Application of chicoric acid in preparation of medicine for improving hematopoietic injury caused by radiation

Abstract

The invention discloses application of chicoric acid in preparation of a medicine for improving hematopoietic injury caused by radiation, and belongs to the technical field of biological medicine. In order to solve the problem that an effective intervention means for hematopoietic system injury caused by ionizing radiation is lacked in the prior art, the invention provides application of chicoric acid in preparation of drugs for improving hematopoietic injury caused by radiation, and shows a dual protection mechanism of chicoric acid acting on a hematopoietic differentiation key regulation node. The hematopoietic function is synergistically improved by regulating EPO and other hematopoietic factors and progenitor cell populations, meanwhile, the anti-oxidation effect is achieved, radiation damage is relieved, definite active ingredients and action targets are provided for preparing the medicine for preventing and treating hematopoietic damage caused by radiation, and the application value of clinical transformation is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of chicoric acid in the preparation of drugs to improve radiation-induced hematopoietic damage. Background Technology

[0002] With the rapid development of nuclear technology and its widespread application in fields such as medicine, agriculture, and aerospace, human exposure to ionizing radiation is increasing, and the corresponding health risks are receiving growing attention. Ionizing radiation, as high-energy rays capable of inducing ionization of matter, primarily causes damage to the body through two pathways: first, it directly acts on biological macromolecules such as DNA and proteins within cells, causing single-strand or double-strand breaks in DNA, leading to cell dysfunction or death; second, it indirectly induces oxidative stress by ionizing water molecules in the body, generating a large number of free radicals, thereby damaging multiple systems such as hematopoiesis, the immune system, and the nervous system. Among these, the actively proliferating hematopoietic system is particularly sensitive to radiation; radiation directly damages hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), causing DNA breaks, cell cycle arrest, and increased apoptosis, leading to hematopoietic failure, manifested as pancytopenia in peripheral blood, and causing clinical symptoms such as anemia, bleeding, and infection.

[0003] Currently, while commonly used chemically synthesized radiation protectants offer some protection, they suffer from significant toxic side effects and inconvenient administration. Existing drugs, such as amifostine, provide some protection, but their protective effect on the hematopoietic system is insufficient and they exhibit marked toxic side effects. Cytokine drugs (such as G-CSF and GM-CSF) are primarily used for symptomatic and supportive treatment after radiation exposure, lacking ideal prophylactic options. Therefore, those skilled in the art are eager to develop highly effective and low-toxicity radiation protectants, particularly by extracting potentially active ingredients from natural medicinal resources, which has become an important research direction in this field.

[0004] Echinacea purpurea is a traditional medicinal plant used in North America and Europe for a long time, often for preventing and treating colds and enhancing immune function. Its pharmacologically active components are complex, among which chicoric acid (CA) is widely considered a key component for its immunomodulatory effects, and has been reported to possess various biological activities such as anti-inflammatory, antioxidant, and immunomodulatory properties. Although the immune-enhancing effects of echinacea are well-known, systematic research on its specific component—chicoric acid—regarding radiation protection, especially its protective effects on the hematopoietic system, is still insufficient. Summary of the Invention

[0005] This invention addresses the lack of effective intervention methods for hematopoietic system damage caused by ionizing radiation in the prior art by providing the application of chicoric acid in the preparation of drugs to improve radiation-induced hematopoietic damage.

[0006] One of the objectives of this invention is to provide the application of chicoric acid in the preparation of drugs to improve radiation-induced hematopoietic damage.

[0007] In a preferred embodiment of the present invention, the hematopoietic damage is caused by ionizing radiation.

[0008] In a preferred embodiment of the present invention, the structural formula of chicoric acid is: .

[0009] In a preferred embodiment of the present invention, the molecular formula of chicoric acid is Ci 22 H 18 O 12 The molecular weight is 474.371.

[0010] In a preferred embodiment of the invention, the drug comprises chicoric acid and pharmaceutically acceptable excipients.

[0011] In a preferred embodiment of the present invention, the dosage form of the drug includes, but is not limited to, injection, capsule or tablet.

[0012] Compared with the prior art, the beneficial effects of the present invention are: the present invention is the first to discover that chicoric acid, the active ingredient of echinacea extract, can significantly improve radiation-induced anemia and thrombocytopenia, and its mechanism of action has the following three synergistic effects: At the hematopoietic progenitor cell level, chicoric acid specifically promotes the recovery of hematopoietic progenitor cells (HPCs), common myeloid progenitor cells (CMPs), and myeloid-erythroid progenitor cells (MEPs) in the bone marrow. Among them, MEPs, as key regulatory nodes capable of bidirectional differentiation into erythroid and megakaryocytic lines, play a decisive role in maintaining the normal production of erythrocytes and platelets. Experiments have confirmed that radiation leads to the depletion of these progenitor cells, and chicoric acid intervention can effectively reverse this damage.

[0013] At the level of hematopoietic regulatory factors, chicoric acid significantly increases the level of erythropoietin (EPO) in the serum of irradiated organisms. As a core cytokine driving erythroid hematopoiesis, the recovery of EPO levels and the restoration of the MEP population synergistically promote the regeneration and differentiation of erythroid cells.

[0014] At the level of anti-oxidative stress, chicoric acid alleviates radiation-induced oxidative stress damage and protects the hematopoietic microenvironment through multiple pathways by scavenging free radicals, reducing the level of malondialdehyde (MDA), a lipid peroxidation product, and enhancing the activity of endogenous superoxide dismutase (SOD).

[0015] This invention reveals the dual protective mechanism of chicoric acid on key regulatory nodes of hematopoietic differentiation. It improves hematopoietic function by regulating hematopoietic factors such as EPO and progenitor cell populations, while exerting antioxidant effects to reduce radiation damage. This provides a clear active ingredient and target for the preparation of drugs to prevent and treat radiation-induced hematopoietic damage, and has clinical translational application value. Attached Figure Description

[0016] Figure 1 The figure shows the effect of chicoric acid on the viability of AHH-1 cells; * indicates P <0.05, ** is P <0.01; the vertical axis represents cell viability, and the horizontal axis represents chicoric acid concentration; Figure 2 The image shows the protective effect of chicoric acid against radiation-damaged AHH-1 cells; *** represents... P <0.001, **** is P <0.0001; compared with the control group, #### was P <0.0001; the vertical axis represents cell viability, and the horizontal axis represents chicoric acid concentration; Control represents the control group, and Radiation represents the radiation control group; Figure 3 Figure 1 shows the effect of Echinacea extract-chicoric acid administration on peripheral blood RBCs and related indicators in irradiated mice; A is the RBC detection result; B is the HGB detection result; C is the HCT detection result; * indicates the difference compared to the model group. P <0.05, ** is P <0.01, *** is P <0.001, **** is P <0.0001; compared with the control group, #### is P <0.0001 (±s, n=7); Figure 4 The graph shows the effect of echinacea extract-chicoric acid on platelet count in irradiated mice; * indicates the effect compared to the model group. P <0.05; compared with the control group, #### was P <0.0001 (±s, n=7); Figure 5 The graph shows the effect of echinacea extract-chicoric acid administration on peripheral blood immune cells in irradiated mice; A is the LYMPH% statistical graph; B is the NEUT% (n=7) statistical graph; compared with the model group, *** is P <0.001, **** is P<0.0001; compared with the control group, #### is P <0.0001 (±s); Figure 6 Single-cell image of mouse bone marrow for flow cytometry gating strategy; Figure 7 The graphs show the effects of echinacea extract-chicoric acid administration on hematopoietic cells in the bone marrow of irradiated mice; A is the CLP statistical graph; B is the HPCs statistical graph; C is the CMP statistical graph; D is the MEP statistical graph; the vertical axis values ​​are the relative values ​​circled in each section, compared with the model group, * indicates... P <0.05, ** is P <0.01, *** is P <0.001, **** is P <0.0001; compared with the control group, # is P <0.05, #### is P <0.0001 (±s); Figure 8 The graph shows the effect of echinacea extract-chicoric acid administration on the serum MDA and SOD levels of irradiated mice; A is the MDA statistical graph; B is the SOD statistical graph; compared with the model group, **** is... P <0.0001; compared with the control group, #### is P <0.0001 (±s); Figure 9 The graph shows the effect of echinacea extract-chicoric acid administration on serum EPO levels in irradiated mice; the vertical axis represents the relative values ​​circled in each section, with *** being the highest compared to the model group. P <0.001, **** is P <0.0001; compared with the control group, #### is P <0.0001 (±s); Figure 10 The image shows the HE staining results of bone marrow from mice in each treatment group 14 days after radiation. Detailed Implementation

[0017] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0019] The chicoric acid used in the following examples was purchased from Chengdu Ephacoemulsification Co., Ltd.; the chicoric acid extract (4% chicoric acid content, yellow-green powder) was purchased from Shaanxi Newfu Co., Ltd.; the Cell Counting Kit-8 (CCK-8) assay kit was purchased from Taoshu Biotechnology Co., Ltd.; the malondialdehyde kit was purchased from Edison Biotechnology Co., Ltd.; the erythropoietin (EPO) ELISA research kit, the mouse interleukin-1β (IL-1β) ELISA research kit, the mouse interleukin-6 (IL-6) ELISA research kit, the mouse tumor necrosis factor-α (TNF-α) ELISA research kit, and the mouse superoxide dismutase (SOD) ELISA research kit were purchased from Jiangsu Enzyme Immunoassay Co., Ltd.; human peripheral blood B lymphocytes (AHH-1) were kindly provided by the laboratory of Professor Zhou Pingkun of the Institute of Radiation Medicine, Academy of Military Medical Sciences; the flow cytometry antibodies used are listed in Table 1.

[0020] Table 1

[0021] All experiments in the following examples were independently repeated at least three times. Data are expressed as mean ± standard deviation. Statistical analysis and graphing were performed using GraphPad Prism (version 10.4.2). One-way ANOVA was used to analyze differences among multiple groups, and t-tests were used for statistical analysis between two groups. P A value <0.05 is considered statistically significant.

[0022] Example 1: 1. Preparation of AHH-1 cells in logarithmic growth phase AHH-1 cells were cultured in suspension in upright culture flasks using RPMI-1640 complete medium (purchased from Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% penicillin and streptomycin. The cell density was maintained at 1×10⁻⁶ cells / year. 6 From 2×10⁻⁶ cells / ml to 2×10⁻⁶ 6Between cells / ml, half the medium should be changed every two days. When changing the medium, first remove part of the supernatant culture medium, and then add fresh culture medium at a ratio of 1:2 or 1:4 for dilution and passage. For cell cryopreservation, take cells in the logarithmic growth phase, collect them by centrifugation, resuspend them in cell cryopreservation solution (RPMI-1640 basal medium: fetal bovine serum: DMSO = 7:2:1), and then store them in an ultra-low temperature freezer at -80℃ for long-term storage.

[0023] 2. CCK-8 assay for cell viability (1) Effect of chicoric acid on cell viability: AHH-1 cells in the logarithmic growth phase were taken and divided into 1×10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well plates. The cells were divided into the following groups: a control group (cell-free, 0 μmol / L chicoric acid), a model group (AHH-1 cells, 0 μmol / L chicoric acid), and chicoric acid treatment groups at different concentrations (AHH-1 cells, 10, 20, 40, 80, 120, 160, 200, 240, 320 μmol / L chicoric acid), with 6 replicates per group. Chicoric acid stock solution was pre-prepared at 80 mM concentration and serially diluted to the desired concentration using RPMI-1640 medium containing 2% fetal bovine serum. After administration, AHH-1 cells were cultured for 48 hours, followed by the addition of 10% CCK-8 solution to each well and incubation in the dark for 3 hours. The absorbance (OD value) of each well was measured using a microplate reader at 450 nm.

[0024] Cell viability (%) = (OD-treated group - OD blank group) / (OD model group - OD blank group) × 100% like Figure 1 As shown, chicoric acid did not exhibit significant toxicity in the concentration range of 10-320 μmol / L, and the viability of AHH-1 cells showed a slight increasing trend with increasing chicoric acid concentration, suggesting that it has a certain promoting effect on lymphocyte viability.

[0025] (2) Effect of chicoric acid on cell viability after irradiation: Based on the determination of the safe concentration range of chicoric acid for AHH-1 cells (10-320 μmol / L), the cells were divided into blank group, model group, radiation control group, radiation model group, and chicoric acid administration groups with different concentrations (10-320 μmol / L); the cells were pretreated with chicoric acid 24 hours before ionizing radiation, followed by 4 Gy 60 After a single Co-γ irradiation, cells were cultured for 48 hours. 10% CCK-8 solution was added to each well, and the cells were incubated in the dark for 3 hours to detect the effect of different concentrations of chicoric acid on cell viability after irradiation.

[0026] Cell viability (%) = (OD drug administration group & OD radiation control group - OD blank group) / (OD model group - OD blank group) × 100% like Figure 2 As shown, compared with the blank control group, the cell viability of the ionizing radiation model group was significantly reduced ( p <0.0001 indicates successful model establishment; compared with the ionizing radiation model group, the 10-320 μmol / L chicoric acid treatment groups alleviated the decrease in cell viability caused by ionizing radiation to varying degrees; indicating that chicoric acid has a certain protective effect against ionizing radiation, with the 120 μmol / L concentration group showing the most significant effect. p <0.0001).

[0027] 3. Animal experiments Fifty male C57BL / 6J mice (weighing 19-22g, SPF grade) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in a special animal room at the Animal Center of the Academy of Military Medical Sciences, with the indoor temperature maintained at 26±0.5℃ and the relative humidity at 50%±5%, and a 12-hour light / dark cycle implemented. The animal room was equipped with an independent air purification system and provided sterile distilled water, SPF grade maintenance feed and corn cob bedding. The mice had free access to food and water.

[0028] Experimental mice were randomly divided into 5 groups: control group (NC), irradiation group (MOD), positive control drug amifostine group, low-dose echinacea extract-chicoric acid group (2.5 g / kg / d), and high-dose group (10 g / kg / d). After 7 days of acclimatization, administration began and continued for 7 days. Afterward, the mice were transferred to the Institute of Radiation Medicine, Academy of Military Medical Sciences. 60 In a Co radiation field, mice were given a single whole-body 4.0 Gy γ-ray irradiation at a dose rate of 52.18 R / min to establish a hematopoietic injury model. After irradiation, the mice were returned to the animal center for continued feeding and were administered the drug for 14 consecutive days.

[0029] (1) Peripheral blood cell count To investigate the effect of chicoric acid on peripheral blood counts in irradiated mice, blood samples were collected from each group of mice via orbital blood collection after administration. The samples were placed in heparin sodium anticoagulant tubes, and peripheral blood cell counts were performed using a Sysmex XN-1000V blood analyzer. The measured parameters included platelets (PLT), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), neutrophils (NEU), and lymphocytes (LYM).

[0030] On day 14 post-irradiation, peripheral blood counts of mice in each group were analyzed. Figure 3 As shown, compared with the control group, the irradiated mice had significantly lower red blood cell count (RBC), hemoglobin concentration (HGB), and hematocrit (HCT). p <0.0001 indicates that the irradiated mice were in anemic state. After intervention with echinacea extract-chicoric acid, the downward trend of the above indicators was effectively reversed in all treatment groups. Among them, the RBC, HGB, and HCT levels in the 10 g / kg dose group recovered particularly significantly, approaching and slightly exceeding the levels of the control group. This indicates that echinacea extract-chicoric acid has an ameliorative effect on irradiation-induced anemia in mice.

[0031] (2) Preparation and phenotypic detection of single-cell systems Bone marrow single-cell system: Femurs of mice in each group were taken, and the attached muscles and connective tissues were removed. The bone marrow cavity was rinsed with 1 mL PBS, and the bone marrow tissue was collected and thoroughly dispersed to prepare a single-cell suspension. Finally, the suspension was filtered through a 70 μm cell sieve.

[0032] Phenotypic detection: The cell suspension was labeled with the corresponding fluorescent antibody and the mouse bone marrow single cell samples were detected by flow cytometry (BD FACSAria™ III). The specific phenotypes analyzed are shown in Table 2.

[0033] Table 2

[0034] Ionizing radiation also caused a sharp decrease in platelet count (PLT) in the model group mice, posing a risk of bleeding. Figure 4 As shown, treatment with echinacea extract-chicoric acid can increase the PLT level in irradiated mice, with the 10 g / kg dose group showing a more significant increase (P<0.05), suggesting that it also has a positive promoting effect on megakaryocytic hematopoiesis.

[0035] Echinacea extract-chicoric acid (2.5 g / kg) has a regulatory effect on the imbalance of peripheral blood immune cell ratios induced by radiation. For example... Figure 5 As shown, compared with the control group, the proportion of lymphocytes (LYMPH%) in the irradiated model group was significantly decreased, while the proportion of neutrophils (NEUT%) was significantly increased. p <0.0001), indicating an immune imbalance. Positive control drugs (aminoflavin) and echinacea extract-chicoric acid (2.5 g / kg) can effectively regulate the imbalance of peripheral blood immune cell ratios caused by irradiation. p <0.0001, p<0.001), correcting the abnormal state of decreased lymphocytes and increased neutrophils. This indicates that echinacea extract-chicoric acid can alleviate the imbalance of peripheral blood immune cells in irradiated mice.

[0036] To further investigate the protective mechanism of echinacea extract-chicoric acid on the hematopoietic system of irradiated mice, this study analyzed the changes in hematopoietic stem / progenitor cells in mouse bone marrow using flow cytometry (see gating strategy). Figure 6 ).like Figure 7 As shown, compared with the control group, the number of common lymphoid progenitor cells (CLP), hematopoietic progenitor cells (HPCs), common myeloid progenitor cells (CMP), and myeloid-erythroid progenitor cells (MEP) in the bone marrow of mice in the irradiation model group was significantly reduced. After intervention with echinacea extract-chicoric acid, the number of the above-mentioned key hematopoietic progenitor cells in each treatment group was restored to varying degrees. Combined with previous results, it is suggested that echinacea extract-chicoric acid may promote the regeneration and differentiation of hematopoietic stem / progenitor cells, thereby promoting the recovery of peripheral blood cells and ultimately promoting the reconstruction of the immune and hematopoietic systems after radiation injury.

[0037] (3) ELISA detection of the levels of related factors in mouse serum All operations were performed according to the instructions, including sample addition, enzyme addition, incubation, washing, color development, and termination. The absorbance of each well was measured sequentially at a wavelength of 450 nm. Specific detection indicators included erythropoietin (EPO) and superoxide dismutase (SOD).

[0038] Malondialdehyde (MDA) detection: The thiobarbituric acid method was used to assess lipid peroxidation levels by detecting the condensation reaction product of MDA and thiobarbituric acid; all experimental procedures were strictly followed in accordance with the kit instructions.

[0039] like Figure 8 As shown, compared with the control group, the serum MDA content of mice in the irradiation model group was significantly increased ( P <0.0001), while SOD activity was significantly reduced ( P <0.0001 indicates a significant increase in oxidative stress levels in mice after irradiation. Intervention with echinacea extract-chicoric acid significantly reversed this trend in all treatment groups: decreasing MDA levels and increasing SOD levels; particularly, the effect was most significant in the 10 g / kg high-dose group. P <0.0001); indicating that echinacea extract-chicoric acid has strong antioxidant capacity and can effectively alleviate radiation-induced oxidative stress.

[0040] like Figure 9 As shown, compared with the control group, the serum EPO level in the irradiated model group mice was significantly decreased ( PThe value of P < 0.0001 indicates that irradiation triggered a significant inflammatory response and inhibited erythroid hematopoietic function. After intervention with echinacea extract-chicoric acid, all treatment groups significantly reduced the levels of the above-mentioned inflammatory factors and increased EPO content, with the 10 g / kg dose group showing the most significant effect (P < 0.0001). This suggests that the extract can improve hematopoietic function by inhibiting the inflammatory response and promoting EPO secretion.

[0041] (4) Pathological analysis of mouse bone marrow Bone marrow tissue from mice in each treatment group was paraffin-embedded and stained with hematoxylin and eosin (HE) 14 days after radiation. The specific steps included: tissue sampling, decalcification, dehydration and clearing, paraffin embedding, sectioning (4 μm thickness), HE staining, and finally, the slide images were acquired using a full-slide digital scanning system.

[0042] like Figure 10 The image shows the HE staining results of bone marrow from mice in each treatment group 14 days after radiation. In the untreated control group (NC), the bone marrow structure of mice was dense and homogeneous, with a large number of granulocytic, erythroid, and megakaryocytic hematopoietic cells. The cells had normal morphology and no vacuoles, necrosis, or adipocyte accumulation, indicating good hematopoietic function. In contrast, the irradiated group (MOD) showed a large number of white vacuolated fat regions in the bone marrow cavity, a significant reduction in hematopoietic cells, and a loose bone marrow structure with increased gaps, but no obvious necrosis or hemorrhage was observed, suggesting that the radiation dose did not cause severe bone marrow necrosis, consistent with the pathological characteristics of the peak hematopoietic suppression period in the middle (14 days) after radiation. In the amifostine positive control group, the number of white vacuolated areas (adipocytes) in the bone marrow of mice was reduced compared to the MOD group, the number of hematopoietic cells recovered, and the density of the bone marrow structure improved, which was better than that of the MOD group. Furthermore, in the echinacea extract-chicoric acid administration group, with increasing doses (especially the 10 g / kg / d group), the hematopoietic cell density in the mouse bone marrow gradually recovered to levels close to those of the NC group, adipocyte infiltration further decreased, and more granulocytic / erythroid precursor cells were visible under high magnification, with the structure gradually returning to normal. This indicates that echinacea extract-chicoric acid has a protective or repairing effect on bone marrow hematopoiesis after radiation exposure, and this effect is dose-dependent; that is, the higher the dose, the more pronounced the protective effect.

[0043] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. Application of chicoric acid in the preparation of drugs to improve radiation-induced hematopoietic damage.

2. The application according to claim 1, characterized in that, The hematopoietic damage was caused by ionizing radiation.

3. The application according to claim 1, characterized in that, The structural formula of chicoric acid is: 。 4. The application according to claim 1, characterized in that, The molecular formula of chicoric acid is C 22 H 18 O 12 The molecular weight is 474.

371.

5. The application according to claim 1, characterized in that, The drug includes chicoric acid and pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that, The dosage forms of the drug include, but are not limited to, injections, capsules, or tablets.

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