Use of aloe-emodin

Abstract

The present application relates to aloe-emodin. The present application provides aloe-emodin for preparing a PARP inhibitor. The aloe-emodin has better inhibitory activity on PARP.

Description

Technical Field

[0001] This invention relates to the application of aloe-emodin. Background Technology

[0002] The PARP (Poly ADP-Ribose Polymerase) protein family consists of 17 members whose function is to catalyze the poly(ADP-ribosylation) of proteins, transferring ADP-ribose to the substrate protein. Automatic PARP PARation triggers the release of bound PARP from DNA, allowing access to other DNA repair proteins to complete the repair. Therefore, PARP binding to the damage site, its catalytic activity, and its eventual release from DNA are all crucial steps in cancer cell responses to DNA damage induced by chemotherapy and radiotherapy.

[0003] Inhibition of PARP family enzymes has been developed as a strategy to selectively kill cancer cells by inactivating complementary DNA repair pathways. PARP1 is one of the most important members of the PARP family, with functions broadly involved in DNA repair, chromatin remodeling, transcriptional regulation, and cellular stress responses. Therefore, many PARP1 inhibitors have been developed, six of which have been approved for anticancer therapy. These marketed PARP1 inhibitors have demonstrated excellent clinical efficacy as sensitizers or in patients with homologous recombination-deficient cancers.

[0004] However, hematological and other toxicities limit the clinical application of these drugs. PARP inhibitors with improved selectivity for PARP1 have been reported to offer improved efficacy and reduced toxicity compared to other clinical PARP1 / 2 inhibitors. Strong selective inhibition of PARP1 leads to PARP1 capture on DNA, resulting in DNA double-strand breaks (DSBs) caused by S-phase replication fork collapse. Modern research indicates that PARP1-DNA capture is an effective mechanism for selectively killing tumor cells with high toxicity reactivity disorder (HRD). Therefore, there is an unmet medical need for effective and safe PARP inhibitors, particularly PARP inhibitors selective for PARP1. Consequently, highly selective PARP inhibitors have become an important direction in medicinal chemistry research.

[0005] Colorectal cancer (CRC) accounts for approximately 10% of all cancer diagnoses and cancer-related deaths worldwide each year. It is characterized by its high insidiousness, rapid progression, and high malignancy. Among these, low rectal cancer, due to its proximity to the anus (5-10 mm), makes sphincter-preserving surgery a pressing need for patients.

[0006] Currently, there are various treatment methods for CRC in clinical practice, including endoscopic treatment, surgical resection, preoperative radiotherapy, systemic therapy, targeted therapy, and immunotherapy. However, the high recurrence and metastasis rates after surgery have prevented an improvement in the cure rate and long-term survival rate of CRC, and the mortality rate remains high. Radiation therapy (RT) is one of the important means of treating CRC. High-energy rays (such as X-rays or gamma rays) kill cancer cells by destroying their DNA and inhibiting their proliferation and metastasis. RT can be used preoperatively, postoperatively, or as a single treatment, and is usually used in combination with chemotherapy to improve the treatment effect. Neoadjuvant therapy for CRC in clinical practice uses radiation to destroy the DNA of cancer cells, killing or preventing their growth and spread, thereby reducing the tumor size to meet surgical indications for resection and making sphincter preservation possible.

[0007] According to statistics, over 70% of cancer patients have received radiotherapy (RT) during their treatment. RT has long been used for the curative and palliative treatment of CRC patients. The effectiveness of RT is influenced by various factors, such as the type and stage of the cancer, as different types and stages of cancer have different sensitivities to RT. Generally, early-stage and smaller cancers respond better to RT. Radiation dose and treatment duration are also important: excessive radiation doses can damage healthy tissues, while too short a treatment duration can also affect the treatment outcome. Essentially, the effectiveness of radiotherapy largely depends on the patient's sensitivity to radiation. The higher the radiosensitivity, the stronger the tumor's killing effect on radiation, and the better the effect of RT. Cancers with lower radiosensitivity may require higher radiation doses or longer treatment durations to achieve the same effect. As an important component of comprehensive treatment for colorectal cancer, preoperative chemoradiotherapy remains the standard treatment strategy for locally advanced rectal cancer (stages II and III).

[0008] However, while radiotherapy brings therapeutic benefits to patients, it also increases its toxic side effects, resulting in a relatively small percentage of patients benefiting, and many problems still need to be addressed. Therefore, screening for substances that can effectively improve the sensitivity of radiotherapy and reduce its toxic side effects is of great significance. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the defects in the prior art and provide an application of aloe-emodin.

[0010] The present invention solves the above-mentioned technical problems through the following technical solution:

[0011] In the first aspect, the use of an aloe-emodin, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of an acceptable salt thereof in the preparation of a PARP inhibitor.

[0012] In this invention, the PARP inhibitor may be a PARP1 inhibitor.

[0013] In this invention, the PARP inhibitor may be a PARP inhibitor used in vitro.

[0014] Secondly, the use of an aloe-emodin, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of an acceptable salt thereof in the preparation of a medicament for treating diseases associated with PARP.

[0015] In this invention, the PARP-related disease may be a PARP1-related disease.

[0016] In this invention, PARP-related diseases can refer to diseases that can be improved or cured by inhibiting PARP.

[0017] In this invention, the PARP-related diseases may be cancer, acute ischemic injury, neurodegenerative diseases, or inflammatory diseases and autoimmune diseases.

[0018] In this invention, the cancer may be colorectal cancer.

[0019] In this invention, the PARP-related disease is cancer, and the drug can be a radiosensitizing drug.

[0020] Thirdly, the use of an aloe-emodin, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of an acceptable salt thereof in the preparation of a medicament for the treatment of cancer, acute ischemic injury, neurodegenerative diseases, or inflammatory diseases and autoimmune diseases.

[0021] In this invention, the cancer may be colorectal cancer.

[0022] In this invention, the drug may be a radiosensitizing drug for cancer radiotherapy, preferably a radiosensitizing drug for colorectal cancer radiotherapy.

[0023] In this invention, the radiation intensity of the radiotherapy can be 1-100 Gy, for example 4 Gy, 8 Gy, 10 Gy, 13 Gy or 16 Gy.

[0024] Terminology Explanation:

[0025] Unless otherwise stated, the definitions of terms recorded in this specification and claims, including definitions as examples, exemplary definitions, preferred definitions, and definitions recorded in tables, can be arbitrarily combined and combined with each other. Such combinations and combinations of definitions shall fall within the scope of this specification. Unless otherwise specified, the terms used in this invention have the following meanings:

[0026] The term “treatment” refers to a therapeutic approach. When a specific condition is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; or (4) slowing the development of the condition or one or more biological manifestations of the condition.

[0027] The term "pharmaceutically acceptable salt" is discussed in Berge et al., "Pharmaceutically acceptable salts," J. Pharm. Sci., 66, 1-19 (1977), and is readily apparent to medicinal chemists. The salt is essentially non-toxic and provides the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism, or excretion. The compounds of this invention may have acidic, basic, or amphoteric groups, and typical pharmaceutically acceptable salts include those prepared by reacting the compounds of this invention with an acid.

[0028] The term "solvate" refers to a substance formed by the combination of a compound and a solvent (including but not limited to water, methanol, ethanol, etc.). Solvates are classified into stoichiometric solvates and non-stoichiometric solvates. Solvates include, but are not limited to, monohydrates.

[0029] The term "solvate of a pharmaceutically acceptable salt" refers to a substance formed by the combination of a compound with a pharmaceutically acceptable acid or base and a solvent (including but not limited to water, methanol, ethanol, etc.). The amount of solvent can be stoichiometric or non-stoichiometric. Solvates of pharmaceutically acceptable salts include, but are not limited to, monohydrochloride monohydrates.

[0030] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0031] The reagents and raw materials used in this invention are all commercially available.

[0032] The positive and progressive effects of this invention are as follows:

[0033] (1) The aloe-emodin of the present invention can be used to prepare a PARP inhibitor for in vitro use;

[0034] (2) The aloe-emodin of the present invention affects the active pocket of PARP, causing PARP to directly lose its enzyme activity, thereby inhibiting the activity of PARP;

[0035] (3) In this invention, since the automatic PAR activation triggers the release of PARP from DNA to allow access to other DNA repair proteins to complete the repair, the binding of PARP to the damage site, its catalytic activity and its final release from DNA are important steps for cancer cells to respond to DNA damage caused by chemotherapy and radiotherapy. Aloe-emodin can competitively bind to the enzyme activity site of PARP, causing it to lose its enzyme activity and inhibiting the formation of PAR, thereby achieving the purpose of radiosensitization. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the CETSA test results at different temperatures in Example 4.

[0037] Figure 2 This is a statistical chart of CETSA test results at different temperatures in Example 4.

[0038] Figure 3 This is a schematic diagram of the Western blot test results for γ-H2AX in Example 5.

[0039] Figure 4 This is a diagram showing the cell cloning results of HCT-116 in Example 5.

[0040] Figure 5 This is a schematic diagram of the immunofluorescence expression results of γ-H2AX in Example 5.

[0041] Figure 6 This is a schematic diagram of the immunofluorescence expression results of CRT in Example 5.

[0042] Figure 7 This is a schematic diagram of the Western blot test results of Cleaved PARP in HCT-116 cells after radiotherapy in Example 5.

[0043] Figure 8 This is a schematic diagram showing the test results of different concentrations of aloe-emodin on Cleaved PARP in HCT-116 cells in Example 5. Detailed Implementation

[0044] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0045] Example 1: In vitro inhibitory effect on PARP1 test

[0046] The specific details of the reagents, raw materials, and instruments used in the examples are listed below:

[0047] 3-AB refers to 3-aminobenzamide.

[0048] Olaparib, chemically named 4-[(3-{[4-(cyclopropylcarbonyl)piperazin-1-yl]carbonyl}-4-fluorophenyl)methyl]phthalocyanine-1(2H)-one, CAS number 763113-22-0.

[0049] Aloe-emodin (AE) has the following structural formula:

[0050]

[0051] 1×PBS refers to phosphate buffer, which consists of phosphate buffers (such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate) and salts (such as sodium chloride and potassium chloride), and its pH value is usually between 7.2 and 7.4.

[0052] Triton X-100, also known as Triton X-100.

[0053] The colorimetric PARP assay kit, model RD / 4677-096-K, includes the following: 20×PARP buffer, a high-concentration buffer specifically formulated for this PARP activity assay kit, which must be diluted 1:20 with distilled water to a 1×PARP buffer before use. The 10×PARP Cocktail and 10× Activated DNA are both reagents included in the kit.

[0054] 1. Test Method: The inhibitory effect of drugs (3-AB, commercially available positive drug Olaparib, or aloe-emodin) on PARP1 activity was determined using a colorimetric PARP assay kit (hereinafter referred to as the kit). The specific steps included:

[0055] (1) Remove the strip wells from the kit and add 50 μL / well of 1×PARP buffer to rehydrate histones. Then, incubate at room temperature for 30 minutes. Pour the 1×PARP buffer out of the wells by tapping them on a paper towel. Keep the wells moist, not completely dry;

[0056] (2) Prepare 5 mM aloe-emodin stock solution with DMSO, and then prepare 5 μM and 10 μM aloe-emodin solutions with 1×PARP buffer. Prepare 1 μM, 5 μM and 10 μM 3-AB solution and 5 μM and 10 μM Olaparib solution with 1×PARP buffer. Set up negative control group, positive control group (positive drug is Olaparib), 3-AB group with different doses, aloe-emodin group and Olaparib group respectively. Add 24 μL of different drugs to each well in sequence.

[0057] (3) Dilute the PARP-HSA enzyme (10 U / μL) in the kit with 1×PARP buffer to 0.5 U / μL, and then add 1 μL of PARP-HSA enzyme (0.5 U / well) to each well. Incubate at room temperature for 10 minutes, and use immediately after preparation.

[0058] (4) Prepare a mixture of 2.5 μL of 10×PARP Cocktail, 2.5 μL of 10×Activated DNA and 20 μL of 1×PARP buffer per well. Add 25 μL of the mixture to each well, so that the final reaction volume is 50 μL. Incubate at room temperature for 60 minutes.

[0059] (5) Discard the supernatant. Wash each well twice with 200 μL of solution (1×PBS + 0.1% (w / v) Triton X-100), then wash twice with 1×PBS. After each wash, make sure to remove all liquid by gently tapping the strip well on a paper towel.

[0060] (6) Dilute the Strep-HRP working solution (10 U / μL) with the 1×Strep-diluent in the kit at a ratio of 1:500, and add 50 μL of Strep-HRP working solution to each well. Incubate at room temperature in the dark for 60 minutes.

[0061] (7) Each well should be rinsed twice with 200 μL of solution (1×PBS + 0.1% (w / v) Triton X-100), and then rinsed twice with 1×PBS. After each rinse, make sure to remove all liquid by gently tapping the strip well on a paper towel.

[0062] (8) Preheat the TACS-sapphire substrate (hereinafter referred to as substrate) in the kit to room temperature. Add 50 μL of substrate to each well and incubate in the dark at room temperature for 15 minutes. Stop the reaction by adding 50 μL of 5% (v / v) phosphoric acid to each well and read the absorbance at 450 nm. Calculate the inhibition rate according to the following formula:

[0063] Inhibition rate (%) = [1 - (OD - negative control) / (positive control - negative control)] × 100

[0064] 2. Test results: The inhibition rate (%) of each drug system at different concentrations is shown in the table below.

[0065]

[0066] The data above show that aloe-emodin can significantly inhibit PARP1 activity, IC50... 50 It can be around 5 μM, with a significant increase at a dose of 10 μM.

[0067] Example 2: Toxicity test of aloe-emodin on cancer cells

[0068] The specific details of the reagents, raw materials, and instruments used in the examples are listed below:

[0069] CCK-8 solution, purchased from Meilun, model MA0218.

[0070] HCT-116 cells and HT-29 cells were cultured in DMEM medium (hereinafter referred to as culture medium) containing 10% (w / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin mixture.

[0071] 1. Testing method:

[0072] (1) HCT-116 and HT-29 cells in the logarithmic growth phase were selected for cell seeding. 100 μL of a solution containing 5 × 10⁻⁶ cells was added to each well of a 96-well plate. 3 HCT-116 cell suspension or containing 1×10 4 The HT-29 cell suspension was cultured in a 37°C incubator for 24 hours.

[0073] (2) A blank group (no cells in blank wells), a control group (with cells, no radiation) and an aloe-emodin group were set up. The control group and the model group were replaced with new culture medium. The aloe-emodin group was replaced with culture medium containing 5 μM and 10 μM aloe-emodin and cultured for 48 h.

[0074] (3) Add 10 μL of CCK-8 solution to each well and incubate at 37℃ for 0.5 h. Measure the absorbance at 450 nm using a microplate reader and calculate the cell viability using the following formula:

[0075] Cell viability = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0076] 2. Test results: The cell survival rate (%) of different concentrations of aloe-emodin is shown in the table below.

[0077] HCT-116 cell viability results

[0078]

[0079] HT-29 cell survival results

[0080]

[0081] The data above show that treating HCT-116 and HT-29 cells with aloe-emodin for 48 h did not significantly reduce their activity, indicating that aloe-emodin alone does not affect the normal proliferation of colorectal cancer cells.

[0082] Example 3: Radiosensitivity test of aloe-emodin on colorectal cancer cells

[0083] The specific details of the reagents, raw materials, and instruments used in the examples are listed below:

[0084] The irradiator, purchased from Radsource Technologies in the United States, has the following parameters: 160 kV, 25 mA, no reflection, 4 Shelf, and can be set to irradiation intensities of 4 Gy, 8 Gy, 10 Gy, 13 Gy, and 16 Gy.

[0085] CCK-8 solution, purchased from Meilun, model MA0218.

[0086] HCT-116 cells and HT-29 cells were cultured in DMEM medium (hereinafter referred to as culture medium) containing 10% (w / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin mixture.

[0087] 1. Testing method:

[0088] (1) HCT-116 and CT-26 cells in the logarithmic growth phase were selected for cell seeding. 100 μL of a solution containing 5 × 10⁻⁶ cells was added to each well of a 96-well plate. 3 HCT-116 cell suspension or containing 5×10 3 The cell suspension of CT-26 was cultured in an incubator at 37°C for 24 hours.

[0089] (2) A blank group (no cells in blank wells), a control group (with cells, no radiation), a model group (with cells, with radiation), an aloe-emodin group, and an Olaparib group were set up. The control group and the model group were replaced with new culture medium. The aloe-emodin group was replaced with culture medium containing 1 μM, 2.5 μM, 5 μM and 10 μM aloe-emodin. The Olaparib group was replaced with 5 μM Olaparib solution. After 30 min, the well plates were sealed with sealing film and placed in an irradiator. They were irradiated with 8 Gy and 10 Gy rays respectively. After irradiation, they were placed in an incubator at 37℃ for 3 h. After replacing with new culture medium, they were cultured for another 48 h.

[0090] (3) Add 10 μL of CCK-8 solution to each well and incubate at 37℃ for 0.5 h. Measure the absorbance at 450 nm using a microplate reader and calculate the cell viability using the following formula:

[0091] Cell viability = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0092] 2. Test results: The cell survival rate (%) of different drugs is shown in the table below.

[0093] HCT-116 cell viability results

[0094]

[0095] CT-26 cell survival results

[0096]

[0097] The data above show that HCT-116 and CT-26 cells were irradiated with 8 Gray and 10 Gray, respectively, and then treated with aloe-emodin and olaparib for 48 h. The results indicated that the activity of HCT-116 and CT-26 cells decreased significantly with increasing dose, suggesting that aloe-emodin can significantly increase the radiosensitivity of colorectal cancer cells.

[0098] Example 4: Binding test of aloe-emodin to PARP1

[0099] The specific details of the reagents, raw materials, and instruments used in the examples are listed below:

[0100] HCT-116 cells were cultured in DMEM medium (hereinafter referred to as culture medium or complete culture) containing 10% (w / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin mixture.

[0101] 1×PBS refers to phosphate buffer, which consists of phosphate buffers (such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate) and salts (such as sodium chloride and potassium chloride), and its pH value is usually between 7.2 and 7.4.

[0102] 1% protease inhibitor, purchased from MCE, model HY-K0010.

[0103] 5× protein loading buffer, purchased from Beyotime, model P0015L.

[0104] 1. Test method: Based on the Cell Thermal Migration Experiment (CESTA), the specific steps are as follows:

[0105] (1) Select HCT-116 cells in the logarithmic growth phase for cell seeding, and add 1800 μL of 5×10⁻⁶ cells to each well of a 6-well plate. 5 The HCT-116 cell suspension was cultured in an incubator at 37°C for 24 h.

[0106] (2) Then, add 200 μL of drug-containing monosodium glutamate (the dosage of positive drug Olaparib and aloe-emodin is 50 μM) to each well, and add only the corresponding concentration of DMSO to the control group. Then incubate in a 37℃ incubator for 5 h.

[0107] (3) Discard the drug-containing supernatant, wash once with 1×PBS buffer, add 200 μL of trypsin, digest in a 37℃ incubator for 3 min, add 1 mL of complete culture to stop digestion, transfer to EP tube, centrifuge at 1000 rpm for 5 min, then wash twice with PBS, discard the supernatant for the last time, add PBS containing 1% protease inhibitor to resuspend the cells, divide into 3 equal portions and transfer to 200 μL PCR tubes of PCR instrument.

[0108] (4) In a PCR instrument, heat at 47, 48, 49, 50, 51, 52 and 53 °C for 3 min, then quickly transfer to liquid nitrogen and repeat 4 freeze-thaw cycles (liquid nitrogen flash freeze ←→ 25 °C thaw) to lyse the cells. After lysis, centrifuge at 20000 rcf for 20 min at 4 °C, and transfer the supernatant to a new 1.5 mL EP tube and add 5× protein loading buffer.

[0109] (5) Finally, the protein was denatured by heating at 95°C for 10 min and analyzed by Western blotting.

[0110] 2. Test Results: According to CETSA results, in HCT-116 cells, the aloe-emodin and olaparib groups showed significantly higher protein thermostability at 49, 50, and 51°C compared to the DMSO group. This indicates that aloe-emodin can bind to PARP1 to increase protein thermostability, similar to the effect of the positive control drug olaparib. Figure 1 and Figure 2 The results showed that aloe-emodin and PARP1 have a good binding affinity.

[0111] Example 5: Aloe-emodin as a DNA damage test for colorectal cancer cells after radiotherapy

[0112] The specific details of the reagents, raw materials, and instruments used in the examples are listed below:

[0113] The γ-H2AX immunofluorescence assay kit was purchased from Beyotime, model C2035S.

[0114] γ-H2AX rabbit monoclonal antibody, purchased from Huabio, model ET1705-97.

[0115] Anti-rabbit 488, purchased from Beyotime, model A0423.

[0116] The nuclear staining solution (DAPI) refers to 4',6-diamidino-2-phenylindole, purchased from Beyotime, model P0131.

[0117] RIPA lysis buffer, purchased from Beyotime, P0013B.

[0118] TBST is composed of 50 mL Tris-HCl (pH 7.5, approximately 50 mM) + 8 g NaCl + 1 mL Tween-20, diluted to 1 L with water.

[0119] ECL chemiluminescent liquid, purchased from Beyotime, P0018S.

[0120] 4% paraformaldehyde fixative solution, purchased from Beyotime, model P0099.

[0121] 1% protease inhibitor, purchased from MCE, model HY-K0010.

[0122] 5× protein loading buffer, purchased from Beyotime, model P0015L.

[0123] The ATP assay kit was purchased from Beyotime, model S0026.

[0124] 1×PBS refers to phosphate buffer, which consists of phosphate buffers (such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate) and salts (such as sodium chloride and potassium chloride), and its pH value is usually between 7.2 and 7.4.

[0125] HCT-116 cells were cultured in DMEM medium (hereinafter referred to as culture medium or complete culture) containing 10% (w / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin mixture.

[0126] The irradiator, purchased from Radsource Technologies in the United States, has the following parameters: 160 kV, 25 mA, no reflection, 4 Shelf, and can be set to irradiation intensities of 4 Gy, 8 Gy, 10 Gy, 13 Gy, and 16 Gy.

[0127] I. Testing Methods: Since radiation therapy (RT) inhibits cancer cell proliferation and metastasis by destroying cancer cell DNA, immunofluorescence and Western blot were used to detect the expression of γ-H2AX protein in HCT-116 cells after radiotherapy, as detailed below:

[0128] 1. The specific steps for detecting the DNA damage marker γ-H2AX using immunofluorescence are as follows:

[0129] (1) Select HCT-116 cells in the logarithmic growth phase for cell seeding. First, add cell spreaders to the bottom of the dish, and then add 100 μL of 5×10⁻⁶ cells to each well of a 6-well plate. 5 HCT-116 cells were cultured in suspension for 24 hours and divided into control group (with cells, no radiation), aloe-emodin group, model group (with cells, with radiation), and irradiated aloe-emodin group and Olaparib group. The culture medium was discarded and the corresponding drug-containing culture medium was added. After 30 minutes, the well plate was sealed with sealing film and placed in the irradiator. It was irradiated with 8 Gy rays. After irradiation, it was placed in an incubator at 37℃ for 3 hours and then cultured with fresh culture medium for 12 hours.

[0130] (2) Discard the supernatant culture medium, add 4% paraformaldehyde fixative, and fix for 15 minutes. The amount of fixative should be enough to cover the sample. For samples in a 6-well plate, usually 500 μL of fixative is added to each well.

[0131] (3) Remove the fixative and wash three times with 1×PBS for 5 minutes each time. Each time you wash, try to remove as much residual liquid as possible, and keep the sample surface slightly moist. Do not let it dry out. After the last wash, remove all the washing solution.

[0132] (4) Stain with the γ-H2AX immunofluorescence assay kit (hereinafter referred to as the kit), add the immunostaining blocking solution in the kit, and block at room temperature for 20 minutes. The amount of immunostaining blocking solution should be enough to cover the sample. Add 500 μL of blocking solution to each well of a 6-well plate.

[0133] (5) Remove the immunostaining blocking solution, add γ-H2AX rabbit monoclonal antibody, and incubate overnight at 4°C. The amount of γ-H2AX rabbit monoclonal antibody should be enough to cover the sample. Usually, 50 μL of γ-H2AX rabbit monoclonal antibody is added to each well of the 6-well plate.

[0134] (6) Carefully aspirate the γ-H2AX rabbit monoclonal antibody into a suitable container, store at 4°C, and keep for future use.

[0135] (7) Wash with 1×PBS 3 times, 10 minutes each time. Each time you wash, try to remove as much residual liquid as possible, and keep the sample surface slightly moist. Do not let it dry out. After the last wash, remove all the washing solution.

[0136] (8) Add anti-rabbit 488 and incubate at room temperature for 1 hour. The amount of anti-rabbit 488 should be enough to cover the sample. Usually, 50 μL of anti-rabbit 488 is added to each well of the 6-well plate.

[0137] (9) Carefully aspirate the anti-rabbit 488 into a suitable container, store at 4°C, and save for future use.

[0138] (10) Wash twice with 1×PBS for 10 minutes each time. During each wash, try to remove as much residual liquid as possible, while keeping the sample surface slightly moist and not drying it out. After the last wash, remove all washing solution.

[0139] (11) Add nuclear staining solution (DAPI) and stain at room temperature for about 5 minutes. The amount of nuclear staining solution (DAPI) should be enough to cover the sample. Usually, 50 μL of nuclear staining solution (DAPI) is added to each well of a 6-well plate.

[0140] (12) Remove the nuclear staining solution (DAPI), wash three times with 1×PBS for 5 minutes each time. During each wash, try to remove as much residual liquid as possible, while keeping the sample surface slightly moist and not drying out. After the last wash, remove all 1×PBS.

[0141] (13) Place the cell smears on a glass slide, mount and air dry. Observe and photograph using confocal microscopy. γ-H2AX staining is green fluorescence, and DAPI staining of the cell nuclei is blue fluorescence.

[0142] 2. The expression of the DNA damage marker γ-H2AX protein was detected using Western blot. The specific steps are as follows:

[0143] (1) Select HCT-116 cells in the logarithmic growth phase for cell seeding, and then add 100 μL of 5×10⁻⁶ cells to each well of a 6-well plate. 5 HCT-116 cells were cultured in suspension for 24 h;

[0144] (2) The group was divided into a control group, a model group, and an irradiated aloe-emodin group and an Olaparib group. The culture medium was discarded and the corresponding drug-containing culture medium was added. After 30 min, the well plate was sealed with a sealing film and placed in an irradiator. The plate was irradiated with 8 Gy rays. After irradiation, the plate was placed in a 37℃ incubator for 3 h and then the culture medium was replaced for 24 h.

[0145] (3) Discard the culture medium, wash twice with 1×PBS, lyse the cells for 20 min with RIPA lysis buffer containing 1% protease inhibitor, obtain the total cell protein, and determine the protein concentration by BCA method.

[0146] (4) Add 5× protein loading buffer. Finally, heat at 95°C for 10 min to denature the protein, separate by SDS-PAGE and transfer to a polyvinylidene fluoride membrane. Then, block the membrane with 5% skim milk at room temperature for 2 h.

[0147] (5) Rinse the membrane three times with TBST (10 min each time), and react the membrane with the diluted primary antibody at 4°C overnight. Rinse the membrane three times with TBST, and react the membrane with the diluted secondary antibody at room temperature for 1 h.

[0148] (6) Rinse the membrane three times with TBST and develop the protein bands on the membrane with ECL chemiluminescent solution.

[0149] 3. The proliferation capacity of colorectal cancer cells was observed using the cell plate colony formation assay. The specific steps are as follows:

[0150] (1) Select cells in good condition that are in the logarithmic growth phase and have a cell density of 80% for plating. Digest and resuspend the cells, and then seed the well-sprayed cell suspension into sterile six-well plates at a rate of 2000 cells per well. Gently shake the plates to mix well and then incubate them in an incubator for 24 h.

[0151] (2) After the cells were completely attached to the wall, they were divided into control group, model group, aloe-emodin group and Olaparib group. After culturing for 24 hours, the culture medium was discarded and replaced with a complete culture medium containing 10 μM aloe-emodin and Olaparib.

[0152] (3) X-ray irradiation was performed using an 8 Gy dose gradient, and culture continued after irradiation. Each group had 3 replicates, and the culture medium was replaced with fresh medium every 3 days.

[0153] (4) After 10 days, discard all the culture medium, wash twice with 1×PBS, then fix the cells with methanol for 10 min, discard the fixative, add 2 ml of crystal violet staining solution to each well, and stain for 20 min. Finally, rinse gently with water, place on the lab bench to dry and take photos.

[0154] 4. The specific steps for observing immunogenic cell death in colorectal cancer cells based on ATP content detection are as follows:

[0155] (1) Select cells in good condition, in the logarithmic growth phase, and with a cell density of 80% for plating. Digest and resuspend the cells, then pipette the cell suspension at 5 × 10⁻⁶. 4 The required number of cells per well were seeded into a sterile 24-well plate, gently shaken to mix, and then incubated in an incubator for 24 hours.

[0156] (2) After the cells were completely attached to the wall, they were divided into a control group, an aloe-emodin group (without irradiation), a model group, an irradiated aloe-emodin group and an Olaparib group. After culturing for 24 h, the culture medium was discarded and replaced with a complete culture medium containing 10 μM aloe-emodin and Olaparib.

[0157] (3) X-ray irradiation was performed using an 8 Gy dose gradient. After irradiation, the culture was continued for 12 h, and the supernatant culture medium was collected.

[0158] (4) To detect ATP content according to the ATP detection kit, first dilute the ATP standard solution with ATP detection lysis buffer to 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM, and use a multi-functional microplate reader with luminometer function to detect the luminescence intensity to prepare a standard curve.

[0159] (5) Add 100 μL of ATP detection working solution to the wells of a black 96-well plate. Let it stand at room temperature for 3-5 minutes to allow all the background ATP to be consumed, thereby reducing the background. Then add 10 μL of sample, mix quickly with a pipette, and after 10 seconds, use a multi-functional microplate reader with luminometer function to detect the luminescence intensity to calculate the ATP content.

[0160] 5. The expression of Cleaved PARP protein in HCT-116 cells was detected using Western blot. The specific steps are as follows:

[0161] (1) Select HCT-116 cells in the logarithmic growth phase for cell seeding, and then add 100 μL of 5×10⁻⁶ cells to each well of a 6-well plate. 5 HCT-116 cells were cultured in suspension for 24 h;

[0162] (2) The group was divided into a control group, an aloe-emodin group (without irradiation), a model group, an irradiated aloe-emodin group and an Olaparib group, and an unirradiated group. The culture medium was discarded and the corresponding drug-containing culture medium was added. After 30 min, the well plate was sealed with a sealing film and placed in an irradiator. The plate was irradiated with 8 Gy rays. After irradiation, the plate was placed in a 37℃ incubator for 3 h and then the culture medium was replaced for 24 h.

[0163] (3) Discard the culture medium, wash twice with 1×PBS, lyse the cells for 20 min with RIPA lysis buffer containing 1% protease inhibitor, obtain the total cell protein, and determine the protein concentration by BCA method.

[0164] (4) Add 5× protein loading buffer. Finally, heat at 95°C for 10 min to denature the protein, separate by SDS-PAGE and transfer to a polyvinylidene fluoride membrane. Then, block the membrane with 5% skim milk at room temperature for 2 h.

[0165] (5) Rinse the membrane three times with TBST (10 min each time), and react the membrane with the diluted primary antibody at 4°C overnight. Rinse the membrane three times with TBST, and react the membrane with the diluted secondary antibody at room temperature for 1 h.

[0166] (6) Rinse the membrane three times with TBST and develop the protein bands on the membrane with ECL chemiluminescent solution.

[0167] II. Test Results:

[0168] 1. The relative abundance of γ-H2AX using different drugs is shown in the table below.

[0169]

[0170] like Figure 3 and Figure 5 As shown, based on immunofluorescence and Western blot results, RT significantly increased the expression of γ-H2AX in HCT-116 cells, indicating that RT can damage DNA structure. Furthermore, administration of aloe-emodin and olaparib further increased γ-H2AX expression after RT, suggesting that aloe-emodin can significantly increase DNA damage, thereby exacerbating cell death. Figure 4 As shown, aloe-emodin significantly inhibited cell proliferation after HCT-116 radiotherapy. These results all indicate that aloe-emodin enhances the radiosensitization effect of colorectal cancer cells.

[0171] 2. The ATP release concentration (nM) of different drugs is shown in the table below.

[0172]

[0173] like Figure 6 and Figure 7 As shown, the tumor microenvironment after cancer cell death was examined, and the results showed that aloe-emodin could cause calreticulin (CRT) to evert to the cell surface and promote the release of ATP. This result suggests that the radiosensitizing effect of aloe-emodin may be due to promoting the immunogenic death of colorectal cancer cells.

[0174] 3. Expression of Cleaved PARP protein in HCT-116 cells using different drugs

[0175] like Figure 7 As shown, the expression of Cleaved PARP in HCT-116 cells increased after radiotherapy, with significant increases in protein concentration in the model group, aloe-emodin group, and Olaparib group. However, Figure 8 As shown, aloe-emodin did not increase the expression of Cleaved PARP in HCT-116 cells because, even though the concentration of aloe-emodin was increased, the protein concentration of Cleaved PARP did not show a significant change in the horizontal direction.

[0176] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. The use of an aloe-emodin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a PARP-related disease, wherein the PARP-related disease is colorectal cancer, and the medicament is a radiosensitizer.

2. Use according to claim 1, characterized in that, It meets one or two of the following conditions: (1) The PARP-related diseases mentioned are PARP1-related diseases; (2) PARP-related diseases are those that can be improved or cured by inhibiting PARP.

3. The use of an aloe-emodin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of colorectal cancer, wherein the medicament is a radiosensitizer.

4. Use according to claim 3, characterized in that, The radiation intensity of the radiotherapy is 1-100 Gy.

5. Use according to claim 4, characterized in that, The radiation intensity of the radiotherapy is 4 Gy, 8 Gy, 10 Gy, 13 Gy or 16 Gy.

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