded in 6-well plates with 2 mL per well, then the culture plates were transferred to 37 ℃ and 5% CO2 incubator overnight. When the cell growth density reached 60%ཞ70% and added corresponding drugs according to the grouping for 24 h. Then, the cells were digested and collected, and washed with cold PBS two times. 300 µL binding buffer was added to resuspended cells and 5 µL FITC Annexin V and PI were added respectively. After reaction for 30 min at room temperature and under dark conditions, the apoptosis rate of cells was detected by flow cytometry.
Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR)
The cells that had been processed were collected and the total RNA was isolated using TRIzol® reagent. The RT reaction was performed using PrimeScript™ RT reagent with gDNA Eraser kit, and quantitative real-time PCR reaction was performed with TB Green® Premix ExTaq™ II kit following the manufacturer's instructions. RT-qPCR analyses were performed with the LightCycler® 480 system. β-actin was used as an internal reference, the relative mRNA expression levels of Akt, Nrf2, and HO-1 were quantified using the 2
‑ΔΔCt method. The Oligonucleotide sequences of specific primers for Akt, Nrf2, and HO-1 as follows:
Gene
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Forward primer sequences (5’−3’)
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Reverse primer sequences (5’−3’)
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Akt
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5’-CATCGTGTGGCAGGATGTGTA−3’
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5’-ACCTGGTGTCAGTCTCAGAGGTG−3’
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Nrf2
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5’-GCACATCCAGACAGACACCAG−3’
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5’-TATCCAGGGCAAGCGACTCAT−3’
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HO-1
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5’-CTGGAGATGACACCTGAGGTCAA−3’
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5’-CTGACGAAGTGACGCCATCTG−3’
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β-actin
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5’-CATCCGTAAAGACCTCTATGCCAAC−3’
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5’-ATGGAGCCACCGATCCACA−3’
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Western blot
The cells that had been processed were collected and lysed with RIPA lysis buffer. Nuclear protein was extracted using the Cytoplasmic-Nuclear protein extraction kit, and protein concentration was determined using a BCA kit. The cells were mixed with 5X loading buffer to denature in boiling water. Each protein sample (30 µg) was subjected to 10% SDS‑PAGE, then the separated proteins were transferred into PVDF membranes. After 2 h of blocking with 5% skimmed milk at room temperature, the membranes were incubated at 4 ˚C overnight with primary antibodies against phospho-Akt (1:2000), Akt (1:1000), Nrf2 (1:1000), HO-1 (1:30000), Lamin B1 (1:1000), and β‑actin (1:50000). β‑actin and Lamin B1 were used as controls. The next day, the membranes were incubated with Goat anti-rabbit IgG-HRP (1:5000) for 1.5 h at room temperature. The bound antibodies were visualized using an enhanced chemiluminescence reagent under the multicolor fluorescence imaging system. Then, the gray values of each blot was measured by ImageJ software.
Immunofluorescence staining
The B16F10 cells were seeded on glass slides in a 6‑well plates, and treated with the indicated drugs. The cells that had been processed were collected and washed 3 times with PBS and fixed with 4% formaldehyde solution for 30 min, then permeated with 0.3% Triton X‑100 for 10 min, and blocked with 5% goat serum for 30 min at 37 ˚C. The cells were then incubated with primary anti‑Nrf2 antibody Nrf2 (1:200) overnight at 4 ˚C. Next day, the cells were washed 3 times with PBS and incubated with FITC goat anti-rabbit IgG (H + L)(1:200) for 1 h at 37 ˚C under dark condition, then the cell nuclei were counterstained with DAPI for 5 min. Finally, the nuclear translocation of Nrf2 was observed using fluorescence microscopy.
Statistical analysis
All data were expressed as mean ± standard deviation (SD). SPSS 22.0 software was used for statistical analysis. Two-way ANOVA followed by post hoc Tukey’s test was performed to evaluate the antioxidation properties of Q, B, QB, and one‑way ANOVA with Tukey’s post hoc test was used to analyze other groups differences. P < 0.05 was considered to be statistically significant.