Identification method and application of traditional Chinese medicine monomer for reversing Venetoclax drug resistance

Abstract

The invention discloses an identification method and application of a traditional Chinese medicine monomer for reversing Venetoclax drug resistance. The traditional Chinese medicine monomer is a monomer component usnic acid in a usnea alcohol extract. The identification method of the usnea alcohol extract comprises the following steps: reversing the drug resistance of the leukemia BCL-2 inhibitor by using the usnea alcohol extract; the invention discloses a method for reversing drug resistance of a leukemia BCL-2 inhibitor by using usnic acid. It can be identified that the usnea diffracta alcohol extract can effectively reverse the drug resistance of AML to a BCL-2 inhibitor; and the prepared traditional Chinese medicine effective monomer usnic acid for reversing the drug resistance of the leukemia cell BCL-2 inhibitor can be used for preparing a sensitizer for the drug resistance of the leukemia cell BCL-2 inhibitor ABT-199.

Description

Technical Field

[0001] The present invention relates to the technical field of identification of traditional Chinese medicine monomers, and in particular to an identification method and application of a traditional Chinese medicine monomer capable of reversing Venetoclax resistance. Background Art

[0002] BCL-2 inhibitors are the cornerstone core drugs in the era of targeted therapy for AML. Their 30% primary resistance and high induced resistance are key issues that currently plague clinical practice. How to further explore new resistance mechanisms and find suitable target / signal axis regulatory drugs is an important link in further improving the clinical efficacy of AML patients, especially elderly and refractory patients. The application of traditional Chinese medicine in the treatment of leukemia has a long history. Cloud fog grass (Usnea) is a common clinical drug. Combined with the previous research basis, we found that its main ingredient "usnic acid" has the effect of overcoming the resistance of leukemia cells to the BCL-2 inhibitor ABT-199 by mediating a comprehensive stress response. It has low in vitro toxicity dose requirements and good in vivo safety, and has good clinical transformation and application potential. The purpose of the present invention is to provide an identification method and use of usnic acid. Summary of the invention

[0003] The purpose of the present invention is to provide a method for identifying a Chinese medicine monomer that can reverse Venetoclax resistance and its application, so as to solve the above-mentioned problems.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] A method for identifying a Chinese medicine monomer that reverses Venetoclax resistance and its application, wherein the Chinese medicine monomer is usnic acid, a monomer component in usnea alcohol extract;

[0006] The identification method of the usnea ethanol extract comprises:

[0007] Method for reversing leukemia BCL-2 inhibitor resistance using Usnea alcohol extract;

[0008] Method of using usnic acid to reverse leukemia BCL-2 inhibitor resistance.

[0009] As a further solution of the present invention: the method of using usnea alcohol extract to reverse the resistance of leukemia BCL-2 inhibitors comprises:

[0010] S1: preparing Usnea aqueous extract;

[0011] S2: preparing Usnea alcohol extract;

[0012] S3: CCK8 assay to detect the proliferation inhibition of ABT-199 resistant leukemia cell line;

[0013] S4: Test of the effect of Usnea extract combined with ABT-199 against ABT-199 resistance in mice;

[0014] S5: Detection of usnic acid content in usnea extract.

[0015] As a further solution of the present invention: the method of using usnic acid to reverse the resistance of leukemia BCL-2 inhibitors comprises:

[0016] Q1: Cell activity detection;

[0017] Q2: Flow cytometry was used to detect the apoptosis level of MOLM-13R and THP-1R cells;

[0018] Q3: Flow cytometry was used to detect the cycle distribution of MOLM-13R and THP-1R cells;

[0019] Q4: Flow cytometry was used to detect mitochondrial membrane potential of MOLM-13R and THP-1R cells;

[0020] Q5: Experiment on the effect of usnic acid combined with ABT-199 against ABT-199 resistance in mice.

[0021] As a further solution of the present invention: In Q5, the experiment of the effect of usnic acid combined with ABT-199 on anti-ABT-199 resistance in mice includes:

[0022] Q51: Grouping and dosing;

[0023] Q52: MOLM-13R-luc cell inoculation;

[0024] Q53: In vivo imaging;

[0025] Q54: Dissection and tissue sampling.

[0026] As a further solution of the present invention: the method of using usnic acid to reverse the resistance of leukemia BCL-2 inhibitors comprises:

[0027] P1: Effects of usnic acid combined with ABT-199 on cell proliferation and apoptosis under ISRIB intervention;

[0028] P2: qRT-PCR detection of the mRNA expression of ISR pathway ATF4, CHOP, and NOXA genes;

[0029] P3: Under the intervention of ISRIB, qRT-PCR was used to detect the effect of usnic acid combined with ABT-199 on the mRNA expression of ATF4, CHOP, and NOXA genes in the ISR pathway.

[0030] As a further solution of the present invention: in P1, under the intervention of ISRIB, the effects of usnic acid combined with ABT-199 on cell proliferation and apoptosis include:

[0031] P11: cell proliferation;

[0032] P12: Cell apoptosis detection.

[0033] As a further solution of the present invention: in P2, qRT-PCR detection of the mRNA expression of ISR pathway ATF4, CHOP, and NOXA genes includes:

[0034] P21: Experimental grouping;

[0035] P21: Quantitative detection.

[0036] As a further solution of the present invention: an application of a Chinese medicine monomer for reversing Venetoclax resistance, wherein the effective Chinese medicine monomer for reversing leukemia cell BCL-2 inhibitor resistance is used to prepare a sensitizer for leukemia cell BCL-2 inhibitor ABT-199 resistance.

[0037] Beneficial effects of the present invention:

[0038] The present invention uses a method for reversing the drug resistance of leukemia BCL-2 inhibitors by using usnea alcohol extract and a method for reversing the drug resistance of leukemia BCL-2 inhibitors by using usnic acid, thereby identifying that usnea alcohol extract and its effective monomer usnic acid can effectively reverse the drug resistance of AML to BCL-2 inhibitors.

[0039] Usnic acid is an effective component in usnea alcohol extract that can reverse the drug resistance of acute myeloid leukemia ABT-199 in vitro and in vivo. The content of usnic acid in usnea alcohol extract detected by HPLC is 82.43 mg / g. The mechanism of usnic acid reversing ABT-199 resistance is to activate the comprehensive stress response, thereby promoting cell apoptosis; and the prepared effective Chinese medicine monomer that reverses the drug resistance of leukemia cells to BCL-2 inhibitors can be used to prepare a sensitizer for the drug resistance of leukemia cells to BCL-2 inhibitors ABT-199. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described below in conjunction with the accompanying drawings.

[0041] Figure 1 The figure shows the inhibitory effect of Usnea extract on the cell proliferation of BCL-2 inhibitor-resistant AML cells; (A, B) CCK8 assay for the cell viability of MOLM-13R (A) and THP-1R (B) cells after treatment with Usnea water extract and alcohol extract for 48 hours;

[0042] Figure 2The figure shows that the usnea alcohol extract combined with ABT-199 inhibits the proliferation of MOLM-13R and THP-1R cells; (A, B) CCK8 detects the cell viability of MOLM-13R (A) and THP-1R (B) cells after 48 hours of treatment with usnea water extract, ABT-199 alone or in combination. (C, D) CCK8 detects the cell viability of MOLM-13R (C) and THP-1R (D) cells after 48 hours of treatment with usnea alcohol extract, ABT-199 alone or in combination;

[0043] Figure 3 The figure shows that Usnea alcohol extract can cooperate with ABT-199 to inhibit the growth of MOLM-13R tumor-bearing nude mice subcutaneous transplanted tumors; (A) During the experimental observation period, the tumor growth curves were drawn by measuring the changes in the volume of nude mouse transplanted tumors in the tumor-bearing control group (Vehicle group), ABT-199 group, Usnea water extract group (UAE group), ABT-199 combined with Usnea water extract group (UAE group), Usnea alcohol extract group (UEE group), and ABT-199 combined with Usnea alcohol extract group (ABT-199+UEE group); (B) The weight changes of nude mice in each group during the experiment; (C) After dissection, the photos of the transplanted tumors in each group of nude mice and the weight of the transplanted tumors in each group of nude mice; (D) The heart, liver, spleen, lung, and kidney indexes of nude mice in each group;

[0044] Figure 4 The following are the contents of usnic acid in the water extract and alcohol extract of usnea: (A) Standard curve of usnic acid; (BC) HPLC chromatograms of usnic acid in the reference substance (B), water extract of usnea (C), and alcohol extract of usnea (D). The number 1 in the figure is usnic acid;

[0045] Figure 5 The figure shows the proliferation inhibition effect of ABT-199 sensitized by non-toxic doses of usnic acid on leukemia resistant strains; CCK8 detected the cell viability of MOLM-13R (A) and THP-1R (B) cells after 48 hours of treatment with usnic acid, ABT-199 alone or in combination;

[0046] Figure 6 The figure shows the effect of non-toxic doses of usnic acid on the apoptosis-inducing effect of ABT-199 on drug-resistant leukemia strains; flow cytometry was used to detect the apoptosis of MOLM-13R (A) and THP-1R (B) cells after 48 hours of treatment with usnic acid, ABT-199 alone or in combination;

[0047] Figure 7 The figure shows the cell cycle arrest of drug-resistant cell lines promoted by usnic acid combined with ABT-199; flow cytometry was used to detect the cell cycle changes of MOLM-13R (A) and THP-1R (B) cells after 48 hours of usnic acid, ABT-199 alone or in combination;

[0048] Figure 8 This is a graph showing that usnic acid combined with ABT-199 exacerbates the decrease in mitochondrial membrane potential of drug-resistant cell lines; flow cytometry was used to detect the effects of usnic acid, ABT-199 alone and in combination on the mitochondrial membrane potential of MOLM-13R (A) and THP-1R (B) cells after 24 hours of action;

[0049] Fig. 9 Figure 1 shows that usnic acid combined with ABT-199 reduces the tumor burden of MOLM-13R tumor-bearing mice. (A, B) Bioluminescence imaging technology was used to detect the tumor burden of MOLM-13R tumor-bearing mice on the 8th day (A) and the 16th day (B); (C, D) Flow cytometry was used to detect the expression ratio of bone marrow CD45+ cells in MOLM-13R tumor-bearing mice on the 21st day; (E) The curve of the change of mouse body weight in the control group, ABT-199 group, usnic acid group and combined group with the number of days of administration; The effect of usnic acid combined with ABT-199 on the liver and kidney function of MOLM-13R tumor-bearing mice; Statistical graph of the biochemical indicators of trough ALT (F), AST (G), BUN (H) and CRE (I) in the serum of NSG mice in each group;

[0050] Fig.10 This is a diagram showing that ISRIB can restore the inhibitory effect of usnic acid combined with ABT-199 on the proliferation of drug-resistant cells; CCK8 experiments evaluate the effect of ISRIB on the cell survival rate of the usnic acid and ABT-199 combined administration group in MOLM-13R (A) and THP-1R (B) cells;

[0051] Fig.11 This is a diagram showing that ISRIB can restore the apoptosis of usnic acid combined with ABT-199 in drug-resistant cells; flow cytometry was used to detect the effect of ISRIB on apoptosis in MOLM-13R (A) and THP-1R (B) cells in the combination group of usnic acid and ABT-199;

[0052] Fig.12 The figure shows the regulatory effect of usnic acid combined with ABT-199 on the ISR signaling pathway; qRT-PCR was used to detect the effects of usnic acid, ABT-199 alone or in combination on the mRNA transcription levels of ATF4, CHOP, and NOXA in MOLM-13R (A) and THP-1R (B) cells at 24 hours;

[0053] Fig.13 ISRIB can restore the up-regulated genes in the ISR pathway caused by usnic acid combined with ABT-199; qRT-PCR detected the effect of ISRIB on the mRNA transcription levels of ATF4, CHOP, and NOXA in MOLM-13R (A) and THP-1R (B) cells treated with usnic acid and ABT-199. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The present invention provides a method for identifying a Chinese medicine monomer that can reverse Venetoclax resistance, wherein the Chinese medicine monomer is usnic acid, a monomer component in usnea alcohol extract;

[0056] The identification method of the usnea ethanol extract includes the following embodiments:

[0057] Embodiment 1

[0058] The in vivo and in vitro efficacy of Usnea alcohol extract in reversing leukemia BCL-2 inhibitor resistance is as follows:

[0059] J1: Preparation of Usnea aqueous extract:

[0060] Take 80g of Usnea medicinal material, decoct it twice with water, add 15 times the volume of water for the first time, decoct for 20 minutes, then add 10 times the volume of water for the second time, decoct for 10 minutes, filter the medicinal solution with gauze after each decoction, combine the two medicinal solutions, and get the Usnea water extract. Put the obtained Usnea water extract into a 60℃ rotary evaporator and evaporate it under reduced pressure to get the Usnea water extract concentrate, i.e., the extract. Spread the extract evenly into a glass dish, cover with plastic wrap, put it into a -80℃ refrigerator, and store it frozen. After the extract in the glass dish is frozen and solidified, dry it in a vacuum freeze dryer to get 4.57g of Usnea water extract freeze-dried powder, with an extraction rate of 5.71%. Use distilled water to prepare a solution of the required concentration and store it in a 4℃ refrigerator;

[0061] J2: Preparation of Usnea alcohol extract:

[0062] Take 80g of Usnea medicinal material, heat and reflux extract with 15 times the amount of 80% ethanol at 75℃ for 4 hours, filter the liquid with gauze, and use a rotary evaporator to reduce pressure, concentrate and dry to obtain Usnea alcohol extract. After freezing the extract in a -80℃ refrigerator, put it in a freeze dryer to dry, and obtain 8.82g of Usnea alcohol extract freeze-dried powder, with an extraction rate of 11.01%. Prepare the freeze-dried powder with distilled water to make a solution of the required concentration and store it in a 4℃ refrigerator;

[0063] J3: CCK8 assay to detect the proliferation inhibition of ABT-199 resistant leukemia cell lines:

[0064] The MOLM-13R and THP-1R cell lines were intervened with 1-256 μg / mL of Usnea water extract and alcohol extract respectively for 48 hours, and CCK8 was used to detect the cell viability. The results showed that with the increase of the dosage concentration, both the Usnea water extract and alcohol extract had different degrees of inhibitory effects on MOLM-13R and THP-1R cell lines. The in vitro proliferation inhibitory effect of Usnea alcohol extract on MOLM-13R and THP-1R cells was much higher than that of the Usnea water extract group, and its IC 50 5.68μg / mL and 7.04μg / mL respectively. Figure 1 As shown;

[0065] Based on the above results, we selected two concentrations of Usnea water extract (4, 6 μg / mL) and ethanol extract (4, 6 μg / mL) and combined them with different concentrations of ABT-199 to act on drug-resistant cells for 48 hours. We then used CCK8 to measure the cell proliferation and calculated the IC 50 To compare the differences in cell survival rates in each group. The results showed that as the concentration of ABT-199 gradually increased from 0.05μM to 16μM, the cell viability of ABT-199-resistant strains gradually decreased. Compared with the ABT-199-treated group, there was no significant change in the cell viability of the ABT-199 combined with Usnea water extract group (4, 6μg / mL), while the cell viability of the resistant cell lines treated with ABT-199 combined with Usnea alcohol extract (6μg / mL) was significantly decreased, and the reversal multiples of resistance in MOLM-13R and THP-1R cells were 4.1 and 4.3, respectively. Figure 2 As shown;

[0066] J4: Test of the effect of Usnea extract combined with ABT-199 on anti-ABT-199 resistance in mice: Mice were divided into groups for drug administration: Vehicle group: 0.9% saline, 0.1mL / 20g; ABT-199 group: 80mg / kg (10% ethanol + 60% Phosal50PG + 30% PEG400 dissolved); UAE group: 2g / kg (dissolved in saline); UAE+ABT-199 group; UEE group: 2g / kg (dissolved in saline); UEE+ABT-199 group. One day before inoculation of cells, the mice were divided into groups according to the above grouping scheme, and the nude mice were gavaged at the same time and place every day, and the administration was continued until the mice were killed.

[0067] Subcutaneous transplantation in mice: Balb / c male mice, 4-6 weeks old, with an initial body weight of 18 to 22 grams. MOLM-13R cells in good growth state were collected, and the cell concentration was adjusted after counting. MOLM-13R cells (5×10 6The cell suspension was subcutaneously inoculated into the left armpit of Balb / c mice using a 1mL sterile syringe on a clean bench. On the second day after inoculation, observe whether there are hard, white nodules, which indicates that the model is successful. The tumor volume was measured every 2 days, and the calculation formula was: Tumor volume (mm3) = (long diameter (mm) × short diameter (mm) 2 ) / 2. 24 hours after the last administration, the nude mice in each group were killed by spinal dislocation, and the tumors and organs of the heart, liver, spleen, lung and kidney were removed and weighed;

[0068] The study found that compared with the model group, the Usnea alcohol extract combined with ABT-199 could significantly inhibit the growth of transplanted tumors in mice and reduce the weight of in vitro tumors. In addition, there were no significant differences in the body weight and organ indexes of mice in each group, indicating that the drug administration had no potential toxicity to mice. Figure 3 As shown;

[0069] J5: Detection of usnic acid content in usnea extract:

[0070] (1) Preparation of reference solution: Accurately weigh an appropriate amount of usnic acid, add methanol to prepare a 212 μg / mL solution, and sonicate to obtain the reference solution.

[0071] (2) Preparation of test solution: Accurately weigh 100 mg of Usnea water extract freeze-dried powder and 10 mg of alcohol extract freeze-dried powder, add 10 mL of 50% methanol solution, ultrasonicate for 15 minutes, and filter with filter paper. Take the filtrate, centrifuge at 12,000 rpm for 15 minutes, and filter with a 0.22 μm filter membrane to obtain the prepared test solution.

[0072] (3) Chromatographic conditions: An Elite column (4.6 mm × 250 mm, column number: E2120840) was used, the mobile phase was 0.1% formic acid (A)-acetonitrile (B), gradient elution, 10-90% B (0-15 min), 10% B (15-25 min). The detection wavelength was 254 nm, the injection volume of the reference and test samples was 10 μL, and the column temperature was 25°C.

[0073] (4) Drawing of standard curve: Accurately pipette the reference solution (212 μg / mL) and dilute it to the concentrations of 106, 53, 21.2, 10.6, 5.3, and 2.12 μg / mL in sequence, with an injection volume of 10 μL; the injection volume for the concentration of 424 μg / mL is 20 μL.

[0074] The regression equation was y=18884x-106957, R=0.9997, and the linearity was good in the range of 2.12μg / mL-424μg / mL. After calculation, the content of usnea alcohol extract was 82.43mg / g, RSD=0.78%, and the content of usnic acid in usnea water extract was 0.49mg / g, RSD=0.48%, indicating good repeatability. Figure 4 shown.

[0075] Embodiment 2

[0076] In vitro and in vivo efficacy study of usnic acid in reversing leukemia BCL-2 inhibitor resistance;

[0077] K1: Cell viability assay

[0078] After 48 hours of treatment with a series of concentration gradients of ABT-199 alone and three concentrations of usnic acid in combination with different concentrations of ABT-199, CCK8 was used to detect the reversal of the resistance of MOLM-13R and THP-1R cell lines to ABT-199 by usnic acid.

[0079] The results showed that the reversal fold of usnic acid (1.5 μM) on MOLM-13R cells was 9.7, and the reversal fold of usnic acid (4 μM) on THP-1R cells was 9.5. Figure 5 As shown;

[0080] K2: Flow cytometry was used to detect the apoptosis level of MOLM-13R and THP-1R cells;

[0081] K21: Experimental Grouping

[0082] MOLM-13R cell line: usnic acid monotherapy group (U group - 1.5 μM), ABT-199 monotherapy group (A group - 1, 2, 4 μM), and ABT-199 combination group (U-1.5 μM combined with A-1, 2, 4 μM groups, respectively);

[0083] THP-1R cell line: usnic acid monotherapy group (U group - 4 μM), ABT-199 monotherapy group (A group - 0.25, 0.5, 1 μM), and the combination of the two drugs group (U-4 μM combined with A-0.25, 0.5, 1 μM groups, respectively);

[0084] K22: Cell plating

[0085] MOLM-13R and THP-1R cells in the logarithmic growth phase were collected by centrifugation and the cell suspension was adjusted to a final concentration of 5 × 10 5 / mL, inoculated into 6-well plates (4mL / well). Different drugs were added to the 6-well plates inoculated with cells and intervened for 48 hours;

[0086] K23: Flow cytometry

[0087] After 48 hours, observe the morphology of cells using an inverted microscope and collect cells from each group. Wash the cells once with pre-cooled PBS by centrifugation, discard the supernatant after centrifugation, add 500 μL of Binding buffer to each tube to resuspend the cells, then add 5 μL of Annexin V-FITC and 10 μL of PI dye solution, incubate at room temperature in the dark for 5 minutes, and then detect on the machine.

[0088] The results showed that compared with the ABT-199 monotherapy group, usnic acid combined with ABT-199 could significantly induce apoptosis of MOLM-13R and TNP-1R cells. Figure 6 As shown;

[0089] K3: Flow cytometry to detect the cycle distribution of MOLM-13R and THP-1R cells

[0090] K31: Experimental Grouping

[0091] MOLM-13R cell line: usnic acid monotherapy group (U group - 1.5 μM), ABT-199 monotherapy group (A group - 1, 2 μM), and ABT-199 combination group (U-1.5 μM combined with A-1, 2 μM groups, respectively);

[0092] THP-1R cell line: usnic acid monotherapy group (U group - 4μM), ABT-199 monotherapy group (A group - 0.5, 1μM), and the two-drug combination group (U-4μM combined with A-0.5, 1μM groups, respectively).

[0093] K32: Cell plating

[0094] MOLM-13R and THP-1R cells in the logarithmic growth phase were taken and the final concentration was 5×10 5 / mL was inoculated into a 6-well plate. Different drugs were added to the 6-well plate inoculated with cells and intervened for 24 hours;

[0095] K33: Flow cytometry

[0096] Collect the cells after administration, wash once with PBS, centrifuge, and discard the supernatant. Add 1 ml DNA Staining solution and 10 μl Permeabilization solution, vortex for 5-10 seconds to mix. Incubate at room temperature in the dark for 30 minutes, select the lowest loading speed, and detect on the flow cytometer;

[0097] The results showed that usnic acid combined with ABT-199 could arrest cells in the G1 phase and induce G1 phase arrest of the cell cycle. Figure 7 As shown;

[0098] K4: Flow cytometry to detect mitochondrial membrane potential in MOLM-13R and THP-1R cells

[0099] K41: Experimental Grouping

[0100] MOLM-13R cell line: usnic acid monotherapy group (U group - 1.5 μM), ABT-199 monotherapy group (A group - 1, 2 μM), and ABT-199 combination group (U-1.5 μM combined with A-1, 2 μM groups, respectively);

[0101] THP-1R cell line: usnic acid monotherapy group (U group - 4 μM), ABT-199 monotherapy group (A group - 0.5, 1 μM), and the combination of the two drugs group (U-4 μM combined with A-0.5, 1 μM group, respectively);

[0102] K42: Cell plating

[0103] MOLM-13R and THP-1R cells in the logarithmic growth phase were taken and the final concentration was 5×10 5 / mL was inoculated into a 6-well plate. Different drugs were added to the 6-well plate inoculated with cells and intervened for 24 hours;

[0104] K43: Flow cytometry

[0105] After the drug intervention, the cells were collected by centrifugation, resuspended in 0.5 mL of culture medium, added to 0.5 mL of JC-10 staining working solution, mixed by inversion several times, and incubated in a cell culture incubator at 37°C for 20 minutes. After the incubation at 37°C, the cells were centrifuged at 4°C (600 g, 4 minutes), the supernatant was discarded, and then washed twice with JC-10 staining (1×) buffer by centrifugation. The cell pellet was resuspended in 200 μL of JC-10 staining buffer and tested on the machine. The data were analyzed by Modify5 software;

[0106] The results showed that in MOLM-13R and THP-1R cells, compared with the control group, the administration of usnic acid and ABT-199 alone at the used concentrations had no effect on the mitochondrial membrane potential of the cells; compared with ABT-199 alone, usnic acid combined with ABT-19 could significantly reduce the mitochondrial membrane potential and cause mitochondrial depolarization, such as Figure 8 shown.

[0107] K5: Effect of usnic acid combined with ABT-199 against ABT-199 resistance in mice

[0108] K51: Animal grouping and drug administration

[0109] Animal grouping: NSG mice were randomly divided into 4 groups: tumor-bearing control group (Vehicle group), ABT-199 group, usnic acid group, and the two-drug combination group, with 6 mice in each group. There was no significant difference in the body weight of mice among the groups.

[0110] Administration: The drug dose was set as ABT-199: 80 mg / kg (dissolved in 10% ethanol + 60% Phosal50PG + 30% PEG400); usnic acid group: 20 mg / kg (dissolved in 5% DMSO + 40% PEG300 + 5% Tween-80 + 50% Saline); Vehicle group: mixed solvent without usnic acid (5% DMSO + 40% PEG300 + 5% Tween-80 + 50% Saline), 0.1 mL / 20 g. On the second day after cell inoculation, the drugs were administered according to the above grouping scheme, and NSG mice were gavaged at the same time and place every two days, and the drugs were continuously administered until the mice were killed.

[0111] K52:MOLM-13R-luc cell seeding

[0112] Select MOLM-13R cells transfected with luciferase in logarithmic growth phase, collect the cell suspension in a 50 mL centrifuge tube, centrifuge and discard the supernatant, resuspend the cells in medium without serum and double antibody, and adjust the cell concentration to 1×10 7 / mL. Select female NSG mice aged 3-5 weeks and inoculate cells in a biosafety cabinet. Pay attention to aseptic operation during the operation. Use a 1mL sterile syringe to draw 200μL of cell suspension, empty the bubbles in the syringe, and after injection through the tail vein, press gently with a cotton swab for a few seconds and put it back in the cage.

[0113] K53: Intravital Imaging of Mice

[0114] Live imaging was performed on the 8th and 16th days after tumor inoculation. The mice were removed from the cage, their abdominal cavity was gently exposed, and the abdominal skin was disinfected with 75% alcohol. The syringe needle with the liquid absorbed was inserted into the abdominal cavity at a 45° angle to the skin. At this time, there was a sense of empty space. The needle was fixed, the needle tip was kept still, and the needle was withdrawn. If there was no reflux, D-luciferin at a concentration of 15 mg / mL (0.2 mL / 20 g body weight) could be slowly injected. Seven minutes after the injection, the mice were placed in an anesthesia box, anesthetized with isoflurane, and placed on an imaging darkbox platform for imaging analysis.

[0115] K54:NSG mouse dissection and tissue collection

[0116] On the 21st day after cell transplantation, NSG mice were killed and dissected. A portion of the blood was used for blood index detection, and the remaining blood was centrifuged and separated to obtain serum. In addition, the five major organs of the heart, liver, spleen, lung, and kidney were taken and weighed.

[0117] Mouse liver function (AST, ALT, CRE, BUN) detection kits were purchased from Nanjing Jiancheng and operated according to the instructions.

[0118] Mouse bone marrow tumor load detection: Take the femur of the mouse, cut off both ends of the bone, use a 1mL syringe to absorb PBS, flush the bone marrow cavity from one end of the bone, and wash each end three times with PBS. Collect the flushed solution into a centrifuge tube, centrifuge at 600g for 5 minutes, discard the supernatant, add 2mL of red blood cell lysis solution to each tube to remove red blood cells. After washing once with PBS, resuspend in 100μL of CD45 antibody diluted in PBS, incubate at room temperature away from light for 20 minutes, and wash twice with 1mL PBS to remove unbound excess antibodies. Finally, resuspend the cells with 300μL PBS, keep at 4℃ away from light, and detect on the machine.

[0119] The results showed that the combination of usnic acid and ABT-199 can effectively reduce the AML tumor burden in the bone marrow of NSG mice. In addition, we also detected the changes in the levels of ALT, AST, BUN and CRE in the serum of NSG mice to further evaluate the possible hepato-renal toxicity of ABT-199 alone, usnic acid alone and the combination of the two drugs. Fig. 9 As shown, there was no significant difference in the liver and kidney function indicators among the mice groups.

[0120] Embodiment 3

[0121] Experimental methods for the mechanism of usnic acid reversing leukemia BCL-2 inhibitor resistance:

[0122] L1: Effects of usnic acid combined with ABT-199 on cell proliferation and apoptosis under ISRIB intervention

[0123] L11: Cell proliferation

[0124] To determine whether activation of the ISR is necessary for usnic acid to overcome ABT-199 resistance, we used ISRIB, a chemical inhibitor of the ISR, to block the downstream effects of eIF2α phosphorylation.

[0125] The MOLM-13R cell line was set up with Ctrl group, usnic acid (1.5μM) single drug group, ABT-199 (1μM) single drug group, ISRIB (2.5, 5, 10μM) single drug groups, usnic acid (1.5μM) + ABT-199 (1μM) group, usnic acid (1.5μM) + ABT-199 (1μM) group combined with ISRIB (2.5, 5, 10μM) at various concentrations.

[0126] The THP-1R cell line was divided into Ctrl group, usnic acid (4μM) single drug group, ABT-199 (0.5μM) single drug group, ISRIB (1, 2, 4μM) single drug groups, usnic acid (4μM) + ABT-199 (0.5μM) group, usnic acid (4μM) + ABT-199 (0.5μM) group combined with ISRIB (1, 2, 4μM) at different concentrations, respectively.

[0127] After 48 hours of drug treatment in each group, CCK8 was used to detect cell proliferation. The CCK8 results showed that ISRIB intervention could restore cell proliferation caused by usnic acid combined with ABT-199. Fig.10 shown.

[0128] L12: Cell apoptosis detection

[0129] The MOLM-13R cell line was set up with Ctrl group, usnic acid (1.5μM) single-drug group, ABT-199 (1μM) single-drug group, ISRIB (10μM) single-drug group, usnic acid (1.5μM) + ABT-199 (1μM) group, and usnic acid (1.5μM) + ABT-199 (1μM) + ISRIB (10μM) combination group.

[0130] The THP-1R cell line was set up with Ctrl group, usnic acid (4μM) single-drug group, ABT-199 (0.5μM) single-drug group, ISRIB (1μM) single-drug group, usnic acid (4μM) + ABT-199 (0.5μM) group, and usnic acid (4μM) + ABT-199 (0.5μM) + ISRIB (1μM) combination group.

[0131] After 48 hours of drug administration, the cells in each group were tested for cell apoptosis using flow cytometer.

[0132] The experimental results showed that the apoptosis rates of MOLM-13R and THP-1R cells induced by the combined administration of usnic acid and ABT-199 were 52.22±3.51% and 58.10±1.21%, respectively. However, under the intervention of ISRIB, the apoptosis rates of MOLM-13R and THP-1R cells induced by the combined administration were reduced to 20.07±5.05% and 26.77±2.04%, respectively. Fig.11 shown.

[0133] L2: qRT-PCR detection of mRNA expression of ISR pathway ATF4, CHOP, and NOXA genes

[0134] L21: Experimental Grouping

[0135] ① Effect of usnic acid combined with ABT-199 on the expression level of cell-related mRNA

[0136] MOLM-13R cell line: usnic acid monotherapy group (U group - 1.5 μM), ABT-199 monotherapy group (A group - 1, 2 μM), and ABT-199 combination group (U-1.5 μM combined with A-1, 2 μM groups, respectively);

[0137] THP-1R cell line: usnic acid monotherapy group (U group - 4μM), ABT-199 monotherapy group (A group - 0.5, 1μM), and the two-drug combination group (U-4μM combined with A-0.5, 1μM groups, respectively).

[0138] ② Effect of integrated stress pathway inhibitor (ISRIB) on the combination of usnic acid and ABT-199

[0139] MOLM-13R cell line: usnic acid monotherapy group (U group - 1.5 μM), ABT-199 monotherapy group (A group - 1 μM), the two-drug combination group (UA - 1 μM group), and each group plus ISR chemical inhibitor (ISRIB - 10 μM), a total of eight groups, incubated together for 24 hours;

[0140] THP-1R cell line: usnic acid monotherapy group (U group - 4μM), ABT-199 monotherapy group (A group - 0.5μM), the two-drug combination group (UA - 0.5μM group), and each group plus ISR chemical inhibitor (ISRIB - 1μM), a total of eight groups, incubated together for 24 hours.

[0141] L22: Quantitative testing

[0142] RNA was extracted using the Trizol-chloroform method, and the concentration and purity of the extracted RNA were detected by Nanodrop. Subsequently, the template RNA was used to prepare a reverse transcription system mixture to obtain cDNA. The SYBR qRT-PCR reaction system was prepared for testing, and β-actin was used as an internal reference to calculate the relative expression of the gene.

[0143] The results showed that usnic acid combined with ABT-199 could significantly increase the mRNA transcription levels of ATF4, CHOP, and NOXA in cells. Fig.11After ISRIB intervention, the increase in the mRNA transcription levels of ATF4, CHOP, and NOXA induced by the combined administration of usnic acid and ABT-199 was significantly inhibited, as shown in Fig.12 and 13 shown.

[0144] Embodiment 4

[0145] The invention discloses an application of an effective Chinese medicine monomer for reversing the drug resistance of leukemia cells to a BCL-2 inhibitor. The effective Chinese medicine monomer for reversing the drug resistance of leukemia cells to a BCL-2 inhibitor ABT-199 is used to prepare a sensitizer for drug resistance of leukemia cells to a BCL-2 inhibitor ABT-199.

[0146] Through the above examples, it is confirmed that usnic acid is an effective component in usnea alcohol extract that can reverse the drug resistance of acute myeloid leukemia ABT-199 in vitro and in vivo. The present invention finds that the mechanism of usnic acid reversing ABT-199 resistance is to activate the comprehensive stress response, thereby promoting cell apoptosis.

[0147] The above is a detailed description of an embodiment of the present invention, but the content is only a preferred embodiment of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for identifying a Chinese medicine monomer that reverses Venetoclax resistance. It is characterized in that The Chinese medicine monomer is usnic acid, a monomer component in usnea alcohol extract; The identification method of the usnea alcohol extract comprises: Method for reversing leukemia BCL-2 inhibitor resistance using Usnea alcohol extract; Method of using usnic acid to reverse leukemia BCL-2 inhibitor resistance.

2. A method for identifying a Chinese medicine monomer that reverses Venetoclax resistance according to claim 1, It is characterized in that The method of using usnea alcohol extract to reverse the drug resistance of leukemia BCL-2 inhibitors comprises: S1: preparing Usnea aqueous extract; S2: preparing Usnea alcohol extract; S3: CCK8 assay to detect the proliferation inhibition of ABT-199 resistant leukemia cell line; S4: Test of the effect of Usnea extract combined with ABT-199 against ABT-199 resistance in mice; S5: Detection of usnic acid content in usnea extract.

3. The method for identifying a Chinese medicine monomer that reverses Venetoclax resistance according to claim 1, It is characterized in that The method of using usnic acid to reverse the drug resistance of leukemia BCL-2 inhibitors comprises: Q1: Cell activity detection; Q2: Flow cytometry was used to detect the apoptosis level of MOLM-13R and THP-1R cells; Q3: Flow cytometry was used to detect the cycle distribution of MOLM-13R and THP-1R cells; Q4: Flow cytometry was used to detect mitochondrial membrane potential of MOLM-13R and THP-1R cells; Q5: Experiment on the effect of usnic acid combined with ABT-199 against ABT-199 resistance in mice.

4. The method for identifying a Chinese medicine monomer that reverses Venetoclax resistance according to claim 1, It is characterized in that In Q5, the experiment of the effect of usnic acid combined with ABT-199 on anti-ABT-199 resistance in mice includes: Q51: Grouping and dosing; Q52: MOLM-13R-luc cell inoculation; Q53: In vivo imaging; Q54: Dissection and tissue sampling.

5. The method for identifying a Chinese medicine monomer that reverses Venetoclax resistance according to claim 1, It is characterized in that The method of using usnic acid to reverse the drug resistance of leukemia BCL-2 inhibitors comprises: P1: Effects of usnic acid combined with ABT-199 on cell proliferation and apoptosis under ISRIB intervention; P2: qRT-PCR detection of the mRNA expression of ISR pathway ATF4, CHOP, and NOXA genes; P3: Under the intervention of ISRIB, qRT-PCR was used to detect the effect of usnic acid combined with ABT-199 on the mRNA expression of ATF4, CHOP, and NOXA genes in the ISR pathway.

6. The method for identifying a Chinese medicine monomer that reverses Venetoclax resistance according to claim 1, It is characterized in that In P1, under the intervention of ISRIB, the effects of usnic acid combined with ABT-199 on cell proliferation and apoptosis include: P11: cell proliferation; P12: Cell apoptosis detection.

7. The method for identifying a Chinese medicine monomer that reverses Venetoclax resistance according to claim 1, It is characterized in that In P2, qRT-PCR was used to detect the mRNA expression of ISR pathway ATF4, CHOP, and NOXA genes, including: P21: Experimental grouping; P21: Quantitative detection.

8. Application of a Chinese medicine monomer for reversing Venetoclax resistance, It is characterized in that The effective Chinese medicine monomer for reversing the drug resistance of leukemia cells to the BCL-2 inhibitor is used for preparing a sensitizer for the drug resistance of leukemia cells to the BCL-2 inhibitor ABT-199.

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