Application of harpagoside in preparation of ionizing radiation injury protection medicine

Abstract

The invention discloses an application of harpagoside in preparation of a medicine for preventing ionizing radiation injury. Research finds that ionizing radiation can cause zebrafish embryonic dysplasia, hematopoietic dysfunction and low athletic ability, and harpagoside can effectively prevent and treat the conditions. The research results indicate that the harpagoside is a novel way for preventing and treating ionizing radiation damage.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of harpagoside in the preparation of ionizing radiation damage protection drugs. Background Art

[0002] Radioactive pollution is a serious hazard. It is not only a public health issue, but also a national security issue. Water is a valuable global resource and one of the important ways for radioactive pollutants to spread. Once radioactive substances enter the water, they will accelerate the spread and expand the scope of pollution. Accidental nuclear leakage and improper handling of radioactive waste may cause water pollution. Therefore, the development of new and effective radiation protection drugs is of great significance to protecting the safety of marine life and even human health.

[0003] Currently, in terms of radiation protection drugs, only amifostine is used clinically to treat indications related to ionizing radiation damage. However, due to its rapid metabolism, serious side effects (can cause vomiting, diarrhea and neurotoxicity, etc.) and inconvenient injection, its application in combating high-dose ionizing radiation damage is largely limited. Scientists here have also been working on developing other new radiation protection drugs. The drugs currently authorized by the US FDA for new drug clinical trials include 5-androst-5-ene-3β,17β-diol (5-AED) / Neumune, genistein (BIO300), CBLB502 (Entolimod), HemaMax (recombinant human interleukin-12, rHuIL-12) and ON01210 (chlorobenzyl sulfone derivative). The drugs in the preclinical research stage include AEOL10150 (meso-porphyrin mimetic), ALXN4100TPO, Palifermin, phosphoinositide-3 kinase (PI3K) inhibitor (LY294002), fibroblast growth factor peptide (fibroblast growth factor peptide, FGF-P), histone deacetylase inhibitor-phenylbutyric acid and geldanamycin analog 17-dimethylamino-ethy-lamino-17-demethoxygeldanamycin (17-DMAG), etc.; there are still some chemical, biological and plant herbal medicine ingredients and other radiation protectors that are still in the primary basic research stage. The research and development of these radiation protectors is still a long way from actual clinical application. Summary of the invention

[0004] In the current situation where the problem of radioactive contamination of water bodies is prominent and there is an urgent need for radiation protection of aquatic organisms, the present invention intends to screen radiation protection agents with high safety and excellent effects from natural plant ingredients from the treasure house of traditional Chinese medicine and evaluate their protective effects, so as to provide protection and rescue for aquatic organisms from ionizing radiation damage, and open up new strategies for maintaining the safety of aquatic organisms and species diversity.

[0005] Specifically, the present invention provides an application of harpagoside, wherein the application includes any one of the following:

[0006] 1) Use in the preparation of drugs for preventing or treating ionizing radiation damage;

[0007] 2) Use in the preparation of drugs for preventing or treating abnormal embryonic development induced by ionizing radiation;

[0008] 3) Use in the preparation of drugs for preventing or treating inflammation induced by ionizing radiation;

[0009] 4) Use in the preparation of drugs for regulating the level of embryonic oxidative stress induced by ionizing radiation;

[0010] 5) Use in the preparation of drugs for preventing or treating hematopoietic damage induced by ionizing radiation;

[0011] 6) Use in the preparation of drugs for preventing or treating ionizing radiation-induced hematopoietic erythroid cell reduction;

[0012] 7) Use in the preparation of drugs for preventing or treating ionizing radiation-induced hematopoietic myeloid cell reduction;

[0013] 8) Use in the preparation of drugs for preventing or treating low motor activity induced by ionizing radiation.

[0014] Furthermore, the effective concentration of harpagoside in the drug is 50 μM or 100 μM.

[0015] Furthermore, the ionizing radiation is gamma-rays.

[0016] Further, the ionizing radiation is 6 Gy or 2 Gy.

[0017] Furthermore, the embryo is a zebrafish embryo.

[0018] Furthermore, the abnormal embryonic development includes short body length, low survival rate and deformity.

[0019] Furthermore, the low exercise activity includes abnormal exercise behavior trajectory, short exercise distance, slow average speed, low activity frequency, and short cumulative activity time.

[0020] Furthermore, the hematopoietic damage is hematopoietic damage of zebrafish.

[0021] Furthermore, the erythropoietic cell reduction or myelopoietic cell reduction is erythropoietic cell reduction or myelopoietic cell reduction of zebrafish.

[0022] The present invention also provides a pharmaceutical composition for preventing or treating ionizing radiation damage, wherein the active ingredient of the pharmaceutical composition is harpagoside.

[0023] The drug of the present invention can be used by preparing a pharmaceutical composition in any manner known in the art. This composition comprises an active ingredient, plus one or more pharmaceutically acceptable carriers. This depends on the mode of administration and the designed dosage form. Inorganic or organic carriers known to those skilled in the art for therapeutic inertness include (but are not limited to) lactose, corn starch or its derivatives, talc, vegetable oils, waxes, fats, polyhydroxy compounds such as polyethylene glycol, water, sucrose, ethanol, glycerol, and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers, and the like can also be added thereto, and these substances are used as needed to help the stability of the formulation or to help improve the activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration. The preparations that can be used in this composition can be in the form of the original compound itself, or optionally in the form of its pharmaceutically acceptable salt. The drug of the present invention can be administered alone, or in various combinations, and in combination with other therapeutic agents. The composition prepared in this way can be administered in any appropriate manner known to those skilled in the art as needed. When using the pharmaceutical composition, a safe and effective amount of the drug of the present invention is administered to humans. The specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skills of skilled physicians.

[0024] The drug of the present invention can be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral administration.

[0025] The administration route of the drug of the present invention is not limited, as long as it can exert the desired therapeutic effect or preventive effect, including but not limited to intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, through the skin, through the pleura, local, inhalation, through the mucosa, skin, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, ​​intrahepatic, intratumoral. In some cases, it can be administered systemically. In some cases, it is administered locally.

[0026] The dosage of the drug of the present invention is not limited as long as the desired therapeutic effect or preventive effect is obtained. The dosage of the therapeutic drug or preventive drug of the present invention can be determined using, for example, the therapeutic effect or preventive effect on the disease as an indicator.

[0027] Other therapeutic agents that may be administered with the medicaments of the present invention include therapeutic agents for the treatment and / or prevention of ionizing radiation.

[0028] The present invention also provides a method for preventing or treating ionizing radiation damage, which comprises the following steps: administering harpagoside to a person in need thereof.

[0029] The term "subject in need" of the present invention includes mammals, including but not limited to humans, cows, horses, cats, dogs, rodents or primates. In some embodiments, the subject is a human.

[0030] The term "treatment" generally relates to treatment and therapy of humans or animals (e.g., in veterinary applications) in which some desired therapeutic effect is achieved, such as inhibiting the progression of a disease, and includes reducing the rate of progression, halting the rate of progression, alleviating symptoms of a disease, ameliorating a disease, and curing a disease. Treatment as a preventative measure (i.e., prophylaxis) is also included. For example, the term "treatment" also includes application to patients who have not yet developed a disease but are at risk of developing a disease.

[0031] As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, weakening any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The results of the evaluation of the protective effect of harpagoside on zebrafish embryos exposed to ionizing radiation and the screening of effective concentrations are shown in the figure; A: zebrafish embryo survival rate; B: zebrafish embryo deformity rate;

[0033] Figure 2 The results of the effect of harpagoside on the development of irradiated zebrafish embryos are shown in Figure 1, where A: developmental body length; B: developmental picture; C: survival rate; D: ratio of each deformity type;

[0034] Figure 3 The results of the graph showing the effect of harpagoside on the inflammation level of zebrafish embryos induced by ionizing radiation; A: TNF-α; B: IL-10;

[0035] Figure 4The results of the graph showing the effect of harpagoside on the oxidative stress level of zebrafish embryos induced by ionizing radiation; A: ROS; B: MDA;

[0036] Figure 5 The results of the figure showing the effect of harpagoside on zebrafish hematopoietic damage induced by ionizing radiation;

[0037] A: Fluorescence image of zebrafish tail; B: Statistics of fluorescence intensity of zebrafish tail;

[0038] Figure 6 The results of O-Dianisidine staining to detect the effect of harpagoside on the reduction of zebrafish directional hematopoietic erythroid cells induced by ionizing radiation are shown in the figure; A: hematopoietic erythroid cell staining picture; B: statistical data of the hematopoietic erythroid cell staining area;

[0039] Figure 7 The results of Sudan Black B staining to detect the effect of harpagoside on the reduction of zebrafish directional hematopoietic myeloid cells induced by ionizing radiation; A: hematopoietic myeloid cell staining picture; B: statistical data of the total amount of hematopoietic myeloid cells;

[0040] Figure 8 A graph showing the effect of harpagoside on the locomotor behavior trajectory of ionizing radiation zebrafish larvae;

[0041] Fig. 9 The results of the effect of harpagoside on the movement behavior of zebrafish larvae exposed to ionizing radiation are shown in the figure; A: moving distance; B: average speed; C: active frequency; D: cumulative duration of activity;

[0042] Fig.10 A graph showing the effect of harpagoside on the levels of thyroid hormones T3 and T4 in the tissues of zebrafish larvae exposed to ionizing radiation; wherein, A: thyroid hormone T3; B: thyroid hormone T4. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under the conditions recommended by the manufacturer.

[0044] Example Study on the protective effect of harpagoside against ionizing radiation

[0045] Experimental Materials

[0046] 1) Harpagoside: purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number 20480.

[0047] 2) Amifostine: purchased from MedChemExpress, product number HY-B0639.

[0048] 3) O-dianisidine: purchased from Sigma-Aldrich, catalog number 119-90-4.

[0049] 4) Sudan Black B: purchased from Sigma-Aldrich, catalog number 199664.

[0050] 5) ELISA kits: ELISA kits for thyroid hormones T3 (TW8864H), T4 (TW5687H) and inflammation-related factors TNF-α (TW58004) and IL-10 (TW10072H) were purchased from Shanghai Tongwei Biotechnology Co., Ltd.

[0051] 6) Reactive Oxygen Species (ROS, #G0163W) and Malondialdehyde (MDA, #G0109W) detection kits were purchased from Suzhou Grace Biotechnology Co., Ltd.

[0052] 7) Wild-type AB zebrafish: AB zebrafish at the peak reproductive stage of 4 to 6 months old, the breeders were purchased from Nanjing EzeRinka Biotechnology Co., Ltd.

[0053] 8) Fluorescently labeled transgenic zebrafish: Myeloid-specific green fluorescently labeled zebrafish, Tg(mpx:EGFP), were purchased from China Zebrafish Resource Center (CZRC).

[0054] 9) 137 Csγ-ray irradiation source: purchased from Atomic Energy of Canada Limited, model is USD Autocell40, and the dose rate is 0.8 Gy / min.

[0055] 10) Stereo fluorescence microscope: Nikon, model SMZ1270.

[0056] 11) Multi-mode microplate reader: purchased from Perkin Elmer, USA, model number is Enspire.

[0057] 12) Zebrafish micro-visual behavior analysis system: DanioScope, Noldus Information Technology.

[0058] 13)Image J software: 1.49X, NIH, http: / / rsb.info.nih.gov / ij / .

[0059] Experimental methods

[0060] 1) Evaluation of the protective effect of harpagoside on zebrafish embryos exposed to ionizing radiation and screening of effective concentrations

[0061] Wild-type AB zebrafish were paired in a 1:1 ratio and their embryos were collected.

[0062] Grouping: The mice were grouped into normal control group, ionizing radiation group (6 Gy), ionizing radiation (6 Gy) + 50 μM harpaoside group, ionizing radiation (6 Gy) + 100 μM harpaoside group and ionizing radiation + 200 μM harpaoside group, with 3 replicates in each group.

[0063] Each group of embryos was cultured in a 24-well plate, with 20 embryos per well immersed in 1 mL Holt Buffer (5 mmol / L NaCl, 0.17 mmol / L KCl, 0.33 mmol / L CaCl2, 0.66 mmol / L NaHCO3, pH 7.2) and cultured in a constant temperature incubator at 28 degrees. At 3 hpf (hours post-fertilization), the corresponding concentration of drugs was added to the 24-well plate, and the ionizing radiation group was irradiated with 6 Gy γ-rays at 4 hpf and cultured. Fresh Holt Buffer was replaced every day, and the corresponding concentration of harpagoside drugs was added. The survival rate of each group of embryos was observed and counted under a stereo microscope at 24 hpf, and the malformation rate of each group of embryos was counted at 72 hpf. Based on this, the protective effect of harpagoside on ionizing radiation zebrafish embryos was preliminarily evaluated and the effective protective concentration was screened.

[0064] 2) Effects of harpagoside on the development of irradiated zebrafish embryos

[0065] Wild-type AB zebrafish were paired in a 1:1 ratio and their embryos were collected.

[0066] Grouping: The animals were divided into normal control group, ionizing radiation group (6 Gy), ionizing radiation (6 Gy) + 50 μM harpaoside group and ionizing radiation (6 Gy) + 0.1 mM amifostine (amifostine was selected as the positive control drug, the same below), with 3 replicates in each group.

[0067] Each group of embryos was cultured in a 24-well plate, with 20 embryos per well immersed in 1 mL of Holt Buffer, and cultured in a constant temperature incubator at 28 degrees. At 3 hpf (hours post-fertilization), two drugs of corresponding concentrations were added to the 24-well plate, and the ionizing radiation group was irradiated with 6 Gy γ-rays at 4 hpf. Fresh Holt Buffer was replaced every day, and the corresponding concentration of harpagoside or amifostine was added. The survival rate of embryos in each group was counted under a stereomicroscope at 3, 24, 48 and 72 hpf. The body length of normal juveniles in each group was counted and photographed at 72 hpf. The ratio of embryos with different deformity grades (grade I: normal developmental morphology; grade II: mild tail curvature; grade III: moderate tail curvature + moderate pericardial edema + moderate yolk cyst, etc.; grade IV: severe tail curvature + severe pericardial edema + severe yolk cyst, etc.) in each group was counted at 72 hpf. The effects of harpagoside on the survival, developmental process and developmental morphology of γ-ray irradiated zebrafish embryos were evaluated.

[0068] 3) Effects of harpagoside on ionizing radiation-induced inflammation in zebrafish embryos

[0069] Wild-type AB zebrafish were paired in a 1:1 ratio and their embryos were collected.

[0070] Grouping: The mice were divided into normal control group, ionizing radiation group (6 Gy), ionizing radiation (6 Gy) + 50 μM harpaoside group and ionizing radiation (6 Gy) + 0.1 mM amifostine group, with 3 replicates in each group.

[0071] Each group of embryos was cultured in a 24-well plate, with 30 embryos per well immersed in 1 mL of Holt Buffer, and cultured in a constant temperature incubator at 28 degrees. At 3 hpf (hours post-fertilization), two drugs of corresponding concentrations were added to the 24-well plate, and the ionizing radiation group was irradiated with 6 Gy γ-rays at 4 hpf. Fresh Holt Buffer was replaced every day, and the corresponding concentrations of harpagoside or amifostine were added. Embryonic tissues of each group were collected at 72 hpf, homogenized in PBS ice bath, and the expression levels of two inflammatory-related factors TNF-α and IL-10 in the tissues were detected using ELISA commercial kits. Based on this, the effect of harpagoside on the level of inflammation induced by ionizing radiation in zebrafish embryos was evaluated.

[0072] 4) Effects of harpagoside on oxidative stress levels in zebrafish embryos induced by ionizing radiation

[0073] Wild-type AB zebrafish were paired in a 1:1 ratio and their embryos were collected.

[0074] Grouping: The mice were divided into normal control group, ionizing radiation group (6 Gy), ionizing radiation (6 Gy) + 50 μM harpaoside group and ionizing radiation (6 Gy) + 0.1 mM amifostine group, with 3 replicates in each group.

[0075] Each group of embryos was cultured in a 24-well plate, with 30 embryos per well immersed in 1 mL of Holt Buffer, and cultured in a constant temperature incubator at 28 degrees. At 3 hpf (hours post-fertilization), two drugs of corresponding concentrations were added to the 24-well plate, and the ionizing radiation group was irradiated with 6 Gy γ-rays at 4 hpf. Fresh Holt Buffer was replaced every day, and the corresponding concentrations of harpagoside or amifostine were added. Embryonic tissues of each group were collected at 72 hpf, homogenized in PBS ice bath, and the levels of two oxidative stress-related markers ROS and MDA in the tissues were detected using a commercial kit. Based on this, the effect of harpagoside on the oxidative stress level of zebrafish embryos induced by ionizing radiation was evaluated.

[0076] 5) Effects of harpagoside on hematopoietic damage induced by ionizing radiation in zebrafish

[0077] The Tg(mpx:EGFP) fluorescently labeled transgenic zebrafish were paired in a 1:1 ratio, and the embryos were collected in Holt Buffer and cultured in a 28-degree constant temperature incubator, with fresh Holt Buffer replaced every day. At 2 days post-fertilization (dpf), zebrafish fry with good development and consistent fluorescence intensity were selected under a stereofluorescence microscope.

[0078] A normal control group, an ionizing radiation group (final screening dose, 2 Gy), an ionizing radiation (2 Gy) + 50 μM harpaoside group, and an ionizing radiation (2 Gy) + 0.1 mM amifostine group were set up, with 3 replicates in each group. The juveniles in each group were cultured in a 24-well plate, and each well contained 20 juveniles immersed in 1 mL Holt Buffer. The juveniles in the ionizing radiation group were irradiated with 2 Gy of γ-rays once, and the ionizing radiation (2 Gy) + 50 μM harpaoside and ionizing radiation (2 Gy) + 0.1 mM amifostine groups were treated with 50 μM harpaoside or 0.1 mM amifostine, respectively, 1 h before irradiation. Fresh Holt Buffer was replaced every day after irradiation, and the corresponding concentration of harpaoside or amifostine was supplemented. At 72 hours post-irradiation (hpi), the whole-body fluorescence images of zebrafish in each group were taken under a stereo fluorescence microscope, and the changes in the whole-body fluorescence intensity of zebrafish in each group were counted using Image J software. The above experiments were used to preliminarily evaluate the effects of harpagoside on ionizing radiation-induced hematopoietic damage in zebrafish.

[0079] 6) O-Dianisidine staining to detect the effect of harpagoside on ionizing radiation-induced reduction of directional hematopoietic erythroid cells in zebrafish

[0080] Weigh 100 mg of O-Dianisidine powder and dissolve it in 70 mL of anhydrous ethanol. Store at 4°C away from light. Add 2 mL of ddH2O, 2 mL of O-Dianisidine solution, 500 mL of 0.1 M sodium acetate solution at pH 4.5, and 100 μL of 30% H2O2 to a 15 mL centrifuge tube in order, mix well and set aside. Pair male and female Tg (mpx: EGFP) fluorescently labeled transgenic zebrafish in a 1:1 ratio, collect the embryos produced in Holt Buffer, and culture them in a 28-degree constant temperature incubator, replacing fresh Holt Buffer every day. At 2 dpf, select zebrafish fry with good development and consistent fluorescence intensity under a stereofluorescence microscope.

[0081] A normal control group, an ionizing radiation (2Gy) group, an ionizing radiation (2Gy) + 50μM harpaoside group, and an ionizing radiation (2Gy) + 0.1mM amifostine group were set up, with 3 replicates in each group. The juveniles in each group were cultured in a 24-well plate, and each well contained 20 juveniles immersed in 1mL Holt Buffer. The juveniles in the ionizing radiation group were irradiated with 2Gy of γ-rays once, and the ionizing radiation (2Gy) + 50μM harpaoside group and the ionizing radiation (2Gy) + 0.1mM amifostine group were treated with 50μM harpaoside or 0.1mM amifostine, respectively, 1h before irradiation. After irradiation, fresh Holt Buffer was replaced every day, and the corresponding concentration of harpaoside or amifostine was supplemented. The juveniles in each group were collected at 3.5dpf, placed in a 1.5mL EP tube, and 500μL of staining working solution was added. The juveniles were stained for 30 to 60min on a shaker at room temperature in the dark. During this period, several fry can be taken out and placed under a microscope to observe the staining effect. After staining, remove the dye solution and wash with PBST three times, 10 minutes each time. Add 1mL 4% paraformaldehyde and fix for more than 2 hours. Wash with PBST three times, 10 minutes each time, and store in 100% glycerol. Collect images under a stereo microscope.

[0082] 7) Sudan Black B staining to detect the effect of harpagoside on the reduction of zebrafish hematopoietic myeloid cells induced by ionizing radiation

[0083] Tg(mpx:EGFP) fluorescently labeled transgenic zebrafish were paired in a 1:1 ratio, and the embryos were collected in Holt Buffer and cultured in a 28-degree constant temperature incubator, with fresh Holt Buffer replaced every day. At 2dpf, zebrafish larvae with good development and consistent fluorescence intensity were selected under a stereofluorescence microscope, and a normal control group, an ionizing radiation (2Gy) group, an ionizing radiation (2Gy) + 50μM harpagoside group, and an ionizing radiation (2Gy) + 0.1mM amifostine group were set up, with 3 replicates in each group. The larvae in each group were cultured in a 24-well plate, and each well contained 20 larvae immersed in 1mL Holt Buffer. The juveniles in the ionizing radiation group were irradiated with 2Gy of γ-rays at once. One hour before irradiation, the ionizing radiation (2Gy) + 50μM harpaoside group and the ionizing radiation (2Gy) + 0.1mM amifostine group were treated with 50μM harpaoside or 0.1mM amifostine, respectively. After irradiation, fresh Holt Buffer was replaced every day, and the corresponding concentration of harpaoside or amifostine was supplemented. The juveniles of each group were collected at 3.5dpf (days post-fertilization), fixed with 4% paraformaldehyde, and shaken at 4℃ overnight. Weigh 0.6g Sudan Black B powder and dissolve it in 200mL of anhydrous ethanol, filter it with filter paper and set aside. The fixed zebrafish juveniles of each group were rinsed with PBST 3 times, 5min each time. Add 500μL Sudan Black B dye solution to the 1.5mL EP tube containing embryos, and stain for 30-60min at room temperature in a shaker in the dark. During this period, several fry can be taken out and placed under a microscope to observe the staining effect. After obvious staining signals are seen, rinse with 100% anhydrous ethanol until the signals are clearly visible. Place each group of fry in 100% glycerol for image acquisition.

[0084] 8) Effects of harpagoside on the motor behavior of irradiated zebrafish larvae

[0085] Wild-type AB zebrafish were paired in a 1:1 ratio and their embryos were collected.

[0086] The animals were divided into groups of normal control, ionizing radiation group (6 Gy), ionizing radiation (6 Gy) + 50 μM harpagoside group and ionizing radiation (6 Gy) + 0.1 mM amifostine, with 3 replicates in each group. Embryos of each group were cultured in 24-well plates, with 30 embryos per well immersed in 1 mL Holt Buffer, and cultured in a constant temperature incubator at 28 degrees. At 3 hpf, two drugs of corresponding concentrations were added to the 24-well plates, and the ionizing radiation group was irradiated with 6 Gy γ-rays at 4 hpf. Fresh Holt Buffer was replaced every day, and harpagoside or amifostine of corresponding concentrations were added. Embryonic tissues of each group were collected at 5 dpf, and 7 to 8 zebrafish larvae with normal morphological development were selected from each group. Each was placed in a 96-well microwell plate, and each well was filled with Holt Buffer. The well plate was placed in a zebrafish micro-visual behavior analyzer for free swimming behavior trajectory collection and analysis. Data were collected for statistical analysis of indicators such as moving distance, average moving speed, active frequency, and active cumulative duration. Embryonic tissues from each group were collected and homogenized in PBS ice bath, and the protein expression levels of thyroid hormones T3 and T4 in the tissues were detected using ELISA commercial kits. The effects of harpagoside on the motor behavior characteristics of zebrafish larvae induced by ionizing radiation were evaluated.

[0087] Experimental Results

[0088] 1) Evaluation of the protective effect of harpagoside on zebrafish embryos exposed to ionizing radiation and screening of effective concentrations

[0089] Ionizing radiation can lead to a decrease in the survival rate of zebrafish embryos at 24 hpf and an increase in the deformity rate at 72 hpf, while 50 μM and 100 μM harpagoside treatment can reverse this trend to varying degrees, with the 50 μM treatment having a better effect. Therefore, harpagoside can play a radiation protective role on zebrafish embryos, and 50 μM is the optimal protective concentration ( Figure 1 ).

[0090] (2) Effects of harpagoside on the development of irradiated zebrafish embryos

[0091] Ionizing radiation can cause the body length of 72hpf zebrafish to shorten and the survival rate of 3-72hpf to decrease, while treatment with harpaoside (50μM) or amifostine (100μM) can increase the body length and improve the survival rate of zebrafish to varying degrees, and amifostine has a relatively better effect ( Figure 2 AC). Ionizing radiation can cause 72hpf zebrafish embryos to have malformation phenotypes of different severity (expressed in different grades), while treatment with harpagoside (50μM) or amifostine (100μM) can increase the proportion of embryos with mild (grade I) malformations and reduce the proportion of embryos with severe (grade IV) malformations, and this trend is more obvious in amifostine treatment ( Figure 2 D).

[0092] 3) Effects of harpagoside on ionizing radiation-induced inflammation in zebrafish embryos

[0093] Ionizing radiation can cause an increase in the level of inflammation in zebrafish embryonic tissues (the protein expression of the pro-inflammatory factor TNF-α is upregulated, and the protein expression of the anti-inflammatory factor IL-10 is downregulated), while harpagoside (50μM) or amifostine (100μM) can reverse this trend to varying degrees, and the reversal effect of amifostine treatment is more significant ( Figure 3 ).

[0094] 4) Effects of harpagoside on oxidative stress levels in zebrafish embryos induced by ionizing radiation

[0095] Ionizing radiation can cause an increase in the levels of ROS and MDA, markers of oxidative stress, in zebrafish embryonic tissues, while harpagoside (50 μM) or amifostine (100 μM) can reverse this trend to varying degrees, and the reversal effect of amifostine treatment is more significant ( Figure 4 ).

[0096] (5) Effects of harpagoside on ionizing radiation-induced hematopoietic damage in zebrafish

[0097] Ionizing radiation can weaken the fluorescence intensity of the tail of Tg(mpx:EGFP) zebrafish, while treatment with harpaoside (50μM) or amifostine (100μM) can restore the fluorescence intensity to a certain extent, and the effect of amifostine treatment is more obvious ( Figure 5 ).

[0098] 6) O-Dianisidine staining to detect the effect of harpagoside on ionizing radiation-induced reduction of directional hematopoietic erythroid cells in zebrafish

[0099] Ionizing radiation can reduce the number of hematopoietic erythroid cells in Tg(mpx:EGFP) zebrafish (most obvious in the heart), while treatment with harpagoside (50μM) or amifostine (100μM) can reverse this trend to some extent ( Figure 6 ).

[0100] 7) Sudan Black B staining to detect the effect of harpagoside on the reduction of zebrafish hematopoietic myeloid cells induced by ionizing radiation

[0101] Ionizing radiation can reduce the number of hematopoietic myeloid cells in Tg(mpx:EGFP) zebrafish (most obvious in the tail), while treatment with harpagoside (50μM) or amifostine (100μM) can reverse this trend to some extent ( Figure 7 ).

[0102] 8) Effects of harpagoside on the motor behavior of irradiated zebrafish larvae

[0103] Ionizing radiation can weaken the movement activity and reduce the behavioral trajectories of zebrafish larvae, while treatment with harpagoside (50 μM) or amifostine (100 μM) can reverse this trend to some extent ( Figure 8 ). According to the statistical data, ionizing radiation can reduce the moving distance, average speed, active frequency and cumulative active duration of zebrafish larvae, while treatment with harpagoside (50μM) or amifostine (100μM) can increase these four indicators to varying degrees ( Fig. 9 ). The results of testing thyroid hormone levels showed that ionizing radiation could significantly reduce the levels of thyroid hormones T3 and T4 in the tissues of larval fish, while treatment with harpagoside (50 μM) or amifostine (100 μM) could increase the levels to varying degrees ( Fig.10 ).

[0104] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. Application of harpagoside, the application comprising any one of the following: 1) Use in the preparation of drugs for preventing or treating ionizing radiation damage; 2) Use in the preparation of drugs for preventing or treating abnormal embryonic development induced by ionizing radiation; 3) Use in the preparation of drugs for preventing or treating inflammation induced by ionizing radiation; 4) Use in the preparation of drugs for regulating the level of embryonic oxidative stress induced by ionizing radiation; 5) Use in the preparation of drugs for preventing or treating hematopoietic damage induced by ionizing radiation; 6) Use in the preparation of drugs for preventing or treating ionizing radiation-induced hematopoietic erythroid cell reduction; 7) Use in the preparation of drugs for preventing or treating ionizing radiation-induced hematopoietic myeloid cell reduction; 8) Use in the preparation of drugs for preventing or treating low motor activity induced by ionizing radiation.

2. The use according to claim 1, characterized in that: The effective concentration of harpagoside in the drug is 50 μM or 100 μM.

3. The use according to claim 1, characterized in that: The ionizing radiation is 6 Gy or 2 Gy.

4. The use according to claim 1, characterized in that: The embryos are zebrafish embryos.

5. The use according to claim 1, characterized in that: The abnormal embryonic development includes short body length, low survival rate and deformity.

6. The use according to claim 1, characterized in that: The low exercise activity includes abnormal exercise behavior trajectory, short exercise distance, slow average speed, low activity frequency, and short cumulative activity time.

7. The use according to claim 1, characterized in that: The hematopoietic injury is hematopoietic injury of zebrafish.

8. The use according to claim 1, characterized in that: The hematopoietic erythroid cell reduction or hematopoietic myeloid cell reduction is the hematopoietic erythroid cell reduction or hematopoietic myeloid cell reduction of zebrafish.

9. A pharmaceutical composition for preventing or treating ionizing radiation damage, wherein the active ingredient of the pharmaceutical composition is harpagoside.

10. The use according to claim 9, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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