Application of dihydromyricetin (DHM) in preparation of anti-hepatoma medicines

[0097] Example 1 Effect of DHM on proliferation of liver cancer cell lines

[0098] 1. In vitro culture of liver cancer cell lines

[0099] 1. Cell source

[0100] Human liver cancer cell lines HepG2, Huh7, SK-HEP-1 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; MHcc97L, MHcc97H, QSG7701, QGY7703 were purchased from Shanghai Baili Biotechnology Co., Ltd.; QGY7701 was purchased from Shanghai Maternal and Child Health Hospital; human normal liver The cell line L-02 was purchased from Science Cell; the mouse liver cancer cell line Hepal-6 was donated by Shanghai Jiaotong University.

[0101] 2. Cell Culture and Subculture

[0102] HepG2, QSG7701, L-02 and Hepal-6 used 1640 medium (Gibco); Huh7, SK-HEP-1, MHcc97L, MHcc97H, QGY7701 and QGY7703 used DMEM medium (Gibco). 10% fetal bovine serum (fetal bovine serum, FBS, Gibco) was added to the medium during cell culture. The above cell lines were all stored at 37°C, 5% CO 2 Culture in an incubator, and when the cells grow to 80-90% of the bottom of the culture dish, they are digested with 0.25% trypsin-EDTA and passaged.

[0103] 2. Effect of DHM on cell proliferation

[0104] Main test materials: Dihydromyricin (Dihydromyrictin, DHM, Sigma) dissolved in dimethyl sulfoxide (DMSO), prepared into a 50mM concentrated solution, stored at -20°C, and diluted to 5, 10, 25, 50, 100 and 150 μM, ready to use; thiazolyl blue (MTT, Sigma) dissolved in PBS, the final concentration is 5 mg/mL, stored at 4 °C, and used within one week after preparation; trypsin (Gibco); PBS; 6-well plate (Nunc) .

[0105] Test method: the cells were digested, centrifuged and resuspended in the corresponding culture medium, with 1×10 4 The density per well was planted in a 96-well plate at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, remove the original culture medium, add freshly prepared culture medium containing 5, 10, 25, 50, 100, and 150 μM DHM respectively to continue the culture, and stop the culture at 12h, 24h, 36h, 48h and 72h of culture respectively , add 20 μL of MTT to each well, and then return to 37 ° C, 5% CO 2 After culturing in the incubator for 4 hours, carefully remove the culture medium containing MTT, add 150 μL of DMSO to each well, carefully shake and mix well, and then detect the absorbance value of each well at OD570nm on a microplate reader. Three replicate groups were set up for each concentration.

[0106] test results:

[0107] DHM has a dose-dependent effect on HepG2 cells. After 36 hours of DHM treatment, the inhibition rate of DHM on HepG2 cells can exceed 10%. After 72 hours of treatment, the inhibition rate of 150μM group can reach 20% ( figure 2 );

[0108] DHM has obvious time-dependent and dose-dependent inhibitory effects on QGY7701, QSG7701 and Hepal-6 cells: after 24 hours of DHM treatment of QGY7701 cells, the inhibition rate of 150 μM group can reach more than 40%, and after 48 hours of treatment of 150 μM group The inhibition rate can exceed 50% ( image 3 ); QSG7701 cells showed dose-dependent and time-dependent cell fragmentation after 24 hours of DHM treatment ( Figure 13 ), cell debris makes the MTT assay show a false positive result of promoting cell growth, ( Figure 4) observed by DAPI staining, it was found that all the cells died in the high-concentration test group with severe fragmentation, that is, the treatment time exceeded 36 hours, and when the drug treatment concentration exceeded 50 μM, the inhibition rate could reach 100%; The effect of DHM on Hepal-6 cells does not seem to be significantly dose-dependent, but the inhibition rate of the 150μM group was significantly higher than that of the other groups. After 48 hours of drug treatment, the inhibition rate reached more than 40% (P<0.05). At 72h, the false positive result of DHM promoting cell proliferation was also shown due to cell death and fragmentation ( Figure 5 ).

[0109] The effect of DHM on Huh7, MHcc97H and MHcc97L cells had no obvious dose-dependent phenomenon, but with the prolongation of treatment time, the inhibition rate of cell proliferation showed an upward trend: when DHM was treated for 12 hours, there was almost no proliferation inhibition in each concentration group, but After 24 hours of treatment, the inhibition rate increased gradually over time, but some test groups of Huh7 and MHcc97L cells showed false positive results of strong proliferation due to severe cell fragmentation. Among them, when MHcc97H was treated with DHM for 72 hours, the inhibition rate of 150 μM group could reach 50%; MHcc97L could have an inhibition rate of more than 30% when treated with DHM for 72 hours; after Huh7 was treated with DHM for 48 hours, the inhibition rate of the test group with a treatment concentration greater than 50 μM was all acceptable. More than 40% ( Image 6 ,7,8).

[0110] DHM had no obvious proliferation inhibitory effect on QGY7703, SK-HEP-1 and L-02, HL7702 ( Figure 9 , 10, 11, 12).

[0111] Example 2 Induction of DHM on apoptosis of liver cancer cells

[0112] 1. Cell culture and passage

[0113] In this experiment, the human liver cancer cell line QGY7701 was purchased from Shanghai Maternal and Child Health Hospital. The culture medium was DMEM culture solution supplemented with 10% FBS. 2 Culture in an incubator, and when the cells grow to 80-90% of the bottom of the culture dish, they are digested with 0.25% trypsin-EDTA and passaged.

[0114] 2. The effect of DHM on the apoptosis of liver cancer cells

[0115] Main test materials: DHM, dissolved in dimethyl sulfoxide (DMSO), prepared as a 50mM concentrated solution, stored at -20°C, diluted to 25, 50, 100 and 150μM when used, ready to use; FITC Annexin Ⅴ Apoptosis Detection Kit Ⅰ (BD Pharmingen); Accutase-Enzyme Cell Detachment Medium (eBioscience); PBS; 60mm petri dish, special sample tube for flow cytometry.

[0116] Test method: Digest the cells into a cell suspension, spread them on a 60mm culture dish at a suitable density, and store them at 37°C, 5% CO 2 Incubate in the incubator for about 24 hours. When the cells grow to about 50% of the bottom of the culture dish, remove the original culture medium and add freshly prepared culture medium containing 25, 50, 100, and 150 μM DHM to continue the culture. After culturing for 24 hours, carefully collect the supernatant and digest the cells with Accutase-Enzyme Cell Detachment Medium, mix the cell suspension with the supernatant and centrifuge, wash the collected cells once with PBS, centrifuge and resuspend in 1×Binding Buffer, adjust Cell density is 1 x 10 6 individual/mL. Pipette 100 μL of cell suspension into the sample tube, add 5 μL Annexin Ⅴ-FITC and 5 μL Propidium Iodide (PI), mix well, and react at room temperature in the dark for 15 minutes, then add 400 μL 1×Binding Buffer to each tube, and use flow cytometry to detect Cell apoptosis.

[0117] Experimental results: The ability of DHM to induce the apoptosis of liver cancer cells was dose-dependent. When the drug was added for 24 hours, the apoptosis rate of cells in each group increased with the increase of drug concentration. When the drug concentration reached 100 μM, the cell apoptosis rate could reach 26.03±4.69 ( Figure 14 ).

[0118] Example 3 Western Blotting Detection of Cell Apoptosis-Related Factor Expression

[0119] 1. Cell culture and passage

[0120] The human liver cancer cell line HepG2 was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; QSG7701 and QGY7703 were purchased from Shanghai Baili Biotechnology Co., Ltd.; the mouse liver cancer cell line Hepal-6 was donated from Shanghai Jiao Tong University. HepG2, QSG7701 and Hepal-6 used 1640 medium (Gibco), and QGY7703 used DMEM medium (Gibco). 10% fetal bovine serum (fetal bovine serum, FBS, Gibco) was added to the medium during cell culture. The above cell lines were all stored at 37°C, 5% CO 2 Culture in an incubator, and when the cells grow to 80-90% of the bottom of the culture dish, they are digested with 0.25% trypsin-EDTA and passaged.

[0121] 2. Expression detection of apoptosis-related factors

[0122] Main test materials: DHM, dissolved in dimethyl sulfoxide (DMSO), prepared into a 50mM concentrated solution, stored at -20°C, diluted to 50 and 100μM when used, ready to use; RIPA lysate (Beyotime); PMSF ; BCA protein concentration assay kit (Beyotime), rabbit anti-human primary antibody (P53,PARP, Caspase3, Caspase8, Caspase9); mouse anti-human primary antibody (β-Tubulin); horseradish peroxidase-conjugated goat Anti-rabbit secondary antibody and goat anti-mouse secondary antibody; acrylamide, SDS, ammonium sulfite, Tris-Base, PVDF membrane, ECL luminescence solution (GE Healthcare).

[0123] Test method: Digest the cells into a cell suspension, spread them on a 60mm culture dish at a suitable density, and store them at 37°C, 5% CO 2 Incubate in the incubator for about 24 hours. When the cells grow to about 60% of the bottom of the culture dish, remove the original culture medium and add freshly prepared culture medium containing 50 μM and 100 μM DHM respectively to continue the culture. Use the culture medium without DHM for the control group. The culture was terminated 24 h after the addition of DHM, the culture medium was removed and washed twice with PBS, and the cells were lysed by adding RIPA lysate containing 1% PMSF. Centrifuge the lysate at 4°C, 12000×g/min for 10 min, collect the supernatant, measure the protein concentration, load the sample with equal amount of protein, separate the protein by 10% SDS-PAGE electrophoresis, transfer to PVDF membrane, and block with 5% skimmed milk powder After washing with TBST (containing 1% Tween20), primary antibodies (P53, PARP, Caspase3, Caspase8, Caspase9) were incubated overnight at 4°C. Wash 5 times with TBST, 5 min each time, incubate with secondary antibody at room temperature for 1 h, wash 5 times with TBST, 5 min each time, use ECL luminescence solution for chemiluminescence and expose in a dark room, and analyze the expression trend of related proteins.

[0124] test results:

[0125] When HepG2 cells were treated with DHM for 24 hours, with the increase of DHM concentration, the main bands and cut bands of P53, Caspase8 and Caspase9 all showed an increasing trend; the expression of PARP showed a decreasing trend; the expression of Caspase3 had no obvious change ( Figure 16 -A).

[0126] When QSG7701 cells were treated with DHM for 24 hours, with the increase of DHM concentration, the cut fragments of P53, Caspase3 and Caspase8, the main band and cut fragments of PARP all showed an increasing trend; the expression of Caspase9 had no significant change ( Figure 16 -B).

[0127] When Hepal-6 cells were treated with DHM for 24 hours, with the increase of DHM concentration, P53, Caspase8 and Caspase9 showed an increasing trend; PARP main band and shear band were weakened with the increase of concentration; the expression of Caspase3 had no significant change ( Figure 16 -C).

[0128] When QGY7701 cells were treated with DHM for 24 hours, with the increase of DHM concentration, P53, Caspase3, Caspase9, and PARP all showed an increasing trend ( Figure 16 -D).