Application of curcumin in preparation of medicine for preventing and / or treating bone tissue radiation injury caused by Ra-223

Abstract

The invention provides an application of curcumin in preparation of a medicine for preventing and / or treating bone tissue radiation injury caused by Ra-223. Through in-vivo experiments, the protection effect of curcumin on bone tissue injury caused by Ra-223 radiation is evaluated. Experimental results show that curcumin can inhibit the change of blood calcium and serum phosphorus content caused by radium chloride, reduce apoptosis of bone tissue cells and inhibit excessive activation of osteoclasts. The invention not only provides an experimental basis for the application of curcumin in the aspect of radiation damage resistance, but also provides a new thought for developing novel anti-radiation drugs and treating skeleton diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of curcumin in preparing a medicine for preventing and / or treating bone tissue radiation damage caused by Ra-223. Background Art

[0002] Curcumin has potential applications in anti-inflammatory, anti-diabetic, anti-cancer, and anti-aging activities. Furthermore, its role in radioprotection is increasingly being demonstrated, as it can reduce radiation-induced DNA double-strand breaks and mitigate the formation of gamma-ray-induced micronuclei. Radium-223 (Ra-223) has a relatively short half-life (11.4 days) and primarily undergoes alpha decay, releasing short-range (100 μm) alpha particles with a high linear energy transfer (LET) radiation signature. Currently, Ra-223 is administered clinically for the treatment of cancer bone metastases in the form of radium dichloride. Ra-223 is absorbed and concentrated in bone, binding to hydroxyapatite and primarily deposited in areas of active bone remodeling. It induces DNA double-strand breaks and complex chromosomal rearrangements in osteoblasts, leading to cell death. However, the potential for radiation damage to the bone marrow during the treatment of cancer bone metastases by Ra-223 limits its application. This is particularly true for patients whose only treatment option is Ra-223, who must implement or develop radiation protection measures to minimize the damage caused by Ra-223. Curcumin's antioxidant properties can scavenge the genomic damage of radiation-generated free radicals, resulting in low toxicity and significant radioprotective effects. Radiation-induced reactive oxygen species (ROS) production is one of the primary causes of radiation damage. Lipid peroxidation caused by ROS can damage proteins, cell membranes, and nucleic acids. Therefore, enhancing the body's antioxidant capacity and mitigating the damaging effects of oxidative stress are key strategies for radiation protection. As a potent antioxidant, curcumin can scavenge free radicals and mitigate the effects of oxidative damage. It has been designated as a Generally Recognized As Safe (GRAS) food by the US Food and Drug Administration (FDA). Curcumin has been used in radiation protection experiments on tissues susceptible to radiation damage, such as the brain, stomach, and pancreas, demonstrating a certain degree of protection. This has contributed to the advancement of curcumin's clinical application in radiation protection. Therefore, applying curcumin to protect bone tissue from radiation damage caused by radionuclide therapy has significant implications for the advancement of radionuclide therapy for patients with bone metastases and holds clinical value. Summary of the Invention

[0003] The purpose of the present invention is to provide a new medical use of curcumin.

[0004] The new medical use of curcumin provided by the present invention is the use of curcumin in preparing a radiation protectant.

[0005] In the application, the radiation protectant may specifically be a Ra-223 radiation protectant.

[0006] Furthermore, the application is the application of curcumin in the preparation of a drug for preventing and / or treating bone tissue radiation damage caused by Ra-223.

[0007] In the application, the drug for preventing and / or treating bone tissue radiation damage caused by Ra-223 has at least one of the following effects: 1) Suppressing the changes in blood calcium and phosphorus levels caused by radium chloride in subjects administered radium chloride; 2) It has a protective effect on the bone tissue of subjects administered radium chloride, specifically by reducing the apoptosis of bone tissue cells; 3) It has an inhibitory effect on osteoclast hyperactivation in subjects administered radium chloride.

[0008] The subject has been administered with curcumin before the administration of radium chloride, the subject has been administered with curcumin while the subject has been administered with radium chloride, or the subject has been administered with curcumin after the administration of radium chloride.

[0009] Through a series of experiments, this study explored the potential role of curcumin in protecting against bone damage caused by radium chloride. The results demonstrate that curcumin, a natural compound with multiple pharmacological activities, exhibits potential in mitigating radium chloride-induced damage to the skeletal system. This discovery not only provides strong support for the application of curcumin in protecting against radiation damage but also offers new insights for the development of anti-radiation drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The effect of radium chloride on the survival of mice. There were 5 mice in each group. n =5.

[0011] Figure 2 The figure shows the tissue absorption dose distribution of mice after administration of radium chloride. There were 3 mice in each group at different activities and different time points. n =3.

[0012] Figure 3 Curcumin inhibits the decrease in blood calcium content caused by radium chloride. Three mice were included in each group at different activity levels and time points. n =3;* p <0.05,*** p <0.001.

[0013] Figure 4 Curcumin inhibits the increase of blood phosphorus content caused by radium chloride. Three mice were included in each group at different activity levels and different time points. n =3;* p <0.05,*** p <0.001.

[0014] Figure 5 Curcumin inhibits the effect of radium chloride on blood phosphorus content. Three mice were included in each group at different activity levels and time points. n =3.

[0015] Figure 6 H&E staining analysis of mouse femur after administration of curcumin combined with radium chloride.

[0016] Figure 7 Analysis of cell apoptosis in mouse femoral tissue after administration of curcumin combined with radium chloride. A, TUNEL staining; B, fluorescence intensity analysis of apoptotic cells. n =3,* p <0.05,*** p <0.001; C, Fluorescence intensity analysis of apoptotic cells in mouse femurs at different time points after administration of curcumin combined with 1 μCi radium chloride.

[0017] Figure 8 TRAP staining analysis of mouse femoral osteoclasts after administration of curcumin combined with radium chloride. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0019] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0020] Example 1: Curcumin protects bone tissue from radiation damage induced by radium chloride Materials and Methods 1.1 Materials Dophigol (radium chloride) is currently used to treat patients with prostate cancer bone metastases. Therefore, male mice were used for this experiment. C57BL / 6 male mice (6 weeks old, male) were purchased from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The experimental animals were housed under a temperature (24 ± 1°C), humidity (60-70%), and light intensity (12h / 12h day / night cycle). They were fed SPF-grade feed (added every 2 days), sterile water (changed every 2 days), and bedding (SPF-grade, changed every 2 days). Mice had free access to food and water daily to ensure proper animal welfare.

[0021] 1.2 Survival rate of mice exposed to radium chloride Dofigo was purchased from China Tongfang. The experimental animals were randomly divided into control group, 1 μCi group, 2 μCi group and 4 μCi group, with 5 mice in each group. The control group was injected with the same volume of normal saline as the 4 μCi group. The drugs in each group were injected through the tail vein, and the survival of the mice was monitored and observed every day.

[0022] 1.3 Curcumin protection experiment grouping Curcumin was purchased from MCE (HY-N0005). Animals were randomly assigned to each group (see Table 1), with five mice per group. Experimental groups and injection activity are shown in Table 1. Day 1 was defined as the day of tail vein injection of radium chloride. The control group received the same volume of saline as the 4 μCi group. Curcumin was administered intraperitoneally for five consecutive days. On the fourth day following curcumin injection, radium chloride was administered via the tail vein. Mice were euthanized and samples were collected for analysis 1, 6, and 12 days after radium chloride injection.

[0023]

[0024] Note: "-" means that this group is not set. There are 3 mice in each group at different activity levels and time points. n =3.

[0025] 1.4 Tissue absorbed dose distribution detection On the 1st, 6th and 12th day after the injection of radium chloride, the heart, liver, spleen, lung, kidney, stomach, intestine, femur, muscle, blood, testicle, brain and thyroid tissues of different groups of mice were separated and weighed using an analytical balance, and the dose distribution of each organ was measured using a fully automatic gamma counter.

[0026] 1.5 Determination of inorganic elements Blood was collected from mice via the orbital cavity and centrifuged at 3000 rpm / min for 15 minutes at 4°C. Serum was then collected and stored at -80°C. Serum total calcium was determined spectrophotometrically. The calcium detection kit was reacted with the sample for 120 seconds, and the absorbance was measured at 660 nm. Serum inorganic phosphorus was determined using the phosphomolybdic acid method. Inorganic phosphorus in serum combines with molybdate to form a phosphomolybdic acid compound, which is then reduced to molybdenum blue using a reducing agent for colorimetric determination. The absorbance was measured at 340 nm. Serum total magnesium was determined using the magnesium reagent method. The magnesium detection kit was reacted with the sample, and the absorbance was measured at 540 nm.

[0027] 1.6 H&E staining Mouse right hind limb femoral tissue was fixed in 4% paraformaldehyde at room temperature for at least one day. After rinsing with tap water, the tissue was dehydrated using a gradient of 70%, 80%, 95%, and 100% alcohol, cleared with xylene, and then paraffinized by immersion in paraffin wax at approximately 58°C. After the wax block cooled, the wax block was placed on a microtome and serially sectioned to a thickness of 5 μm. The sections were then unfolded in 42°C warm water, mounted flat on a glass slide, and baked at 60°C for 1 hour. Finally, the tissue was dewaxed using xylene and various concentrations of alcohol. After dewaxing, the tissue was stained with hematoxylin and eosin. After mounting, the sections were observed under a microscope, photographed, and data analyzed.

[0028] 1.7 TUNEL staining TUNEL staining was performed according to the kit instructions. Paraffin sections of bone tissue were dewaxed and hydrated, then treated with permeabilization solution. TdT enzyme solution and fluorescein-labeled dUTP were mixed to prepare the TUNEL reaction mixture. The TUNEL reaction mixture was added dropwise to the sample, evenly covering the sample. The sample was incubated in a humidified chamber at 37°C for 1 hour and then washed with PBS. POD was added dropwise to the sample to allow HRP to specifically bind to the fluorescein antibody. The sample was incubated in a humidified chamber at 37°C for 30 minutes and washed with PBS. DAB substrate was added dropwise to the sample to react with HRP to produce a color reaction, thereby labeling apoptotic cells.

[0029] 1.8 Tartrate-resistant acid phosphatase staining (TRAP staining) Paraffin sections of bone tissue were dewaxed in xylene and dehydrated with anhydrous ethanol, then washed with PBS. According to the TRAP kit instructions, acetate buffer, hexazofuchsin (mixed in equal proportions with sodium nitrite solution immediately before use), and naphthol AS-BI phosphate solution were mixed in the specified proportions and filtered to prepare the TRAP working solution. Sections were placed in a humidified chamber, and the TRAP working solution was dripped over the sections. The sections were then incubated at 37°C in the dark for 1 hour. After the reaction, the sections were thoroughly washed with deionized water and counterstained with hematoxylin. A positive result was observed as a purple-red precipitate in the osteoclast cytoplasm, while a negative result indicated the absence of this precipitate. Osteoclast activity was assessed by comparing staining intensity and osteoclast counts across the sections.

[0030] 2. Results and Discussion 2.1 Effects of different radium chloride activities on mouse survival Figure 1 The figure shows a survival analysis of mice exposed to different radium chloride activity levels. As the activity increased, the survival rate of mice gradually decreased. The 4 μCi group experienced the most significant drop in survival, with all mice dead by day 9 after administration. In the 2 μCi group, only 20% of the mice were still alive by day 12 after administration, demonstrating that radium chloride is severely toxic to mice, affecting survival and that survival is dose-dependent.

[0031] 2.2 Tissue absorbed dose distribution of radium chloride The tissue absorbed dose distribution of a radiopharmaceutical refers to the radioactive dose received by various tissues during the absorption, distribution, metabolism, and excretion of the radiopharmaceutical in the body. This distribution is crucial for understanding the efficacy and toxicity of the drug and for developing appropriate radiotherapy plans. Figure 2 Figure A shows the relative absorption capacity of different organs and tissues for the cumulative dose of radium chloride. On day one after administration, the femur absorbed the highest dose among the heart, liver, spleen, lung, kidney, stomach, intestine, femur, muscle, blood, brain, testis, and thyroid. Femoral absorption reached 6.66 Gy, 23.17 Gy, and 46.75 Gy in the 1 μCi, 2 μCi, and 4 μCi groups, respectively. These results indicate that the absorbed dose in the femur is proportional to the administered radium chloride activity.

[0032] Figure 2Figure B shows the tissue absorbed doses at different times after administration of 1 μCi of radium chloride. The cumulative absorbed dose to the femur was time-dependent, reaching 6.66 Gy, 14.84 Gy, and 27.71 Gy on days 1, 6, and 12, respectively. These results indicate a time-dependent deposition of radium chloride in femoral tissue. Therefore, personalized treatment plans can be developed based on the distribution of radium chloride in tissues and organs and the dose-response effect in bone tissue to improve therapeutic efficacy and minimize side effects.

[0033] 2.3 Protective effect of curcumin on blood calcium, blood magnesium and blood phosphorus levels in mice injected with radium chloride Blood calcium, magnesium, and phosphorus are important mineral elements in the human body, playing a vital role in maintaining normal physiological functions. Calcium is involved in bone formation, muscle contraction, nerve impulse transmission, and blood coagulation. As a cationic calcium mimetic, Ra-223 is absorbed and concentrated in the bones, where it binds to hydroxyapatite, affecting changes in blood calcium levels to a certain extent. Figure 3 Figure A shows the effect of different activities of radium chloride on serum calcium levels one day after administration. There was no difference in calcium levels between the control group and the curcumin group. The serum calcium level in the radium chloride group decreased, and curcumin administration in advance could restore the blood calcium level (2 μCi, p <0.05; 4 μCi, p <0.001). Figure 3 Figure B shows the changes in serum calcium levels at different times after administration of 1 μCi of radium chloride. Curcumin can increase blood calcium levels on days 1, 6, and 12 after administration (days 6 and 12, p <0.05). The above results indicate that curcumin can alleviate the decrease in blood calcium content caused by radium chloride.

[0034] Blood phosphorus has many important physiological functions in the human body. It participates in the composition and metabolism of many important substances in the body, and participates in the energy generation, storage, release and transfer process as well as maintaining the acid-base balance of body fluids. It is one of the important trace elements in the human body. Figure 4 Figure A shows the effect of different activities of radium chloride on serum phosphorus levels after one day of administration. There was no difference in phosphorus levels between the control group and the curcumin group. The serum phosphorus level in the radium chloride group increased, and curcumin administration in advance could reduce the serum phosphorus content (1 μCi, p <0.05; 2 μCi, p <0.05; 4 μCi, p <0.001). Figure 4 Figure B shows the changes in serum phosphorus levels at different times after administration of 1 μCi of radium chloride. Curcumin can reduce the serum phosphorus content on days 1, 6, and 12 after administration (days 1, 6, and 12,p <0.05). Phosphorus in bones exists primarily as inorganic phosphate, which, along with calcium, constitutes the primary mineral component of bone. When bones are damaged or affected by disease, this can lead to increased phosphorus release from the bones, which in turn increases blood phosphorus levels. Curcumin helps maintain bone integrity by promoting bone formation and inhibiting bone resorption. This may reduce phosphorus release, thereby helping to maintain normal blood phosphorus levels.

[0035] Magnesium ions, as activators, participate in various energy metabolisms in the body and have multiple physiological functions. They inhibit the proliferation of smooth muscle cells and endothelial cells and have a positive effect on improving abnormal bone mineral metabolism, atherosclerosis and vascular calcification. Figure 5 A shows the effect of different activities of radium chloride on serum magnesium ion levels after one day of administration. Figure 5 Figure B shows changes in serum magnesium levels at different times after administration of 1 μCi of radium chloride. The results show no significant differences in the effects of radium chloride on serum magnesium levels at different activity levels and time points. While serum calcium and phosphate levels changed, magnesium levels remained unaffected, further demonstrating that radiation damage from radium chloride is primarily concentrated in the bones.

[0036] 2.4 Protective effect of curcumin on radium chloride-induced bone damage Figure 6 The results of H&E staining of femoral tissue are shown. Compared with the control group, the femurs of mice in the radium chloride administration group and the curcumin combined with radium chloride administration group showed varying degrees of bone tissue damage. The cancellous bone is composed of trabeculae arranged in the bone, which is distributed inside the bone. It can be observed that the blue-purple area in the bone cavity of the tissue gradually becomes lighter, while the bone marrow cavity and cancellous space contain bone marrow, and its blue-purple color also gradually becomes lighter. This is due to the binding of hematoxylin to the broken DNA inside. As the nuclear fragmentation causes the cytoplasm to increase, the red staining increases due to the binding of protein substances released by the damaged cells with eosin. Compared with the administration of radium chloride alone, the blue-purple area in the bone cavity of the curcumin combined with radium chloride administration group is darker, but as time goes by, there is no obvious difference in staining, indicating that curcumin may reduce the production of cell micronuclei. The TUNEL experiment was used to analyze the cell apoptosis in bone tissue. Figure 7 A shows the TUNEL staining results of femoral tissue, and 7B shows the fluorescence intensity analysis results of apoptotic cells. Figure 7 C is the fluorescence intensity analysis result of apoptotic cells at different times in the 1 μCi administration group. The results showed that radium chloride induced apoptosis of bone tissue cells in a dose-dependent and time-dependent manner, while curcumin inhibited the occurrence of cell apoptosis (2 μCi, p <0.05; 4 μCi, p <0.001).

[0037] 2.5 Inhibitory effect of curcumin on radium chloride-induced osteoclast hyperactivation Osteoclasts (OCs) are the only multinucleated cells in the body with bone-resorbing activity. They play a crucial role in bone development, growth, repair, and reconstruction, working synergistically with osteoblasts to maintain bone homeostasis. Radium and calcium belong to the same element family and share similar physical and chemical properties. Therefore, Ra-223 can be absorbed into bone as a calcium analog, participating in bone metabolism and accumulating in areas of active bone metabolism, leading to overactivation of osteoclasts and impairing bone repair and reconstruction. Figure 8 Figure 2 shows TRAP staining of femoral osteoclasts. Compared with the control group, the radium chloride group resulted in overactivation of femoral osteoclasts, whereas the curcumin combined with radium chloride group inhibited osteoclast activation. Compared with radium chloride alone, the curcumin combined with radium chloride group inhibited osteoclast activation in femoral tissue. Over time, osteoclast activation in the 1 μCi group gradually increased. The inhibitory effect of the curcumin combined with 1 μCi group on osteoclast activation in femoral tissue increased over time. Overactivation of osteoclasts significantly accelerates bone resorption, potentially leading to excessive loss of bone matrix. Overactivation of osteoclasts may also impair bone repair. During bone repair, osteoblasts are required to form new bone tissue to fill the defect. However, overactivation of osteoclasts may lead to excessive bone clearance, interfering with the normal activity of osteoblasts and impairing bone repair.

[0038] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Application of curcumin in the preparation of radiation protectants.

2. The use according to claim 1, characterized in that: In the application, the radiation protectant is Ra-223 radiation protectant.

3. The use according to claim 2, characterized in that: The application is the application of curcumin in the preparation of a medicine for preventing and / or treating bone tissue radiation damage caused by Ra-223.

4. The use according to claim 2, characterized in that: The drug for preventing and / or treating radiation damage to bone tissue caused by Ra-223 inhibits changes in blood calcium and blood phosphorus levels caused by radium chloride in a subject to which radium chloride is administered.

5. The use according to claim 2, characterized in that: The drug for preventing and / or treating bone tissue radiation damage caused by Ra-223 has a protective effect on the bone tissue of a subject administered with radium chloride, specifically reducing the apoptosis of bone tissue cells.

6. The use according to claim 2, characterized in that: The drug for preventing and / or treating bone tissue radiation damage caused by Ra-223 has an inhibitory effect on the excessive activation of osteoclasts in subjects administered with radium chloride.

7. The use according to any one of claims 4 to 6, characterized in that: The subject has been administered with curcumin before the administration of radium chloride, the subject has been administered with curcumin while the subject has been administered with radium chloride, or the subject has been administered with curcumin after the administration of radium chloride.

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