CN111643488A - Application of bakuchiol in improving anti-tumor effect of chemotherapeutic drugs - Google Patents

Abstract

The invention discloses the application of bakuchiol in improving the antitumor effect of chemotherapeutic drugs. Bakuchiol increases the antitumor effect of chemotherapeutic drugs, while reducing the immune damage of the chemotherapeutic drugs, and the synergistic effect of bakuchiol and chemotherapeutics promotes tumor growth. The related expression of apoptosis factors in cells can promote the apoptosis of tumor cells, and bakuchiol can reduce the damage to leukocytes caused by chemotherapy drugs. It provides a basis for clinical trials and a new way for the development of new drugs.

Description

Application of bakuchiol in improving the antitumor effect of chemotherapeutic drugs

technical field

The invention relates to the technical field of medicine, in particular to the application of bakuchiol self-microemulsion in improving the antitumor effect of cyclophosphamide.

Background technique

Malignant tumor is one of the three major causes of human death, and it is a major health problem faced by all countries in the world. In recent years, anticancer drugs have developed rapidly, and finding new drugs or drug combinations for malignant tumors has become a research goal.

Chemotherapeutic drugs can act on different aspects of tumor cell growth and reproduction, inhibit or kill tumor cells, and are one of the main methods for treating tumors at present. At present, the commonly used chemotherapeutic drugs in clinic include cyclophosphamide, cisplatin, mitomycin, vincristine and so on. However, anti-tumor chemotherapy drugs have different degrees of toxic and side effects, and some serious toxic and side effects are the direct reasons for limiting the dose or use of drugs. While killing tumor cells, they also kill cells in normal tissues, especially the vigorously growing blood and lymphoid tissue cells in the human body. These cells and tissues are an important immune defense system of the human body and destroy the human immune system. , cancer may develop rapidly, with serious consequences.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of application of bakuchiol in improving the antitumor effect of chemotherapeutic drugs in view of the technical problem that chemotherapeutic drugs have myelosuppression and other toxic and side effects in the prior art.

The technical scheme adopted for realizing the purpose of the present invention is:

Application of bakuchiol in improving the antitumor effect of chemotherapeutic drugs.

In the above technical solution, bakuchiol increases the anti-tumor effect of the chemotherapeutic drug, and simultaneously reduces the immune damage of the chemotherapeutic drug.

In the above technical solution, bakuchiol and chemotherapeutic drugs act synergistically to promote the expression of apoptosis factors in tumor cells, and promote tumor cell apoptosis, and at the same time, bakuchiol reduces the damage to leukocytes caused by chemotherapeutic drugs.

In the above technical scheme, bakuchiol and chemotherapeutic drugs act synergistically to up-regulate the expression of Caspsse3, Caspase8, JNK and Bax, and down-regulate the expression of Bcl-2 in tumor tissue.

In the above technical solution, the chemotherapeutic drug is cyclophosphamide, cisplatin, mitomycin or vincristine.

In the above technical scheme, the mass ratio of the bakuchiol to the chemotherapeutic drug is (75-240): (10-40).

In the above technical scheme, the bakuchiol adopts a bakuchiol self-microemulsion, and the mass ratio of the bakuchiol self-microemulsion to the chemotherapeutic drug is (250-800): (10-40).

In the above technical scheme, the bakuchiol self-microemulsion is prepared by the following method: selecting bakuchiol as the oil phase, adding an emulsifier and a co-emulsifier, and placing it in a water bath at 37±2°C at 100±5rpm Stir to combine.

In the above technical solution, the emulsifier is cremophor RH 40 or Cremophor EL 35, and the co-emulsifier is Labrasol.

In the above technical solution, the mass ratio of bakuchiol, emulsifier and co-emulsifier is (1-4):(3-5):(2-4).

Compared with the prior art, the beneficial effects of the present invention are:

1. Bakuchiol is a natural medicinal ingredient extracted from the seeds of Bakuchiol, a natural medicine. It does not have the serious side effects of chemotherapy drugs, and its dosage form is improved. Compared with the original Bakuchiol, it has a relatively high oral bioavailability. Utilization. The experiment confirmed that the combination of cyclophosphamide and bakuchiol self-microemulsion has strong anti-tumor effect.

2. This cell in vitro experiment uses tumor cell S180, bakuchiol as a therapeutic drug, and its cell viability is measured by cck8 to verify the anti-tumor effect of bakuchiol. And through the S180 cell line to induce tumor formation in mice, and then grouped and administered (CTX, CTX+BAK self-microemulsion, BAK self-microemulsion) treatment, the number of white blood cells, tumor tissue HE staining and Tunel tissue staining were detected by blood routine. Western blotting assay showed that the self-microemulsion formulation of bakuchiol could increase the antitumor effect of cyclophosphamide. The invention provides a basis for clinical experiments and a new way for the research and development of new drugs by studying bakuchiol self-microemulsion dosage forms to increase the anti-tumor effect of cyclophosphamide.

3. The combined use of bakuchiol self-microemulsion and cyclophosphamide can reduce the damage of CTX to the immune system, and the effect is very significant.

4. The combined use of bakuchiol self-microemulsion and cyclophosphamide enhanced the expression of apoptotic factors in mouse tumor cells and promoted the apoptosis of tumor cells.

Description of drawings

Figure 1 shows the growth of tumors in M group, CTX group and CM group (measured on the 14th day after modeling).

Figure 2 shows the tumor weights of the M group, the CTX group and the CM group (measured on the 14th day after modeling).

Figure 3 shows the changes of tumor diameter in the M group, the CTX group, and the CM group.

Figure 4 shows the number of leukocytes in the M group, the CTX group, and the CM group.

Figure 5 is the pathological result of HE staining (measured on the 14th day after modeling).

Figure 6 shows the results of TUNEL staining (measured on day 14 after modeling).

Figure 7 is the expression of proteins in mouse tumor tissue (measured on day 14 after modeling).

Figure 8 is a graph of cell viability when NVB, NVB and different concentrations of BAK are used together.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

The reagents and manufacturers used in the following examples:

Bakuchiol self-microemulsion is prepared by the following method:

Select bakuchiol (bakuchiol) as oil phase, cremophor RH 40 as emulsifier, Labrasol as co-emulsifier, in proportion (quality) as bakuchiol:RH40:labrasol:=3:4:3, put under 37 ℃ water bath condition Stir and mix at 100 rpm.

Bakuchiol was purchased from Chengdu Purfield Biotechnology Co., Ltd.

Cyclophosphamide was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

Polyoxyethylene (40) hydrogenated castor oil (26126575L0), BASF, Germany

Caprylic acid macrogol glyceride (126715), French company Gafase

Sesame sesame oil, Shandong Luhua Group Co., Ltd.

Normal saline (190610011), Shandong Kelun Pharmaceutical Co., Ltd.

DMEM high glucose medium (06-1055-57-1ACS), BI company

Fetal Bovine Serum (04-001-1ACS), BI

Penicillin/streptomycin (MA0110), Dalian Meilun Biotechnology Co., Ltd.

Pancreatin (03-050-1A), BI

Rabbit anti-mouse monoclonal antibody Bax(2772), Cell Signaling Technology

Rabbit anti-mouse monoclonal antibody Bcl-2 (3498), Cell Signaling Technology

Rabbit anti-mouse monoclonal antibody Caspase-3 (9662), ABCAM

Rabbit anti-mouse monoclonal antibody Caspase-8 (4790), Cell Signaling Technology

Rabbit anti-mouse monoclonal antibody JNK(9252), Cell Signaling Technology

Rabbit anti-mouse monoclonal antibody β-actin (4970), Cell Signaling Technology

Phosphatase inhibitor cocktail (14E15B83), Tianjin Bright Technology Co., Ltd.

RIPA tissue lysate (R0020), Beijing Soleibao Technology Co., Ltd.

Anti-Rabbit Detection Module (DM001), ProteinSimple, USA

Standard Pack 1 (PS-ST01-8), ProteinSimple, USA

PBS buffer (G0002), Google Bio

Proteinase K, Roche

Membrane rupture fluid (G1204), Google Bio

Xylene (10023418), Sinopharm Chemical Reagent Co., Ltd.

Anhydrous ethanol (100092683), Sinopharm Chemical Reagent Co., Ltd.

HE Stain Set (G1003), Servicebio

Tunel kit (11684817910), Roche

Test animals and tumor strains used in the following examples:

Healthy male Kunming mice, 3-4 weeks old, weighing 17-19 g, 70 mice, SPF grade (license number: SCXK Jing-2016-0011), were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The above animals were kept in the Institute of Radiation Medicine, Chinese Academy of Medical Sciences. The room temperature was 20 to 25°C, and the relative humidity was 40 to 60%. Standard feeding methods were adopted.

S180 mouse ascites tumor cells were purchased from Wuhan Proceeds Biotechnology Co., Ltd.

S180 cell culture method:

①S180 sarcoma cells, DMEM complete medium containing 10% FBS + 1% double antibody, cultured at 37°C, 5% CO ₂ incubator.

②When the cell concentration in the culture flask reaches 80%-90%, add 2-3ml of trypsin to digest, put it in the incubator, and digest for 3 minutes; observe it under a microscope, when the cells are loosely connected, add 2ml of complete medium, The digestion was terminated by pipetting and transferred to a centrifuge tube.

③Put the centrifuge tube in a centrifuge, centrifuge for 5 min at 4°C, 1000 rpm, and discard the supernatant medium. Take 5ml of PBS to resuspend the cells, pipette 10ul onto the cell counting plate, count the cells under an inverted microscope, dilute them appropriately with pre-cooled PBS, adjust the number of cells, and make the cell concentration up to 6×10 ⁷ /mL cell suspension.

Kunming rat modeling method:

A 3-4 week old Kunming mouse was taken and depilated with depilatory cream at 9 cm ² under the mouse's armpit. On the second day, the exposed skin was sterilized with alcohol cotton balls 2-3 times, 0.1 mL of the above tumor cell suspension was drawn with a 1 mL sterile syringe, the mouse was fixed with the left hand, and the syringe was held with the right hand, and injected at 3 cm below the armpit of the mouse.

Example 1

40 Kunming male mice weighing 18-19g were selected and randomly divided into 4 groups, namely the control group, the model group, the cyclophosphamide group (CTX), and the combination group (CM). Tumor modeling was performed according to the Kunming mouse modeling method described above. The specific administration time and method are as follows:

①Control group: 0.2ml of normal saline was given by gavage every day.

②Model group (M): 0.2ml of normal saline was given by gavage every day.

③ Cyclophosphamide group (CTX): On the second day of modeling, each mouse was intraperitoneally injected with 20 mg/kg cyclophosphamide, once every three days.

④Combination group (CM): Bakuchiol self-microemulsion 400 mg/kg (containing bakuchiol 120 mg/kg) was given by gavage every day; 20 mg/kg cyclophosphamide was intraperitoneally injected into each mouse, every three days. Dosing once.

⑤ Bakuchiol group (BAK): Bakuchiol self-microemulsion 400mg/kg (containing bakuchiol 120mg/kg) was given by gavage every day.

1.1 Measurement of tumor diameter and tumor growth inhibition rate

When the mouse tumor grows to a certain volume, as shown in Figure 1, the tumor diameter of the mouse, the longest diameter (a) and the shortest diameter (b), are measured with a vernier caliper every other day, and the volume calculation formula: volume = a ² The formula of *b ² was used to calculate the tumor volume; on the day of sampling, the isolated tumor tissue was washed with saline and accurately weighed. Tumor inhibition was calculated as follows: Inhibition rate (%) = (tumor weight of control mice - tumor weight of treated mice) x 100/tumor weight of control mice.

As shown in Figure 2, compared with the M group, the tumor weight of the mice in the CTX group was significantly reduced. The tumor weight of mice in the CM group was lower than that in the CTX group.

After modeling, along with tumor growth, tumor diameters were measured on day 6, day 8, day 10, day 12, and day 14, respectively. The results are shown in Table 1 and Figure 3 below. It can be seen from the results that the tumor growth of mice administered with CTX, single-administered bakuchiol self-microemulsion or CTX+bakuchiol self-microemulsion obviously does not have any untreated mice. The tumors of mice with CTX + bakuchiol self-microemulsions grew faster, and the tumors of mice treated with CTX + bakuchiol self-microemulsions were significantly slower than those of mice treated with CTX alone and bakuchiol alone. By measuring the tumor weight on the 14th day, it was also known that bakuchiol could promote the inhibitory effect of cyclophosphamide on tumor growth.

Table 1 Measurement of tumor diameter on the 6th day, the 8th day, the 10th day, the 12th day and the 14th day

In the table, * means P<0.05, ** means P<0.01, and *** means P<0.001.

1.2 Determination of white blood cells

After the model was established, 1ml of blood was collected from the eye fundus of the mice on the 0th, 9th and 15th days of administration, respectively, into an anticoagulation tube, and blood routine analysis was performed within 2h in a MEK-6318K blood cell analyzer (Nihon Kohden Kogyo Co., Ltd.). , and the detection index is the white blood cell count (WBC).

As shown in Figure 4, with the increase of tumor growth time after modeling, the number of routine blood leukocytes in the model group showed a decreasing trend. Compared with the model group, the number of leukocytes in the CTX group also showed a decreasing trend, but the bakuchiol self-microemulsion group and the CM group alone could obviously alleviate the decrease in leukocytes, and compared with the CTX group The number of white blood cells increased significantly. It can be seen that the use of the chemotherapeutic drug CTX damages the immune system, and bakuchiol can reduce this damage, and the effect is extremely significant.

1.3 Specimen collection

Blood samples: On the 15th day after modeling, the mice were collected by removing their eyeballs, placed in a 1.5mL EP tube, and allowed to stand at room temperature for 3 hours until the serum and plasma were clearly stratified. Centrifuge at 3500 rpm for 10 min, aspirate the supernatant, and store it in a -20°C refrigerator for later use.

Tissue samples: After the mice were sacrificed, the tumor sites in the armpits of the mice were cut out, placed on ice, and the tumor tissues were separated along the subcutaneous mucosa. Half of the tumor was fixed in 4% paraformaldehyde, and the other half was stored at -80°C Refrigerator spare.

1.4 H&E staining of tumor tissue pathology

(1) Dewax the paraffin sections to water, and set the dehydration procedure as follows: sequentially put the sections into xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% alcohol for 5 min, and wash with distilled water.

(2) Hematoxylin staining: Sections were stained with hematoxylin staining solution for 3-5 minutes, washed with tap water, differentiated with differentiation medium, washed with tap water, returned to blue with blue solution, and rinsed with running water.

(3) Eosin staining: sections were dehydrated in 85% and 95% graded alcohol for 5 minutes, and then stained in eosin staining solution for 5 minutes.

(4) Dehydration and sealing: put the slices into dehydrated ethanol Ⅰ 5min-dehydrated ethanol Ⅱ 5min-dehydrated ethanol Ⅲ 5min-xylene Ⅰ 5min-xylene Ⅱ 5min in turn, seal the slice with neutral resin after finishing, and place it in a cool place to dry . Observe and photograph under microscope.

As shown in Figure 5, the tumor cells in the model group grew in clusters. The nucleus was dyed blue-purple, the texture was pink, and the tissue cells were closely arranged. The color of the CTX group and the bakuchiol self-microemulsion group alone was lighter than that of the model group. When the cells were senescent, they would gradually become eosinophilic and become red, and their arrangement was looser than that of the model group. In addition to such phenomena, the CM group also had Homogeneous red-stained nuclear staining can be seen in necrotic tissue without structural material, and in some cells, chromatin can be seen condensed into clumps with no cytoplasm around or an empty halo. The tumor tissue apoptosis in the bakuchiol self-microemulsion and cyclophosphamide group was stronger than that in the cyclophosphamide single-use group and the bakuchiol-self-microemulsion group alone.

1.5 TUNEL staining of tumor histopathology

(1) Dewaxing paraffin sections to water: Place the sections in xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and distilled water for washing.

(2) Repair: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the liquid from flowing away), drop the proteinase K working solution in the circle to cover the tissue, and incubate in a 37°C incubator for 30 minutes. The slides were washed 3 times in PBS (pH 7.4) with shaking on a destaining shaker, 5 min each time.

(3) Membrane rupture: After the slices are slightly dried, drip the membrane rupture working solution in the circle to cover the tissue, incubate at room temperature for 20 minutes, place the slides in PBS (pH 7.4) and shake and wash 3 times on a decolorizing shaker. 5min times.

(4) Add reagent 1 and 2: according to the number of slices and tissue size, mix an appropriate amount of reagent 1 (TdT) and reagent 2 (dUTP) in the Tunel kit at a ratio of 2:29, add it to the circle to cover the tissue, and place the slice flat in a wet place In the box, incubate in a water bath at 37°C for 2 hours, and add a small amount of water to the wet box to maintain humidity.

(5) DAPI counterstained nuclei: the sections were washed three times with PBS (PH7.4) for 5 min each time. After removing PBS, DAPI staining solution was added dropwise to the circle, and incubated at room temperature for 10 min in the dark.

(6) Cover slides: The slides were placed in PBS (PH7.4) and washed 3 times with shaking on a destaining shaker, 5 min each time. After drying, the sections were mounted with anti-fluorescence quenching mounting medium.

(7) Microscopic examination and photographing: The slices were scanned panoramically under the CaseViewer software and images were collected.

As shown in Figure 6, there are few green parts in the model group, and the whole is blue, so the number of apoptotic cells in the tissue is small. The apoptosis of the CTX group was stronger than that of the model group, and the color of the CM group was significantly greener than that of the CTX group, so the apoptosis was more significant. While the bakuchiol self-microemulsion group alone had stronger fluorescence green intensity than the CM group, the blue area was larger than that of the CM group.

1.6 Detection of protein expression

1.6.1 Protein extraction

Take out the tumor sample and transfer it to liquid nitrogen. Use scissors soaked in enzyme-free water to cut 30-50 mg of tumor tissue into a 1.5 mL shell tube, add 300-500 ul of RIPA protein lysis solution, and use a tissue crusher to completely crush the tissue to tissue foam. , placed on ice for complete lysis for 30min. The lysed samples were centrifuged at 12,000 g for 10 min, and the supernatant was taken.

1.6.2 Determination of protein concentration

(1) Preparation of BCA working solution: Mix reagent A and reagent B in the BCA kit evenly according to the ratio of A:B=50:1. This solution is prepared as it is.

(2) Preparation of standard solution: according to the gradient dilution, the gradient concentration of protein standard product is 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 0 μg/mL.

(3) Add 2 μL of each sample to the 96-well plate, and then add 18 μL of lysis solution; take 20 μL of the above standard substances of different concentrations and add them to other wells; add 180 μL of BCA working solution to both sample wells and standard product wells, and incubate at 37°C for 30 minutes .

(4) Measurement of absorbance: OD value was detected at 562 nm, and a standard curve was prepared according to the obtained value.

Put the standard protein protein with concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 0 μg/mL into the microplate reader according to the sample steps to measure the OD value at 562 nm, and the OD value and its corresponding concentration Put it into excel to make a standard curve. Then calculate the protein concentration of the sample according to the standard curve. The standard curve is a straight line with positive correlation.

1.6.3 Expression of related proteins in tumor tissue

(1) Preparation of DTT: Take DTT powder and add 40 μL of deionized water, blow and beat evenly;

(2) Preparation of 5×Master Mix (ie Loading Buffer): Add 20μL of DTT solution and 20μL of 10×Sample Buffer to Master Mix powder, pipetting evenly;

(3) Preparation of Ladder (ie marker, molecular weight standard): Add 16 μL of ionized water, 2 μL of DTT solution, and 2 μL of 10×Sample Buffer to Ladder powder, pipette evenly, suck out about 10 μL and put it into an empty 0.6 mL tube to be denatured;

(4) Preparation of sample: Assuming that the sample concentration is y, the following is the volume required to prepare one well:

(4) Put the prepared Ladder and the sample together at 95°C for denaturation for 5 minutes; after the denaturation, take out the Ladder and the sample, and cool on ice for 5 minutes. After cooling, vortex to mix, centrifuge briefly, and put on ice for later use;

(5) Preparation of primary antibody: The primary antibody is diluted with Antibody Diluent II and placed on ice for use;

(6) Preparation of luminescent solution: take 200 μL of each of Lumino-S and Peroxide, vortex to mix, and put them on ice for use;

(7) Take out the sample plate, add the prepared reagents to the plate in turn as required, and after adding, centrifuge at 2500rpm for 5min;

(8) Put the bubble-free board into the machine for automatic electrophoresis and development. The relative protein expression levels of cleaved Caspase-3, Caspase-8, Bcl-2, Bax and JNK were analyzed after correction with β-actin in the same samples according to the intensities of the peaks.

As shown in Figure 7, the test results showed that the combined use of bakuchiol and cyclophosphamide up-regulated the expression of Caspsse3, Caspase8, JNK, and Bax, and down-regulated the expression of Bcl-2 in tumor tissue. The combined effect of bakuchiol self-microemulsion and cyclophosphamide was more obvious than that of the CTX group and the bakuchiol self-microemulsion group alone. The combined use of bakuchiol self-microemulsion and cyclophosphamide enhanced the expression of apoptotic factors in mouse tumor cells.

When bakuchiol is used in combination with other chemotherapy drugs, such as cisplatin, mitomycin, and vincristine, similar experimental results are obtained. Bakuchiol can increase cisplatin, mitomycin, vincristine It has anti-tumor effect, promotes tumor cell apoptosis, and reduces the inhibitory effect of the above chemotherapy drugs on leukocyte growth.

As shown in Figure 8, NVB is vinorelbine at a concentration of 0.01 umol/L, cells are A549 (human non-small cell lung cancer cells), and CCK8 is used to detect cell viability after drug reaction. 0.01umol/L of NVB was used in combination with 0umol/L, 0.5umol/L and 1,5umol/L of BAK (bakuchiol) respectively. It can be seen from the results that when the concentration of BAK is greater than 0.5umol/L, the Significantly reduces the viability of cancer cells.

The above are only the preferred embodiments of the present invention. It should be noted that, for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. These improvements and Retouching should also be regarded as the protection scope of the present invention.

Claims (10)

1. Application of bakuchiol in improving antitumor effect of chemotherapeutic drugs is provided.

2. The use of claim 1, wherein bakuchiol increases the anti-tumor effect of the chemotherapeutic agent while decreasing the immunological damage of the chemotherapeutic agent.

3. The use as claimed in claim 1, wherein the bakuchiol acts synergistically with the chemotherapeutic to promote expression of apoptosis factor associated with tumor cells and promote apoptosis of tumor cells, and wherein the bakuchiol reduces damage to leukocytes by the chemotherapeutic.

4. The use as claimed in claim 1, wherein the psoralen and the chemotherapeutic agent act synergistically to up-regulate expression of Caspase 3, Caspase8, JNK, Bax and down-regulate expression of Bcl-2 in tumor tissues.

5. The use of claim 1, wherein the chemotherapeutic agent is cyclophosphamide, cisplatin, mitomycin or vincristine.

6. The use of claim 1, wherein the mass ratio of the bakuchiol to the chemotherapeutic agent is (75-240): (10-40).

7. The use as claimed in claim 1, wherein the bakuchiol is a bakuchiol self-microemulsion, and the mass ratio of the bakuchiol self-microemulsion to the chemotherapeutic agent is (250- & 800): (10-40).

8. The use of claim 7, wherein the bakuchiol self-microemulsion is prepared by: selecting bakuchiol as oil phase, adding emulsifier and co-emulsifier, placing in water bath at 37 + -2 deg.C and stirring at 100 + -5 rpm.

9. Use according to claim 8, wherein the emulsifier is Cremophor RH40 or Cremophor EL 35 and the co-emulsifier is Labrasol.

10. The use of claim 7, wherein the mass ratio of the bakuchiol to the emulsifier to the co-emulsifier is (1-4): (3-5): (2-4).

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