Application of activator in promotion of uranium element discharge

Abstract

The invention relates to application of an activating agent in promotion of uranium element discharge. Specifically, the activating agent is prepared from rifampicin, erythromycin, progesterone, aldosterone, bilirubin, cholate vinblastine, doxomicin, adriamycin, paclitaxel, bosentan, ambrisentan, digoxin, verapamil, tonavir, amprenavir, indinavir, hypericum perforatum juice, curcumin, atorvastatin beclomethasone, budesonide, divaricasone carbamazepine and caffeine. And phenytoin. The use dosage of the activating agent is 10 to 60 mg / kg. According to the method, the accumulation amount of uranium in important organs such as bones and kidneys can be remarkably reduced, so that the potential toxic influence on the key organs is reduced.

Description

Technical Field

[0001] The present invention relates to the field of uranium element discharge, and in particular to the application of an activator in promoting the discharge of uranium elements. Background Art

[0002] Uranium is a radioactive element used as fuel in nuclear power plants. However, if it leaks, it poses a significant threat to the environment and human health. Internal uranium contamination occurs when uranium enters the body through the respiratory tract, digestive tract, skin, and wounds, where it accumulates, particularly in the kidneys and bones, making it difficult to excrete. Long-term chemical damage and internal radiation exposure can lead to irreversible organ damage, such as kidney failure, osteosarcoma, and even death. Given the serious threat posed by internal uranium contamination to human health, the development of effective uranium excretion enhancers is urgent. Uranium excretion enhancers bind to uranium in the body, forming soluble compounds, thereby promoting its excretion and reducing its retention time and toxicity. Current research on uranium excretion enhancers focuses primarily on chelation through coordination chemistry, encompassing two main categories: small molecule chelators and nanochelators. Small molecule chelators, such as diethylenetriaminepentaacetic acid (DTPA), have been used clinically to enhance the excretion of actinides, but their effectiveness against uranium is limited. Nano-chelating agents have the advantages of slow metabolism and relatively low toxicity, but the research is still in its infancy, and there are problems such as complex preparation and high cost. In addition, some new uranium excretion promoters are under research, such as hydroxypyridone (HOPO) ligands, but the excretion-promoting effect and mechanism of these ligands still need to be further verified and optimized. Uranium is both a heavy metal (chemical toxicity) and a radionuclide (α radiation), and its chemical toxicity and radiation damage problems need to be solved at the same time, while other heavy metals only need to deal with chemical toxicity. Uranium is often present in the body as uranyl ions (UO2 2+ ) forms, and its water solubility, charge distribution and coordination characteristics are similar to those of other heavy metal ions (such as Cd 2+ , Pb 2+ ) are significantly different. Therefore, the research background for promoting the excretion of uranium internal contamination is complex and urgent. With the continuous development of the nuclear energy industry and the increasing prominence of nuclear safety issues, multi-faceted research on promoting the excretion of uranium is of great significance for protecting public health and environmental safety. Therefore, it is necessary to further strengthen basic research, explore the mechanism of action and preparation methods of new uranium excretion promoters, and promote their clinical translation and application. Summary of the Invention

[0003] Based on the above problems, this application deeply explores the mechanism of promoting the excretion of uranium contamination from a new perspective, which is essentially different from the traditional excretion promotion method based on chelating agents that is currently widely used. Its core concept is to effectively promote the excretion of uranium by precisely regulating and activating the expression level of P-glycoprotein in the body of the organism. The excretion promotion method proposed in this application shows a high clearance rate of uranium, which means that within the same treatment cycle, uranium can be more thoroughly removed from the body, providing patients with better treatment effects and higher safety. Compared with traditional chelating agent excretion promotion methods, this method not only avoids the side effects that may be caused by non-specific binding to other metal ions, but also achieves more accurate and efficient uranium excretion promotion by targeted activation of P-glycoprotein expression, opening up a new path for the treatment of uranium contamination.

[0004] The present invention provides an application of an activator in promoting the discharge of uranium elements.

[0005] Furthermore, the activator includes at least one of rifampicin, erythromycin, progesterone, aldosterone, bilirubin, bile salt vinblastine, daunorubicin, doxorubicin, paclitaxel, bosentan, ambrisentan, digoxin, verapamil, tonavir, amprenavir, indinavir, St. John's wort, curcumin, atorvastatin, beclomethasone, budesonide, dimethoate, carbamazepine, caffeine, and phenytoin.

[0006] Furthermore, the dosage of the activator is 10-60 mg / kg.

[0007] Furthermore, the method for promoting the discharge of uranium elements comprises the following steps:

[0008] 1) administering the activator according to any one of claims 1 to 3 to a uranium-exposed organism;

[0009] 2) Promote the excretion of uranium from the body through urine or bile by activating P-glycoprotein;

[0010] 3) Regularly monitor the expression level of P-glycoprotein and dynamically adjust the dosage of the activator to maintain the ovulation-inducing effect.

[0011] Furthermore, in step 2), the activator induces P-glycoprotein activation, enhances the conformational change of the P-glycoprotein-specific transport channel, opens the transport channel, and allows uranium to enter the transport channel and be transported outside the cell, thereby enhancing the transport function of uranium and excreting uranium from the body through urine or bile.

[0012] Furthermore, the P-glycoprotein includes P-glycoprotein in the kidney and liver.

[0013] Furthermore, the method is applied in the study of uranium biological transport and detoxification mechanism.

[0014] The present invention also provides a medicine for preventing and treating diseases caused by uranium, which comprises the activator involved in the above application and a pharmaceutically acceptable carrier.

[0015] Furthermore, the diseases caused by uranium include diseases caused by uranium poisoning due to uranium contamination.

[0016] Furthermore, the diseases caused by uranium poisoning include acute uranium poisoning, chronic uranium poisoning, uranium poisoning-induced kidney damage or uranium poisoning-induced bone damage, leukemia, lung cancer, skin cancer, and radiation-related cancers. Compared with the existing technology, the present invention has the following advantages:

[0017] 1. This invention achieves more precise and efficient uranium excretion by specifically activating P-glycoprotein expression, opening up a new path for the treatment of uranium contamination. It not only significantly accelerates uranium metabolism in the body and effectively shortens its retention time, but also significantly reduces and minimizes uranium accumulation in vital organs such as bones and kidneys, thereby alleviating potential toxic effects on these critical organs.

[0018] 2. High Efficiency: By activating P-glycoprotein, the present method significantly accelerates the elimination of uranium from the body. As a key transport protein on the cell membrane, P-glycoprotein possesses highly efficient transport capabilities, rapidly transporting uranium from within cells to the outside for excretion through urine or bile. This efficient transport mechanism significantly shortens the retention time of uranium in the body, thereby reducing its potential harm to organisms.

[0019] 3. Safety: Low side effects: Compared with traditional chelating agent-induced excretion methods, this new excretion-induced mechanism avoids the side effects that may be caused by non-specific binding to other metal ions. As a naturally occurring transport protein in the human body, the activation process of P-glycoprotein is relatively safe and not prone to drug resistance, thereby reducing the risks and discomfort during treatment. Protecting key organs: This mechanism can significantly reduce and minimize the accumulation of uranium in important organs such as bones and kidneys, thereby alleviating the potential toxic effects on these key organs. This is of great significance for protecting the health of organisms and maintaining their normal physiological functions.

[0020] 4. Broad application prospects: emergency treatment and long-term management: This new excretion-promoting mechanism can be used not only for the emergency treatment of uranium poisoning, but also for the preventive treatment of people with long-term exposure. By regularly monitoring the expression level of P-glycoprotein and adjusting the dosing regimen, long-term and effective management of uranium-exposed people can be achieved, reducing the risk of uranium poisoning. Heavy metal biotransport research: P-glycoprotein, as an important transport protein on the cell membrane, plays an important role in the biological transport and detoxification mechanism of heavy metals. This new excretion-promoting mechanism provides a new perspective and ideas for studying the function and mechanism of action of P-glycoprotein in heavy metal transport, which will help promote research progress in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Binding and dissociation curves of different concentrations of uranyl nitrate and P-gp;

[0022] Figure 2 Figure 1: Rifampicin's protective effect against uranium-induced cytotoxicity. Panel A: Dose-effect curve of the rifampicin intervention group; Panel B: Dose-effect curve of the tadalafil intervention group; Panel C: Effect of changes in P-gp activity on 24-hour cell viability of HK-2 cells exposed to uranium. Compared with the group exposed to uranyl nitrate alone, *P < 0.05, **P < 0.01, ***P < 0.001.

[0023] Figure 3 Effect of rifampicin on uranium excretion in uranium-exposed HK-2 cells. A: Intracellular uranium content at different time points; B: Uranium content in cell supernatant at different time points; C: Percentage of cellular uptake at different time points. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the uranyl nitrate-only exposure group.

[0024] Figure 4 Effects of uranium exposure on cell cycle, apoptosis, mitochondrial membrane potential, and reactive oxygen species. HK-2 cells were pretreated with RFP (rifampicin) or TQR (tadalaquine) and then treated with 600 μM uranium for 24 hours. A: Statistical analysis of cell apoptosis results by flow cytometry; B: Statistical analysis of cell mitochondrial membrane potential results by flow cytometry; C: Statistical analysis of cell reactive oxygen species results by flow cytometry; D: Statistical analysis of cell cycle results by flow cytometry. Compared with the control group, # P<0.05, ## P<0.01, ### P<0.001; compared with the uranyl nitrate exposure group only, *P<0.05, **P<0.01, ***P<0.001;

[0025] Figure 5Rifampicin promotes uranium excretion by inducing P-gp expression (Western blot, IHC, and semiquantitative analysis). Figure A: P-gp protein expression in liver and kidney. Figures BC: Semiquantitative analysis of protein expression in Figure A. Figure D: Immunohistochemistry results of P-gp expression in liver and kidney. Figure E: Semiquantitative analysis of protein expression in Figure D. *P < 0.05, **P < 0.01, ***P < 0.001.

[0026] Figure 6 Dynamic monitoring of uranium distribution and clearance in blood and tissues (ICP-MS detection) Figure A: uranium content in plasma; Figure B: uranium content in femur; Figure C: uranium content in kidney; Figure D: uranium content in liver;

[0027] Figure 7 Dynamic monitoring of uranium excretion in urine and feces (ICP-MS detection). Panel A: Cumulative uranium content in urine; Panel B: Cumulative uranium content in feces; *P < 0.05, **P < 0.01, ***P < 0.001;

[0028] Figure 8 Uranium exposure caused liver and kidney damage, which was significantly improved by rifampicin (organ coefficients and pathological analysis). Figure, A: Kidney organ coefficient; B: Liver organ coefficient; C: Kidney pathological analysis (HE staining); Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; Compared with the group exposed to uranyl nitrate only, *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 9 Uranium exposure leads to liver and kidney damage, which is significantly improved by rifampicin (biochemical indicators). Figure A: plasma total protein (TP) content; B: plasma alkaline phosphatase (AKP) content; C: plasma alanine aminotransferase (ALT) content; D: plasma aspartate aminotransferase (AST) content; E: plasma urea nitrogen (BUN) content; F: plasma creatinine (CRE) content, compared with the control group. # P<0.05, ## P<0.01, ### P<0.001; compared with the uranyl nitrate exposure group only, *P<0.05, **P<0.01, ***P<0.001;

[0030] Figure 10Effects of immediate and delayed sodium bicarbonate administration after single or multiple exposures on the clearance of uranium and protection against uranium nephrotoxicity in mice. (a) Schematic diagram of the experiments in which sodium bicarbonate was administered immediately and delayed after single or multiple exposures to uranium in BALB / c male mice. (b) U concentrations in renal tissue and urine over the final 24 hours of mice exposed to uranium, followed by immediate and delayed sodium bicarbonate treatment. n = 3 mice for the control group, the single-dose uranium exposure group, the single-dose uranium exposure group plus immediate sodium bicarbonate treatment group, and the single-dose uranium exposure group plus delayed sodium bicarbonate treatment group. n = 4 mice for the multiple-dose uranium exposure group plus delayed sodium bicarbonate treatment group. (c) CRE and BUN levels in mice following immediate and delayed sodium bicarbonate treatment after single-dose uranium exposure. n = 7 for the control group, n = 3 for the uranium exposure group and the uranium exposure group plus sodium bicarbonate treatment group. (d) Representative H&E staining of the S1, S2, and S3 segments of the proximal tubules in the renal cortex of mice following single-dose uranium exposure and immediate and delayed sodium bicarbonate treatment. Images have the same scale bar (20 μm). (e) Quantitative analysis of pathological damage of the proximal tubules, with necrotic or detached cells in the S1, S2, and S3 segments of the proximal tubules.

[0031] Figure 11 Example 6 Flowchart of uranium efflux by cells. Specific implementation methods

[0032] To make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below. However, it should be understood that the description herein is only used to explain this application and is not intended to limit the scope of this application.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended solely for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are commercially available, and the characterization methods involved can be found in the relevant descriptions in the prior art and will not be further elaborated herein.

[0034] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0035] Example 1

[0036] This embodiment provides an application of an activator in promoting the discharge of uranium elements.

[0037] As a further preferred embodiment, the activator is selected from at least one of rifampicin, erythromycin, progesterone, aldosterone, bilirubin, bile salt vinblastine, daunorubicin, doxorubicin, paclitaxel, bosentan, ambrisentan, digoxin, verapamil, tonavir, amprenavir, indinavir, St. John's wort, curcumin, atorvastatin, beclomethasone, budesonide, dimethoate, carbamazepine, caffeine, and phenytoin.

[0038] P-glycoprotein (P-gp) is an ATP-dependent transporter protein widely distributed throughout the body. It belongs to the ATP-binding cassette (ABC) transporter superfamily. P-gp utilizes the energy generated by ATP hydrolysis to facilitate the transmembrane transport of various substances against their concentration gradient. This transport function plays an important role in maintaining intracellular homeostasis and protecting cells from exogenous harmful substances. The primary function of P-gp is to facilitate the transmembrane transport of various substances.

[0039] This application is mainly to significantly increase the expression level of P-gp in mice by pre-using a P-glycoprotein (P-gp) activator. This strategy cleverly utilizes the function of P-gp as an important transport protein on the cell membrane, effectively promoting the excretion of uranium. Experimental results show that in a mouse model exposed to uranium poisoning, the amount of uranium accumulated in mice pre-treated with rifampicin in advance was significantly reduced compared to the untreated group. This discovery not only reveals the key role of P-gp in uranium metabolism and excretion, but also further verifies that increasing the expression level of P-gp through drug intervention can be used as an effective means to reduce the risk of uranium contamination. For example, the use of rifampicin as a P-gp activator not only provides new ideas for the prevention and treatment of uranium poisoning, but also demonstrates its great potential in reducing the potential harm of uranium to organisms.

[0040] As a further preferred embodiment, the dosage of the activator is 10-60 mg / kg, for example, 1, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mg / kg or any value between any two values.

[0041] As a further preferred embodiment, the pharmaceutical preparation includes oral administration, and the dosage form for oral administration is selected from oral liquid, powder, tablet, granule, capsule, pill, syrup, suspension or emulsion.

[0042] Example 2

[0043] This embodiment provides a method for promoting uranium discharge based on the use of the activator described in Example 1 in preparing a method for promoting uranium discharge, comprising the following steps:

[0044] 1) administering the activator described above to the uranium-exposed organism;

[0045] 2) Promote the excretion of uranium from the body through urine or bile by activating P-glycoprotein;

[0046] 3) Regularly monitor the expression level of P-glycoprotein and dynamically adjust the dosage of the activator to maintain the ovulation-inducing effect.

[0047] As a further preferred embodiment, in step 2), the activator is used to induce P-glycoprotein activation, thereby enhancing the conformational change of the P-glycoprotein-specific transport channel, opening the transport channel, allowing uranium to enter the transport channel and be transported outside the cell, thereby enhancing the transport function of uranium, and then excreting uranium from the body through urine or bile.

[0048] As a further preferred embodiment, the P-glycoprotein includes P-glycoprotein in kidney and liver.

[0049] As a further preferred embodiment, the method is applied in the study of uranium biological transport and detoxification mechanism.

[0050] The working principle of the present application is based on the transport function of P-glycoprotein (P-gp). P-gp is an important transport protein located on the cell membrane, which has the function of transporting substances from the cell to the extracellular space. Under normal circumstances, P-gp participates in the transport process of various substances, including drugs, metabolites, etc. In the present application, by activating P-gp, it can be transported from the cell to the extracellular space, and eventually discharged from the body through urine or feces. The activation of P-gp can be achieved by specific activators, such as rifampicin. By pre-treating mice with a certain dose of activator in advance, P-gp is induced to activate or change its conformation, thereby enhancing its transport function. When P-gp is activated, its transport channel opens, and the uranium element is able to enter the transport channel and be transported to the extracellular space. In this process, the transport function of P-gp is fully utilized, thereby achieving effective discharge of the uranium element.

[0051] This application proposes a novel uranium excretion-promoting strategy, which effectively promotes the excretion of uranium by activating P-glycoprotein (P-gp) in the body. This strategy utilizes P-gp's function as a key transport protein on the cell membrane. By increasing its activity and expression level, it significantly enhances the cell's ability to transport uranium, thereby accelerating the elimination of uranium from the body.

[0052] It also has a good uranium removal effect: experimental results show that after mice were pre-treated with P-gp activators (such as rifampicin), the accumulation of uranium in their bodies was significantly reduced after exposure to uranium. This discovery not only verifies the key role of P-gp in uranium metabolism and excretion, but also demonstrates the great potential of this method in reducing the risk of uranium contamination and the potential harm of uranium to organisms. Furthermore, as a naturally occurring transport protein in the human body, P-gp's activation process is relatively safe and not prone to drug resistance.

[0053] Example 3

[0054] A medicine for preventing and treating diseases caused by uranium, characterized in that it comprises the activator involved in the above application and a pharmaceutically acceptable carrier.

[0055] As a further preferred embodiment, the disease caused by uranium includes a disease caused by uranium poisoning due to internal contamination of uranium.

[0056] As a further preferred embodiment, the diseases caused by uranium poisoning include acute uranium poisoning, chronic uranium poisoning, uranium poisoning-induced kidney damage or uranium poisoning-induced bone damage, leukemia, lung cancer, skin cancer and radiation-related cancers.

[0057] Acute uranium exposure: Inhaling or ingesting large doses of uranium compounds, particularly soluble uranium compounds, over a short period of time can cause systemic illness, primarily kidney damage. Symptoms may appear within hours to days and include fatigue, loss of appetite, dizziness, headache, nausea, vomiting, icteric sclera, hepatomegaly, and pain in the liver area. In severe cases, symptoms of acute renal failure, such as oliguria or anuria, elevated serum creatinine (BCr) and blood urea nitrogen (BUN), elevated potassium, and metabolic acidosis, may occur. Long-term, low-dose exposure to uranium compounds can lead to chronic poisoning. Early symptoms are subtle, but gradually develop into kidney damage (such as chronic toxic nephritis), gastrointestinal and other digestive system dysfunction (such as loss of appetite and gastrointestinal motility disorders), and nervous system disorders (such as headache, dizziness, fatigue, insomnia, and memory loss). Furthermore, inhalation of uranium compounds such as uranium hexafluoride (UF6), in addition to kidney and liver damage, can also cause respiratory irritation, such as chest pain, shortness of breath, cough, and cyanosis, due to hydrolysis of uranium hexafluoride in the respiratory tract. In severe cases, pulmonary edema may develop, leading to symptoms such as irritability, difficulty breathing, and coughing up thin white or pink sputum. Severe contamination of the body surface by acidic uranium compound solutions may result in chemical burns to the skin. Long-term exposure to uranium may cause anemia, manifesting as pale complexion and fatigue.

[0058] Example 4

[0059] Prepare a series of gradient concentrations of uranyl nitrate solutions (1.1, 3.3, 11, 33, 111 μM). Purify and dilute the target protein P-gp to an appropriate concentration (such as 50 μg / mL) for subsequent immobilization. Use a molecular interaction instrument (BLI) to immobilize the P-gp protein on the surface of the NTA sensor through a nickel-histidine (His) tag affinity immobilization method. The sensor needs to be activated before use to ensure the effective binding of surface nickel ions to the histidine tag. Before the binding and dissociation kinetics experiment, use a buffer solution (such as PBST) for 10 minutes to balance until the baseline is stable to ensure uniform protein immobilization and no nonspecific adsorption. The NTA sensor with immobilized protein or unimmobilized protein is bound to uranyl nitrate solutions of different concentrations for 60 seconds and dissociated for 60 seconds. Monitor the changes in the resonance signals in the binding stage (binding of uranyl nitrate to P-gp) and the dissociation stage (dissociation during buffer washing) in real time, and record the binding curve. Figure 1 .

[0060] like Figure 1 As shown in Table 1, it is generally believed that the affinity constant KD (M) between small molecules (molecular weight < 2KDa) and proteins is between 10 -3 -10 -6 It is believed that there is a strong affinity between the protein and the small molecule.

[0061] Table 1: Binding parameters of uranyl nitrate to P-gp

[0062] Conc.(μM) Response KD(M) ka(1 / Ms) kdis(1 / Ms) 1.1 -1.620E-03 2.127E-05 7.473E03 1.589E-01 3.3 0.0124 2.127E-05 7.473E03 1.589E-01 11 0.0346 2.127E-05 7.473E03 1.589E-01 33 0.066 2.127E-05 7.473E03 1.589E-01 111 0.0865 2.127E-05 7.473E03 1.589E-01

[0063] Example 5

[0064] To screen drug pretreatment doses, cells were seeded at a density of 4000 cells / well in 96-well plates (100 μL / well). After adherence, gradient concentrations of rifampicin solution (0, 62.5, 125, 250, 500, 800 μM) and tadalaquine solution (0, 1.3, 3.9, 7.81, 15.625, 31.25, 62.5 μM) were added, with three replicates in each group. The treatment was continued for 24 hours to evaluate cytotoxicity.

[0065] Based on the dose screening results, cells were pretreated with either 150 μM rifampicin (REP+U) or 1 μM tadalaquine (TQR+U) for 30 minutes. After removing the supernatant, cells were treated with fresh culture medium containing uranyl nitrate (100, 200, 400, 600, or 800 μM) for 24 hours. The uranium-exposed group (U) received uranyl nitrate alone and was exposed to the corresponding uranium solution without drug pretreatment.

[0066] After uranium exposure, 10 μL of CCK-8 reagent was added to each well, incubated at 37°C for 4 h, and the absorbance (OD value) was measured at 450 nm using a microplate reader.

[0067] The cell inhibition rate was calculated as follows: inhibition rate (%) = [(OD blank group - OD experimental group) / OD blank group] × 100%, where the blank group was a control without cell culture medium, and the experimental group contained basal culture medium and drug treatment.

[0068] Experimental results

[0069] Cytotoxicity assessments of the rifampicin and tadalaquine intervention groups showed that after 24 hours of exposure, the IC50 of rifampicin against HK-2 cells was 313 μM, while the IC50 of tadalaquine was 12 μM, indicating that both had significant cytotoxicity at higher concentrations. To reduce the impact of the drug's own toxicity on the experimental results, subsequent experiments used 150 μM rifampicin and 1 μM tadalaquine as treatment concentrations. The experimental results showed that under conditions of uranium exposure (100-800 μM) for 24 hours, administration of 150 μM rifampicin 30 minutes in advance significantly improved cell survival. Compared with 80.71±3.41%, 73.76±0.80%, 73.18±2.12%, 72.75±3.22%, and 69.33±0.44% in the uranium-only exposure group, the survival rates in the rifampicin-treated group increased to 93.54±13.43%, 105.25±5.47%, 103.10±6.58%, 84.93±6.58%, and 75.32±2.78%, respectively. In contrast, the survival rates in the 1 μM tadalafil-treated group decreased to 71.04±1.07%, 66.05±0.52%, 62.77±2.04%, 56.67±8.62%, and 59.22±0.79%.

[0070] The above results show that if Figure 2 As shown, rifampicin can activate P-gp-mediated transport function to reduce the cytotoxicity of uranyl nitrate. This dose-dependent effect is consistent with the toxic threshold characteristics of uranium in renal cells, that is, there is no obvious cell damage at low concentrations, while high concentrations (>100μM) cause a significant decrease in survival rate.

[0071] Example 6

[0072] Cells in logarithmic growth phase were taken and 1×10 5Cells were seeded at a density of 100 μL / well in a 24-well plate. 500 μL of complete culture medium was added to each well and cultured at 37°C, 5% CO₂ for 24 hours until cells fully adhered. The supernatant was discarded, and cells were pretreated for 30 minutes with either 150 μM rifampicin (in the rifampicin treatment group (REP+U)) or 1 μM tadalaquine (in the tadalaquine treatment group (TQR+U)). After pretreatment, the supernatant was removed, and 500 μL of uranium-containing medium (600 μL) was added to each well. Culture was continued for another 4 hours to ensure efficient uranium uptake. The uranium-containing medium was then discarded and replaced with 500 μL of fresh medium to initiate uranium efflux. To monitor uranium efflux kinetics, supernatant and cell samples were collected at 15, 30, 60, and 120 minutes after treatment. When collecting the supernatant, use pre-cooled 200μL PBS (pH 7.4) to gently rinse the cell surface to remove non-specifically bound uranium ions, and the eluate is combined with the original supernatant as the cell efflux portion. After the cell sample is rinsed with PBS, 500μL 70% concentrated nitric acid (v / v) is added for lysis, and the well plate is washed with 200μL PBS buffer, and the two are combined to form the remaining uranium portion in the cell. Both the effluxed uranium and the remaining uranium in the cell are digested at a constant temperature of 90°C for 2 hours to completely release the uranium component, and all samples are fixed to 8mL, and the uranium concentration is quantitatively detected by ICP-MS. The efflux rate = uranium content in the supernatant / (uranium content in the cell + uranium content in the supernatant) × 100%. The specific process is as follows. Figure 11 shown.

[0073] The uranium efflux process of HK-2 cells was dynamically monitored by ICP-MS. The uranium endocytosis process is very rapid. Therefore, after 4 hours of uranium uptake by the cells, fresh culture medium was replaced, an efflux model was constructed, and the efflux process was dynamically monitored within 120 minutes. The experimental results are shown in Figure 2. Figure 3 As shown, at 120 minutes, the supernatant uranium levels in the uranium-exposed group, the P-gp inducer rifampicin pre-treatment group, and the P-gp inhibitor tadalafil intervention group reached 2.51±0.16mg, 3.41±0.20mg, and 1.90±0.18mg, respectively, while the corresponding intracellular uranium levels decreased to 1.33±0.07mg, 1.20±0.01mg, and 1.76±0.09mg. In addition, the efflux rate in the rifampicin intervention group reached 73.93±1.17% at 120 minutes, significantly higher than the 65.17±2.22% in the control group and 51.86±1.27% in the tadalafil group (P<0.01).

[0074] like Figure 3 The results show that P-gp activation significantly enhances uranium efflux. The decreasing trend of intracellular uranium content is negatively correlated with the accumulation of uranium in the supernatant in a time-dependent manner, suggesting that this process conforms to the characteristics of active transport.

[0075] In existing technologies, at the optimal dosing concentration of 3 ppm uranyl and 25 ppm DTPA, DTPA only removes 6.54% of uranium from cells. Furthermore, DTPA is highly cytotoxic, with cell survival rates remaining below 25% within a concentration range of 25-300 ppm.

[0076] Example 7

[0077] 1. Apoptosis

[0078] To quantitatively distinguish the effects of changes in P-gp activity on live cells, early apoptotic cells, late apoptotic cells, and necrotic cells in HK-2 cells exposed to uranium, Annexin V-FITC and propidium iodide (PI) double staining combined with flow cytometry was used for analysis. The specific experimental steps are as follows:

[0079] Cell culture and collection: Cells in the logarithmic growth phase were selected and seeded in 6-well plates. After treatment with an endocytosis inhibitor for 30 minutes, they were treated with 600 μM uranyl nitrate for 24 hours. The culture supernatant was combined with the cell suspension obtained by digestion with 0.25% EDTA-free trypsin, and the cells were centrifuged at 1200 rpm for 5 minutes to collect the cell pellet.

[0080] Cell washing: Gently wash the cells twice with pre-cooled 1× PBS buffer. Centrifuge at 300 g for 5 minutes after each wash to remove residual culture medium components.

[0081] Cell resuspension and counting: Dilute 5× binding buffer to 1×, resuspend the cell pellet in 1 mL of the diluted buffer, count the cells, and adjust the cell density to 1×106 cells / mL.

[0082] Staining: Take 100 μL of cell suspension (about 1×105 cells) and place it in a 1.5 mL Eppendorf tube, add 5 μL of Annexin V-FITC and PI, and incubate at room temperature for 15 minutes in the dark.

[0083] Flow cytometry: Immediately after staining, add 400 μL of pre-cooled 1× binding buffer to terminate the reaction and detect by flow cytometry within 1 hour.

[0084] 2. Reactive oxygen species

[0085] (1) Probe dilution: DCFH-DA was diluted with serum-free MEM medium to a final concentration of 10 μmol / L.

[0086] (2) Cell pretreatment: After removing the culture medium, the cells were digested with 0.25% trypsin and gently washed once with 2 mL of PBS buffer (pH 7.4).

[0087] (3) Probe incubation: Add 1 mL of DCFH-DA working solution to each well of a six-well plate and incubate at 37°C in the dark for 20 minutes, gently shaking the culture plate every 5 minutes.

[0088] (4) Washing: Discard the probe working solution and wash three times with pre-cooled PBS. Centrifuge at 2500 rpm for 5 minutes after each wash to completely remove uninternalized probe.

[0089] (5) Preparation of cell suspension: Resuspend the cell pellet in 500 μL PBS, gently pipette to form a single-cell suspension and filter through a 40 μm cell sieve to ensure a cell density of 1 × 106 cells per ml.

[0090] (6) Flow cytometry: Flow cytometry was used to detect and analyze the mean fluorescence intensity to quantify the ROS level.

[0091] 3. Mitochondrial membrane potential:

[0092] To investigate the mechanism of action of the mitochondrial-dependent apoptotic pathway in uranium-induced apoptosis in HK-2 cells and to evaluate the interventional effect of changes in P-gp activity on mitochondrial dysfunction, we used the JC-1 fluorescent probe combined with flow cytometry to investigate changes in mitochondrial membrane potential (ΔΨm) in different treatment groups. The specific experimental procedures are as follows:

[0093] (1) Reagent preparation:

[0094] PBS buffer (1×): Thaw the pre-chilled PBS stock solution (10×) in a 37°C water bath and dilute it with deionized water in a 1:9 ratio.

[0095] Preparation of JC-1 working solution: Take JC-1 mother solution (200 μM) equilibrated at room temperature, add it to complete culture medium at a volume ratio of 1% (10 μL / 1 mL), mix well, and store in the dark.

[0096] Cell Harvest: After trypsinization, cells were centrifuged at 300g for 5 minutes to collect the pellet and washed twice with pre-chilled PBS (1×) to remove residual culture medium. For the positive control, CCCP (50mM stock solution) was added to the basal medium to a final concentration of 50μM (1μL / 1mL) and incubated at 37°C for 5 minutes to establish a mitochondrial membrane potential depolarization model.

[0097] (3) Cell staining: 1 mL of JC-1 working solution was added to cells in each treatment group and incubated at 37°C in the dark for 15 minutes. JC-1 emits red fluorescence (590 nm) when present in aggregate form within mitochondria. When ΔΨm decreases, the monomeric form increases, and the proportion of green fluorescence (530 nm) increases. This property can quantitatively reflect mitochondrial membrane integrity.

[0098] (4) Detection and Analysis: After staining, cells were washed twice with PBS, resuspended in 500 μL of PBS, and immediately subjected to flow cytometry. ΔΨm levels were calculated by the ratio of the fluorescence intensity of the green and red fluorescence channels. A decreased ratio indicated mitochondrial dysfunction.

[0099] 4. Cell cycle

[0100] (1) Cell culture and treatment: Cells in the logarithmic growth phase were selected and seeded in 6-well plates at a density of 5 × 105 cells / ml. After the cells adhered overnight, they were pretreated with rifampicin (150 μM) or tadalafil (1 μM) for 30 minutes to regulate P-gp activity. Subsequently, the cells were incubated with culture medium containing 600 μM uranyl nitrate for another 24 hours.

[0101] (2) Cell collection and washing: After removing the culture medium, digest the cells with 0.25% trypsin-EDTA solution and incubate at 37°C for 3 minutes. Add an equal amount of complete culture medium to terminate the digestion, transfer the cell suspension to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and gently resuspend the cell pellet in pre-chilled PBS (pH 7.4). Repeat the washing twice to completely remove the residual culture medium.

[0102] (3) Cell staining: Prepare the staining solution in the following proportions: add 25 μL of propidium iodide and 2.5 μL of RNase solution to every 500 μL of MEM medium and mix thoroughly. Add 0.5 ml of the above-prepared staining solution to each sample and incubate at 4°C in the dark for 30 minutes.

[0103] (4) Flow cytometry: After staining, vortex the cells for 10 seconds to disperse them and filter through a 200-mesh nylon mesh to remove cell aggregates. The proportions of cells in the G0 / G1 phase, S phase, and G2 / M phase were detected and analyzed by flow cytometry.

[0104] 5. Experimental results: Figure 4 As shown,

[0105] 1) Apoptosis

[0106] Flow cytometry analysis showed that after 24 hours of exposure to 600 μM uranyl nitrate, the proportion of early apoptosis in HK-2 cells increased significantly to 6.54±0.96%, the proportion of late apoptosis rose to 7.26±0.11%, and the total apoptosis rate reached 13.80±1.07%. Pretreatment with the P-gp activator rifampicin significantly reduced the total apoptosis rate to 9.90±2.35%. However, addition of the P-gp inhibitor tadalafil dramatically increased the total apoptosis rate to 51.83±2.40% (P<0.01 vs. other groups), indicating that P-gp inhibition significantly exacerbates uranium-induced cytotoxicity.

[0107] 2) Reactive oxygen species

[0108] After 24 hours of exposure to 600 μM uranium, intracellular ROS levels in HK-2 cells increased dramatically to 43.23 ± 1.02%, a 20.46-fold increase compared to the control group. Pretreatment with rifampicin significantly reduced ROS to 15.77 ± 0.68%, only 36.47% of the group exposed to uranium alone. However, the P-gp inhibitor tadalafil further exacerbated oxidative stress, increasing ROS levels to 45.3 ± 0.53%, significantly higher than the group exposed to uranium alone.

[0109] 3) Cell cycle:

[0110] To further investigate the effects of changes in P-gp activity on the cell cycle of HK-2 cells exposed to uranium, cells were treated with various endocytosis inhibitors and then treated with uranium solution (600 μM) for 24 hours before analysis. The G2 / M phase is the final stage of DNA replication and mitosis. As shown in the figure, uranium exposure resulted in a decrease in the proportion of cells in the G2 / M phase. However, no significant improvement in the G2 / M phase was observed across all treatment groups.

[0111] 4) Mitochondrial membrane potential:

[0112] Uranium is a heavy metal that is highly toxic and bioaccumulative. It can enter cells and accumulate in organelles (such as mitochondria), interfering with the normal function of mitochondria, leading to energy metabolism disorders and cell death. The dynamic changes of mitochondrial membrane potential were detected by JC-1 fluorescent probe. The results showed that JC-1 aggregates in the control group cells showed typical orange-red fluorescence (red / green fluorescence ratio was 30.03±0.87%). After 24 hours of exposure to 600μM uranium, the depolarization ratio of mitochondrial membrane potential increased significantly to 65.57±0.67% (P<0.001), an increase of 2.18 times compared with the control group, and the green fluorescence intensity was significantly enhanced, indicating that uranium exposure caused damage to mitochondrial membrane integrity. Pretreatment with the P-gp inducer rifampicin 30 minutes before uranium exposure significantly reduced the proportion of mitochondrial membrane potential depolarization to 49.0±2.20% (a 25.2% decrease compared to the uranium control group, P<0.001), and the red-to-green fluorescence ratio recovered to 75.95% of the control group, indicating that rifampicin effectively antagonized uranium-induced mitochondrial damage. Conversely, pretreatment with the P-gp inhibitor tadalafil further increased the red-to-green fluorescence ratio in the uranium-exposed group to 81.43±1.23% (a 2.71-fold increase compared to the control group, P<0.001), suggesting that inhibition of P-gp function exacerbates uranium-induced mitochondrial toxicity.

[0113] Example 8 (Animal Experiment)

[0114] 1. Experimental Animal Preparation and Grouping

[0115] Animal selection: BALB / c male mice, SPF grade, weighing 18-22 g, were randomly divided into the following groups (n=6 per group):

[0116] Control group (control): 0.9% saline was administered orally, without uranium exposure.

[0117] Uranium exposure group (U): uranyl nitrate poisoning (uranyl nitrate 2 mg / kg, tail vein injection).

[0118] Rifampicin intervention group (RFP+U): After continuous oral administration of rifampicin (40 mg / kg) for 10 days, uranyl nitrate poisoning (uranyl nitrate 2 mg / kg, tail vein injection) was performed.

[0119] Tadaliquine intervention group (TQR+U): Tadaliquine (10 mg / kg) was administered orally for 5 consecutive days, followed by uranyl nitrate poisoning (2 mg / kg uranyl nitrate, injection into the tail vein).

[0120] Rearing conditions: SPF environment, free access to food and water, 12-h day and night cycle, and 1 week of acclimatization before the experiment.

[0121] 2. Drug Treatment and Uranium Exposure

[0122] Rifampicin gavage:

[0123] Rifampicin powder was dissolved in 0.9% saline by ultrasonication to prepare a suspension with a concentration of 4 mg / mL solution, and the suspension was administered orally at a rate of 40 mg / kg, once a day for 10 consecutive days.

[0124] Tadaliquine gavage:

[0125] Tadaliquine powder was dissolved in 0.9% saline by ultrasonication to prepare a suspension with a concentration of 1 mg / mL solution, and 10 mg / kg was administered orally once a day for 5 consecutive days.

[0126] Uranyl nitrate poisoning:

[0127] Uranyl nitrate was dissolved in physiological saline, sterilized by filtration, and injected into the tail vein at a dose of 2 mg / kg.

[0128] 3. Uranium concentration detection

[0129] Sample preparation:

[0130] Tissue samples (liver, kidney, and femur) were microwave-digested and then diluted to volume with ultrapure water.

[0131] Blood samples were diluted directly.

[0132] Detection method: Inductively coupled plasma mass spectrometry (ICP-MS) is used to determine the uranium content in μg / g or μg / mL.

[0133] 4. Rifampicin-induced P-gp activation in the liver and kidneys of mice

[0134] Mice in the rifampicin intervention group (RFP+U) were gavaged daily with 40 mg / kg rifampicin solution (dissolved in 0.9% saline) for 10 consecutive days. The control group (control) was gavaged with an equal volume of saline at the same frequency. 24 hours after the last administration, all experimental animals were euthanized by cervical dislocation, and kidney and liver tissues were quickly isolated. Tissue samples were rinsed with pre-chilled PBS and divided into two parts for processing. Approximately 100 mg of tissue was immediately fixed in 4% paraformaldehyde for 24 hours for immunohistochemistry. The remaining tissue was quickly frozen in liquid nitrogen and transferred to a -80°C freezer for storage. Total protein was subsequently extracted using RIPA lysis buffer (containing protease inhibitors) for Western blotting analysis.

[0135] 5. Uranium metabolism in mice

[0136] To investigate the effect of P-gp on uranium metabolism, mice were euthanized at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours after uranium exposure in the rifampicin, tadalafil, control, and uranium-exposed groups. Whole blood (anticoagulated with heparin), liver, kidney, and femur tissue were collected, rinsed with saline, and weighed. Feces and urine were collected from other mice at 2, 6, 12, 24, 48, and 72 hours after uranium exposure. After accurate weighing, 1 mL of 70% nitric acid solution was added, and the samples were digested in a 90°C water bath for 2 hours until completely dissolved. The volume was then made up to 10 mL with ultrapure water, and uranium content was determined by ICP-MS.

[0137] 6. Detection of liver and kidney pathology indicators

[0138] The rifampicin-treated group received oral gavage with 0.9% saline as the vehicle for 10 consecutive days; the tadalaquine-treated group received the same route of administration for 5 days. The control and uranium-exposed groups received 0.9% saline instead. One hour after the last dose, the animals in all three groups were injected with uranyl nitrate (2 mg / kg, dissolved in 0.9% saline) via the tail vein, while the control group received an equal volume of saline. After 72 hours of exposure, whole blood (anticoagulated with heparin) was collected by enucleation and centrifuged at 3000 × g for 15 minutes to obtain plasma. Liver and kidney tissues were rapidly dissected, rinsed with saline, and weighed, and organ coefficients were recorded. Plasma samples were analyzed for creatinine (Cr), blood urea nitrogen (BUN), total protein (TP), alkaline phosphatase (AKP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Renal tissues were fixed with 4% paraformaldehyde and paraffin sections were prepared for analysis of pathological changes using hematoxylin-eosin (HE) staining.

[0139] Experimental results:

[0140] like Figure 5 As shown, continuous oral administration of rifampicin (40 mg / kg) for 10 days significantly increased P-gp expression in the liver and kidneys of mice. P-gp protein expression in the liver and kidneys of mice in the rifampicin-treated group was significantly higher than that in the control group (Figures A / D, ***P<0.001). Semiquantitative analysis (Figures B / C / E) showed an approximately 2-3-fold increase in P-gp expression, suggesting that rifampicin may enhance drug efflux by inducing P-gp.

[0141] like Figure 6 As shown, plasma uranium concentrations drop rapidly after administration. Uranium accumulates significantly in the femur after 24 hours (Panel B), suggesting that bone is a long-term storage site for uranium. Uranium levels in the liver and kidneys peak within 6 hours (Panels C and D), followed by a slow elimination process. Residual levels in the kidneys are high, indicating that the kidneys are the primary excretor of uranium.

[0142] The physiological process of uranium excretion through the kidneys leads to its specific accumulation in the kidney tissue of mice. The results of ICP-MS quantitative analysis are as follows Figure 6-7 As shown, 24 hours after acute uranium exposure (2 mg / kg), kidney uranium concentrations (7.48 ± 0.76 μg / g) were significantly higher than those in the femur (4.58 ± 0.46 μg / g) and liver (0.055 ± 0.024 μg / g), consistent with the kidney's role as the primary organ for uranium excretion. Significant differences in the metabolic rate of uranium in plasma were observed between groups. Rifampicin-induced P-gp activation significantly accelerated systemic uranium metabolism. Two hours after exposure, plasma uranium concentrations in the rifampicin-treated group decreased from 0.036 ± 0.003 μg / mL in the uranium-exposed group and 0.063 ± 0.009 μg / mL in the tadalafil-treated group, indicating that plasma uranium concentrations had reached a low level. Tissue distribution kinetics showed that the uranium content in the kidney of the P-gp activated group decreased by 26.48% (5.50±1.20μg / g vs 7.48±0.76μg / g in the uranium exposure group) and 44.72% (3.21±1.04μg / g vs 5.81±1.81μg / g in the uranium exposure group) at 24 and 72 hours, respectively; the uranium content in the liver decreased by 61.74% (0.02±0.007μg / g vs 0.06±0.02μg / g in the uranium exposure group) and 76.58% (0.02±0.005μg / g vs 0.07±0.012μg / g in the uranium exposure group), respectively; and the uranium content in the femur decreased by 35.61% (2.95±0.66μg / g in the uranium exposure group), respectively. vs 4.58±0.46μg / g in the uranium exposure group) and 34.67% (3.13±1.00μg / g vs 4.80±0.69μg / g in the uranium exposure group). Figure 7 The results of fecal and urinary accumulation showed that the cumulative excretion percentage of uranium in urine within 72 hours in the P-gp activator rifampicin intervention group was 53.60±9.72%, which was higher than the 36.14±4.72% in the model group and 24.04±2.74% in the P-gp inhibitor group, and were 1.48 times and 2.23 times that of the uranium exposure group and the P-gp inhibitor tadalafil intervention group, respectively; the cumulative excretion percentage of uranium in feces within 72 hours in the P-gp activator rifampicin intervention group was 14.91±1.32%, which was higher than the 11.53±1.95% in the uranium exposure group and 8.4±0.91% in the P-gp inhibitor tadalafil intervention group, and were 1.29 times and 1.78 times that of the uranium exposure group and the P-gp inhibitor tadalafil intervention group, respectively.

[0143] Activation of P-gp alleviated uranium-induced liver and kidney damage in BALB / c mice. Figure 8-9As shown, 72 hours after uranium exposure, the kidneys of mice exposed to uranium and tadalaquine showed significant swelling compared to the control group, with organ indexes increasing to 1.10 times (P < 0.01) and 1.18 times (P < 0.001), respectively. The degree of kidney swelling in the rifampicin group was 1.03 times (P > 0.05), with no statistically significant difference. Analysis of liver organ indexes showed that uranium exposure caused liver atrophy, with the indexes in the uranium-exposed and tadalaquine-treated groups decreasing to 0.75 and 0.71 times that of the control group, respectively. The degree of atrophy in the rifampicin group was 0.91 times (P < 0.01), significantly improved compared to the uranium-exposed group. Histopathological observations revealed that the glomeruli in the renal cortex of the control mice were densely arranged and structurally intact. No obvious pathological changes, such as inflammation, necrosis, hemorrhage, or edema, were observed. The uranium-exposed group showed coagulative necrosis of the renal tubules, with effacement of epithelial cell nuclei, pink proteinaceous mucus exudates within the tubular lumen, and severe destruction of tissue morphology and structure. In the tadalafil-treated group, proteinaceous mucus exudates were observed within the glomeruli, accompanied by extensive cellular edema and vacuoles, extensive tubular necrosis, and loss of tubular structure. In contrast, the rifampicin + uranium-exposed group showed only mild localized edema of renal tubular epithelial cells.

[0144] Plasma biochemical indicators showed that BUN, Cr, AKP, ALT, and AST levels in uranium-exposed mice were significantly elevated (P<0.05), reaching 5.75-fold, 2.18-fold, 1.56-fold, 1.61-fold, and 1.29-fold, respectively, compared to the control group. TP levels were reduced to 0.88-fold (P<0.001). Tadaliquine's inhibitory effect further exacerbated liver and kidney damage, with BUN, Cr, AKP, ALT, and AST levels increasing to 10.35-fold, 2.63-fold, 1.68-fold, 1.95-fold, and 1.45-fold, respectively, compared to the control group. TP levels were further reduced to 0.87-fold (P<0.001). Rifampicin induced improvement of this damage, with TP content restored to 0.93 times that of the control group (P<0.05), and BUN, Cr, AKP, ALT, and AST decreased to 2.92 times, 1.24 times, 1.16 times, 1.21 times, and 1.07 times that of the control group, respectively, which were 49.11%, 43.03%, 25.90%, 24.96%, and 17.00% lower than those in the uranium exposure group.

[0145] Table 2: Effects of P-gp activation on pharmacokinetic parameters in uranium-exposed mice (n=3)

[0146] U RFP+U TQR+U <![CDATA[t 1 / 2h (h)]]> 1.23±0.56 0.67±0.09 1.86±0.09 <![CDATA[AUC 0-∞ (h×ng / mL)]]> 1180.6±227.31 1073.89±246.15 2047.12±73.35 <![CDATA[AUC 0-t (h×ng / mL)]]> 1188.2±230.56 1083.85±248.28 2088.61±82.27 Vd(L / kg) 2.91±1 1.84±0.46 2.57±0.13 CL (L / h / kg) 1.73±0.37 1.92±0.47 0.96±0.04

[0147] Comparative Example

[0148] Animal selection: BALB / c male mice, SPF grade, weighing 18-22 g. The effects of immediate and delayed administration of sodium bicarbonate on the clearance of U and protection against U nephrotoxicity were investigated after single or multiple exposures.

[0149] The specific groups are as follows:

[0150] Control group (control): 0.9% saline was administered orally, without uranium exposure.

[0151] Uranium exposure group (Normal saline): uranyl nitrate poisoning (uranyl nitrate 2 mg / kg, tail vein injection).

[0152] Sodium bicarbonate (NaHCO3immedidte): Uranyl nitrate poisoning (2 mg / kg uranyl nitrate, tail vein injection) plus immediate oral administration of 1.0 g / kg sodium bicarbonate can induce urine alkalinization (pH 8-9).

[0153] Sodium bicarbonate (NaHCO3 24h-delay): Uranyl nitrate poisoning (2 mg / kg uranyl nitrate, tail vein injection) plus oral administration of 1.0 g / kg sodium bicarbonate 24 hours later can induce urine alkalinization (pH 8-9).

[0154] Sodium bicarbonate (NaHCO3 5-delay): Uranyl nitrate poisoning (80 μg / kg uranyl nitrate, injected into the tail vein for 5 consecutive days) plus oral administration of 1.0 g / kg sodium bicarbonate can induce urine alkalinization (pH 8-9).

[0155] like Figure 10 As shown in Figures a and b, immediate administration of 1.0 g / kg sodium bicarbonate induced urinary alkalinization (pH 8-9), significantly reduced renal U content in mice exposed to a single, highly toxic dose of uranium (2.0 mg / kg) and increased 24-hour urinary U excretion, consistent with studies demonstrating uranium clearance by sodium bicarbonate in uranium-contaminated rats. However, delayed single doses of sodium bicarbonate, administered 24 hours or 5 days later, had no effect on U clearance in mice exposed to a single, highly toxic dose of 2.0 mg / kg or a daily low-dose of 80 μg / kg for 5 consecutive days. Multiple doses of the chelator 5LIO-1-Cm-3,2-HOPO, administered 24 hours later, induced a 61.4-65.0% reduction in renal U in mice. Clearly, a single, 24-hour delayed administration of ML-SA1 resulted in the same efficiency in degrading 5LIO-1-Cm-3,2-HOPO as multiple, 24-hour delayed administrations. More importantly, even a single 5-day delayed administration of ML-SA1 significantly removed U from the kidney and increased U excretion in the urine of mice after multiple-dose U exposure, although the efficiency of U removal in the kidney was lower compared with that of ML-SA1 administered with a 24-h delay.

[0156] As uranium levels in the kidneys decrease (Figure b), immediate administration of sodium bicarbonate improves renal proximal tubular function impairment. In Figure c, 48 hours after a single, highly toxic dose of 2.0 mg / kg uranium intoxication, CRE and BUN levels were significantly reduced, and renal pathological damage in the S1, S2, and S3 segments of the proximal tubules was significantly alleviated. In contrast, delayed administration of sodium bicarbonate for 24 hours and 5 days was ineffective in alleviating renal tubular damage, consistent with the results that a single, highly toxic dose of 2.0 mg / kg uranium or a low-dose of 80 μg / kg daily for 5 consecutive days had no effect on renal urinary urinary excretion in mice (Figure b).

[0157] Compared with urine alkalinization with sodium bicarbonate and activation of P-gp expression with rifampicin, rifampicin significantly accelerated uranium excretion in the rifampicin group. Within 72 hours, uranium excretion in urine and feces was 2.28-fold and 1.29-fold higher than that in the uranium-exposed group, respectively, and uranium accumulation in the kidney and liver decreased by 44.72% and 76.58%, respectively. Sodium bicarbonate was effective only when administered immediately (urinary excretion increased after a single high-dose uranium exposure), but delayed administration for 24 hours or 5 days had no significant effect on uranium clearance and had little effect on the excretion of uranium deposited in bone (such as tetravalent uranium). Rifampicin significantly alleviated uranium-induced liver and kidney damage, with renal swelling returning to control levels (no statistical difference), and plasma BUN, Cr, ALT, and AST levels decreased by 25-49% compared with the uranium-exposed group. Sodium bicarbonate only partially ameliorated renal tubular damage when administered immediately; delayed administration failed to alleviate pathological damage and may have increased renal burden due to sodium overload. Rifampicin, by activating endogenous P-gp, avoids the side effect of nonspecific binding to other metal ions and remains effective against already deposited uranium (such as in bone). Sodium bicarbonate relies on urine alkalinization (pH 8-9), requiring strict medical control. It is also ineffective against already deposited uranium and may exacerbate renal impairment at high doses.

[0158] Experimental conclusion:

[0159] Rifampicin upregulates P-gp expression in the liver and kidneys, enhancing uranium excretion and reducing its accumulation in key organs. Uranium accumulates primarily in the bones and kidneys, and long-term exposure can cause liver and kidney damage. However, rifampicin significantly alleviates toxicity. This study provides a theoretical basis for detoxification strategies for uranium poisoning, such as using P-gp inducers to reduce uranium toxicity.

[0160] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. Application of an activator in promoting the discharge of uranium.

2. The application according to claim 1, characterized in that The activator includes at least one of rifampicin, erythromycin, progesterone, aldosterone, bilirubin, bile salt vinblastine, daunorubicin, doxorubicin, paclitaxel, bosentan, ambrisentan, digoxin, verapamil, tonavir, amprenavir, indinavir, St. John's wort, curcumin, atorvastatin, beclomethasone, budesonide, dimethoate, carbamazepine, caffeine, and phenytoin.

3. The application according to claim 1, characterized in that The dosage of the activator is 10-60 mg / kg.

4. The application according to claim 1, characterized in that The method for promoting the discharge of uranium comprises the following steps: 1) administering the activator according to any one of claims 1 to 3 to a uranium-exposed organism; 2) Promote the excretion of uranium from the body through urine or bile by activating P-glycoprotein; 3) Regularly monitor the expression level of P-glycoprotein and dynamically adjust the dosage of the activator to maintain the ovulation-inducing effect.

5. The application according to claim 4, characterized in that: In step 2), the activator induces P-glycoprotein activation, enhances the conformational change of the P-glycoprotein specific transport channel, opens the transport channel, and allows uranium to enter the transport channel and be transported outside the cell, thereby enhancing the transport function of uranium and excreting uranium from the body through urine or bile.

6. The application according to claim 5, characterized in that The P-glycoprotein includes P-glycoprotein in the kidney and liver.

7. The use according to claim 4, characterized in that: Application of the described method in the study of the biological transport and detoxification mechanism of uranium.

8. A drug for preventing and treating diseases caused by uranium, characterized in that: The invention comprises the activator involved in the application according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

9. The use according to claim 1, characterized in that: The diseases caused by uranium include diseases caused by uranium poisoning due to internal contamination of uranium.

10. The drug according to claim 8, characterized in that The diseases caused by uranium poisoning include acute uranium poisoning, chronic uranium poisoning, uranium poisoning-induced kidney damage or uranium poisoning-induced bone damage, leukemia, lung cancer, skin cancer and radiation-related cancers.

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