Use of n-acetylcysteine in the preparation of a medicament for the treatment of uranium poisoning

Abstract

The application belongs to the field of new use of medicines, and more particularly relates to application of N-acetylcysteine in preparation of medicines for treating uranium poisoning. The application applies N-acetylcysteine to treatment of uranium poisoning for the first time, and experiments show that the medicine can significantly reduce the content of intracellular ROS and MDA after uranium poisoning, improve the level of GSH and CAT, and reshape the oxidation-reduction balance; meanwhile, the medicine can effectively relieve DNA damage, significantly restore the RNA synthesis and protein translation level inhibited by uranium, and protect the integrity of ribosome structure. The application fills the technical gap of ribosome protection in intervention of uranium poisoning, has the significant advantages of targeted precision, no toxic side effects, and both antioxidant and repair functions, and is suitable for nuclear accident emergency and occupational exposure protection.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceuticals, and more specifically, relates to the use of N-acetylcysteine ​​in the preparation of medicaments for treating uranium poisoning. Background Technology

[0002] Uranium, as an important radioactive heavy metal nuclide, is widely used in nuclear power generation, nuclear industry manufacturing, national defense, geological exploration, and other fields, holding an irreplaceable strategic position in national economic and defense construction. With the continuous development of the global nuclear energy industry, the increasing frequency of nuclear facility operation and maintenance, and the expansion of nuclear waste disposal scenarios, the health risks to humans caused by occupational uranium exposure and environmental uranium contamination have become increasingly prominent, becoming a key research topic in the fields of nuclear toxicology, public health safety, and emergency medicine.

[0003] Uranium exposure in humans is characterized by its diverse and highly concealed pathways. It can enter the human body through various means, including inhalation of uranium-containing dust and aerosols, ingestion of contaminated water and food, and skin / mucous membrane contact. There is no clearly defined specific exposure threshold scenario; routine occupational activities, sudden nuclear leaks, and environmental contamination can all lead to varying degrees of uranium accumulation in the body. Studies have shown that the kidneys are the primary target organ for uranium toxicity in the human body. Acute high-dose uranium exposure can directly induce acute kidney injury, clinically manifested as renal tubular necrosis, a sharp decline in renal function, proteinuria, and hematuria—typical pathological features. It can also indirectly affect multiple bodily systems, including the liver, bones, and blood system, causing systemic metabolic disorders and functional impairment.

[0004] Once uranium enters a cell, it can rapidly induce a massive surge and abnormal accumulation of reactive oxygen species (ROS), completely disrupting the dynamic balance between oxidation and antioxidation within the cell and triggering a severe oxidative stress response. Excessive ROS continuously attack the cell membrane lipid structure, inducing severe lipid peroxidation, damaging cell membrane integrity and permeability, leading to cell structural damage and functional disorders. Simultaneously, excessive ROS continuously consume important antioxidants such as superoxide dismutase and glutathione, completely destroying the cell's endogenous antioxidant system, causing the cell to lose its autonomous antioxidant and anti-damage capabilities, forming a vicious cycle of oxidative damage. Furthermore, uncontrolled oxidative stress further attacks cellular genetic material, causing DNA double-strand breaks, base damage, and RNA structural disruption, interfering with the normal replication and transmission of cellular genetic information, and inducing cellular gene mutations and metabolic disorders.

[0005] In the field of clinical intervention and protection against uranium exposure injury, there are currently no dedicated, efficient, and safe targeted intervention drugs or protective measures, either domestically or internationally. Existing clinical intervention methods heavily rely on traditional metal chelating agents, such as diethylenetriaminepentaacetic acid (DTI) and calcium edetate sodium. The basic mechanism of action of these drugs is to chelate free uranium ions in the body to form soluble complexes, promoting the excretion of uranium ions through urine and body fluids, thereby reducing the accumulation of uranium in the body and achieving initial detoxification intervention. However, long-term clinical application and experimental research have revealed that traditional metal chelating agents have many inherent technical defects and cannot meet the comprehensive protection and treatment needs of uranium exposure injury, exhibiting significant technical shortcomings.

[0006] First, the drug's targeting selectivity is extremely poor. Traditional chelating agents lack the ability to specifically recognize uranium ions. While chelating uranium ions in the body, they also non-specifically chelate various essential trace elements such as calcium, magnesium, zinc, and iron, leading to electrolyte imbalances, trace element deficiencies, and systemic metabolic abnormalities. Second, the drug itself has significant organ toxicity. Long-term or high-dose use will increase the metabolic burden on the liver and kidneys, inducing secondary liver and kidney damage. For liver and kidney function already damaged by uranium toxicity, this will cause a "damage superposition" negative effect, significantly increasing the clinical treatment risk. Third, and most importantly, traditional metal chelating agents can only remove free uranium ions from body fluids. They cannot target and protect damaged ribosomes, repair inhibited RNA synthesis and protein translation functions, or block uranium-mediated ribosomal stress and apoptosis / pyroptosis pathways. They can only achieve "detoxification" and cannot achieve "damage protection, repair, and anti-cell death," making it difficult to fundamentally curb the cytotoxicity and organ damage process of uranium. Summary of the Invention

[0007] The purpose of this invention is to provide the application of N-acetylcysteine ​​in the preparation of medicaments for treating uranium poisoning.

[0008] This invention provides the application of N-acetylcysteine ​​in the preparation of medicaments for treating uranium poisoning.

[0009] This invention uses N-acetylcysteine ​​as the active ingredient. On one hand, as a classic antioxidant, it effectively blocks the oxidative damage cascade by scavenging excess ROS, restoring the activity of endogenous antioxidant enzymes (GSH / CAT), and inhibiting lipid peroxidation (MDA). On the other hand, this invention is the first to discover that this compound can specifically reverse the inhibitory effect of uranium on the ribosomal transcription and translation system, thereby restoring the ability to synthesize new RNA and the level of newly generated proteins, fundamentally rescuing damaged cells and blocking apoptosis and pyroptosis pathways. This concept abandons the limitations of "simple detoxification" and realizes the transformation from "detoxification and organ protection" to "comprehensive repair of cell function," truly achieving both symptomatic and root-cause treatment without interfering with the body's normal trace element metabolism. It provides a pioneering technical path for highly efficient and low-toxicity intervention in uranium poisoning.

[0010] Furthermore, the drug is used to reduce the amount of ROS generated in cells after uranium poisoning.

[0011] Furthermore, the drug is used to increase intracellular GSH levels after uranium poisoning.

[0012] Furthermore, the drug is used to increase intracellular CAT levels after uranium poisoning.

[0013] Furthermore, the drug is used to reduce intracellular MDA levels after uranium poisoning.

[0014] Furthermore, the drug is used to alleviate cellular DNA damage following uranium poisoning.

[0015] Furthermore, the drug is used to enhance cellular RNA synthesis after uranium poisoning.

[0016] Furthermore, the drug is used to restore cellular protein synthesis levels after uranium poisoning.

[0017] Furthermore, the drug is made from the N-acetylcysteine ​​with a pharmaceutically acceptable carrier and / or excipient.

[0018] The beneficial effects of this invention are as follows: This invention addresses the shortcomings of traditional uranium exposure intervention chelating agents, such as poor targeting, secondary liver and kidney toxicity, and inability to protect ribosomes or repair damage to RNA and protein synthesis. Based on the toxic mechanisms of uranium oxidative stress imbalance, ribosome dysfunction, and programmed cell death, it provides a safe intervention scheme with significant technical advantages and application value. This invention fills the technological gap in ribosome protection in the field of uranium poisoning intervention for the first time, effectively protecting the structural integrity of ribosomes, reversing uranium-induced RNA synthesis and protein translation inhibition, and blocking ribosome stress-induced apoptosis and pyroptosis pathways, thus curbing uranium-induced cytotoxic damage at its source. Simultaneously, this invention significantly inhibits abnormal ROS accumulation in cells, restores the body's oxidative and antioxidant balance, reduces lipid peroxidation and nucleic acid damage, and comprehensively blocks the multi-level cascade damage effects mediated by uranium. Compared to the drawbacks of traditional chelating agents that non-specifically chelate trace elements and easily cause secondary liver and kidney damage, this invention is precisely targeted, has no toxic side effects, does not interfere with the body's normal physiological metabolism, and has significantly improved safety. This invention combines detoxification and organ protection with damage repair, enabling both symptomatic and radical treatment of uranium exposure damage. It effectively reduces the risk of long-term complications such as kidney damage and organ fibrosis, and is applicable to various scenarios such as nuclear accident emergency response and occupational exposure protection. It provides a new and feasible technical approach for efficient and low-toxicity intervention in uranium poisoning. Attached Figure Description

[0019] Figure 1The images show the CCK8 results, where A represents cell viability statistics after 24 hours of uranium exposure, B represents cell viability statistics after 48 hours of uranium exposure, and C represents cell viability statistics after 72 hours of uranium exposure.

[0020] Figure 2 The images show ROS fluorescence images of HK-2 cells exposed to uranium for 24 hours under a 10x objective lens. A shows the fluorescence of HK-2 cells exposed to different concentrations of uranium nitrate (0 μM, 200 μM, 400 μM and 800 μM, 800 μM + NAC co-incubation) for 24 hours. B shows the quantitative analysis results from ImageJ.

[0021] Figure 3 The images show ROS fluorescence after 48 hours of uranium exposure. In the image, A shows the fluorescence of HK-2 cells exposed to different concentrations of uranium nitrate (0 μM, 200 μM, 400 μM and 800 μM, 800 μM + NAC co-incubation) for 48 hours. In the image, B shows the quantitative analysis results from ImageJ.

[0022] Figure 4 The images show the levels of oxidative stress-related indicators. The images, in order, are: GSH, CAT, and MDA levels after 24 h and 48 h of treatment with different concentrations of uranium nitrate (0 μM, 200 μM, 400 μM, and 800 μM, 800 μM + NAC co-incubation). Specifically, A represents the GSH level after 24 h of treatment with different concentrations of uranium nitrate; B represents the CAT level after 24 h of treatment with different concentrations of uranium nitrate; C represents the MDA level after 24 h of treatment with different concentrations of uranium nitrate; D represents the GSH level after 48 h of treatment with different concentrations of uranium nitrate; E represents the CAT level after 48 h of treatment with different concentrations of uranium nitrate; and F represents the MDA level after 48 h of treatment with different concentrations of uranium nitrate.

[0023] Figure 5 The images are from a comet experiment. The images, in order, show the fluorescence of DNA damage after treatment with different concentrations of uranium nitrate (0 μM, 200 μM, 400 μM, and 800 μM, and co-incubation with NAC at 800 μM) for 48 hours.

[0024] Figure 6 The images show EU staining patterns, where A is the fluorescence image of newly synthesized RNA after treatment with different concentrations of uranium nitrate (0 μM, 200 μM, 400 μM and 800 μM, 800 μM and NAC co-incubated) for 48 h, and B is the imageJ quantitative analysis image.

[0025] Figure 7 The diagram shows the puromycin incorporation experiment. After co-incubation with 800 μM uranium nitrate, 800 μM uranium nitrate and NAC for 48 h, the protein synthesis was investigated using Western blotting. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0027] Example 1 I. CCK-8 assay for cell viability.

[0028] 1. Experimental Methods Seeding plate: Add 1×10 to each well of the 96-well plate 4 One hundred microliters of HK-2 cell suspension were added to each sample. The blank control group received only complete culture medium without cell suspension. Each sample group was prepared in triplicate. After adding the cell suspension, the samples were placed in an incubator and incubated until adherence occurred.

[0029] Sample preparation: Discard the culture medium, wash with PBS, add only 100 μL of DMEM to the Control group, add the corresponding concentration of uranium nitrate solution (diluted with DMEM) to the experimental groups, and pretreat the 800 μM + NAC group with 10 mmol of NAC for 15 minutes before co-incubation with 800 μM uranium nitrate. Incubate the plates in a constant temperature incubator for 24 h, 48 h, and 72 h, respectively.

[0030] Measurement: Aspirate the liquid from each well, recover the uranium nitrate solution, and wash twice with PBS. Add 100 μL of CCK-8 preparation solution to each well, wrap with aluminum foil to protect from light, and incubate in a constant temperature incubator for 1 hour. Detect the absorbance at 450 nm using a microplate reader.

[0031] 2. Experimental Results The changes in HK-2 cell viability after exposure to uranium nitrate for 24 h, 48 h, and 72 h were detected using the CCK-8 assay. The experimental results are as follows: Figure 1 As shown, the survival rate of HK-2 cells gradually decreased with increasing uranium concentration. Cell viability was lowest after 24h, 48h, and 72h of uranium exposure at high concentrations (800μM). This indicates that uranium has significant toxicity to HK-2 cells, and this toxicity is dose- and time-dependent. Pretreatment with NAC significantly increased the viability of HK-2 cells after co-incubation with uranium for 24h and 48h.

[0032] II. ROS detection.

[0033] 1. Experimental Methods Seeding plate: Add 2×10 to each well of a 12-well plate 5One mL of cell suspension was added to each sample, with three replicates per sample group. After adding the cell suspension, the samples were placed in a constant temperature incubator and incubated until adherence occurred.

[0034] Sample preparation: Discard the culture medium, wash with PBS, add only 1 mL of DMEM to the control group, and add the corresponding concentration of uranium nitrate solution (diluted with DMEM) to the experimental group. The 800 μM + NAC group was pretreated with 10 mmol of N-acetylcysteine ​​(NAC) for 15 minutes, followed by co-incubation with 800 μM uranium nitrate. After all reagents were added, the plates were incubated in a constant temperature incubator for 24 h or 48 h.

[0035] Preparation of working solution: Dilute DCFH-DA solution with DMEM to 10 μM and use in the dark.

[0036] Pretreatment before measurement: Aspirate the liquid from each well, recover the uranium nitrate solution, and wash twice with PBS.

[0037] Measurement: Add 1 mL of 10 μM DCFH-DA solution to each well, wrap with aluminum foil to protect from light, and incubate in a constant temperature incubator for half an hour. Remove the liquid from the wells, wash five times with DMEM to thoroughly remove the fluorescent probe, observe under an inverted microscope, and perform quantitative processing of the images.

[0038] 2. Experimental Results Figure 2 To expose HK-2 cells to different concentrations of uranium nitrate for 24 hours, Figure 3 After 48 hours of exposure, ROS was detected using the fluorescent probe DCFH-DA. The experimental results are as follows: Figure 2 and Figure 3 As shown, statistical analysis of ROS fluorescence intensity revealed that intracellular ROS levels were significantly higher after uranium treatment compared to the control group. After 24 hours of exposure, as the uranium treatment concentration gradually increased from 200 μM to 800 μM, the ROS content exhibited a gradient upward trend. NAC pretreatment significantly reduced ROS production. After 48 hours of exposure, ROS production further increased, also showing a concentration gradient upward trend. NAC pretreatment significantly reduced ROS production. This indicates that uranium can induce an increase in ROS production, and this effect is clearly dose-dependent and has some time-dependent characteristics. NAC can alleviate this abnormal ROS production.

[0039] III. Physiological Indicator Testing.

[0040] 1. Experimental Methods (1) Measurement of GSH content Seed culture: Plant 5 × 10⁶ seeds in a 10 cm diameter petri dish. 6 Each cell was placed in a constant temperature incubator overnight to allow it to adhere to the culture vessel.

[0041] Sample preparation: Discard the culture medium, wash with PBS, add only 10 mL of DMEM to the control group, and add the corresponding concentration of uranium nitrate solution (diluted with DMEM) to the experimental group. The 800 μM + NAC group was pretreated with 10 mmol of NAC for 15 minutes, and then co-incubated with 800 μM uranium nitrate. After all reagents were added, the plates were incubated in a constant temperature incubator for 24 h or 48 h.

[0042] Pretreatment before measurement: Aspirate liquid from each culture dish, recover uranium nitrate solution, and wash twice with PBS. Add 1 mL of lysis buffer, scrape cells off using a cell scraper, and collect them into 1.5 mL centrifuge tubes. Place in a freezer at -80°C, repeat freeze-thaw three times, then set the centrifuge speed to 12000g at 4°C and centrifuge for 10 minutes. After centrifugation, collect the supernatant for analysis. The assay uses a reduced glutathione (GSH) assay kit (Cat: BC1175), purchased from Solarbio.

[0043] (2) CAT content Seed culture: Plant 5 × 10⁶ seeds in a 10 cm diameter petri dish. 6 Each cell was placed in a constant temperature incubator and allowed to adhere to the culture vessel.

[0044] Sample preparation: Discard the culture medium, wash with PBS, add only 10 mL of DMEM to the control group, and add the corresponding concentration of uranium nitrate solution (diluted with DMEM) to the experimental group. The 800 μM + NAC group was pretreated with 10 mmol of NAC for 15 minutes, and then co-incubated with 800 μM uranium nitrate. After all reagents were added, the plates were incubated in a constant temperature incubator for 24 h or 48 h.

[0045] Pretreatment before measurement: Aspirate liquid from each culture dish, recover uranium nitrate solution, and wash twice with PBS. Add 1 mL of lysis buffer, scrape cells off using a cell scraper, and collect them into 1.5 mL centrifuge tubes. Place in a freezer at -80°C, repeat freeze-thaw three times, then set the centrifuge speed to 8000g at 4°C and centrifuge for 10 minutes. After centrifugation, collect the supernatant for detection. The detection was performed using a catalase (CAT) activity assay kit (Cat: BC0205), purchased from Solarbio.

[0046] (3) MDA content Seed culture: Plant 5 × 10⁶ seeds in a 10 cm diameter petri dish. 6 Each cell was placed in a constant temperature incubator and allowed to adhere to the culture vessel.

[0047] Sample preparation: Discard the culture medium, wash with PBS, add only 10 mL of DMEM to the control group, and add the corresponding concentration of uranium nitrate solution (diluted with DMEM) to the experimental group. The 800 μM + NAC group was pretreated with 10 mmol of NAC for 15 minutes, and then co-incubated with 800 μM uranium nitrate. After all reagents were added, the plates were incubated in a constant temperature incubator for 24 h or 48 h.

[0048] Pretreatment before measurement: Aspirate liquid from each culture dish, recover uranium nitrate solution, and wash twice with PBS. Add 1 mL of lysis buffer, scrape cells off using a cell scraper, and collect them into 1.5 mL centrifuge tubes. Place in a freezer at -80°C, repeat freeze-thaw three times, then set the centrifuge speed to 12000g at 4°C and centrifuge for 5 minutes. After centrifugation, collect the supernatant for detection. The malondialdehyde (MDA) content detection kit (TBARS assay) (Cat: BC6415) was purchased from Solarbio.

[0049] 2. Experimental Results HK-2 cells were exposed to different concentrations of uranium nitrate (0 μM, 200 μM, 400 μM, and 800 μM) for 24 h and 48 h, respectively. The levels of reduced glutathione (GSH), catalase (CAT), and malondialdehyde (MDA) in the cells were then measured. The experimental results are as follows: Figure 4 As shown, after 24 hours of uranium nitrate treatment, compared with the control group, intracellular GSH content and CAT activity were both decreased, while MDA content gradually increased with increasing uranium nitrate concentration. After the addition of NAC, GSH content rebounded, CAT activity recovered somewhat, and MDA content decreased. After 48 hours of treatment, the trends of GSH, CAT, and MDA were consistent with those after 24 hours.

[0050] The results showed that uranium nitrate exposure induced oxidative stress in HK-2 cells, leading to GSH depletion, inhibition of CAT activity, and significant MDA accumulation. NAC treatment effectively alleviated this condition. Considering the changes in enzymes and ROS levels related to the oxidative stress system, uranium exposure caused an imbalance in the cellular redox system, which NAC could alleviate.

[0051] IV. Detection of cellular DNA damage degree by comet electrophoresis.

[0052] 1. Experimental Method: Seed culture: Plant 5 × 10⁶ seeds in a 10 cm diameter petri dish. 6 Each cell was placed in a constant temperature incubator and allowed to adhere to the culture vessel.

[0053] Sample preparation: Discard the culture medium, wash with PBS, add only 10 mL of DMEM to the control group, and add the corresponding concentration of uranium nitrate solution (diluted with DMEM) to the experimental group. The 800 μM + NAC group was pretreated with 10 mmol of NAC for 15 minutes, and then co-incubated with 800 μM uranium nitrate. After all reagents were added, the plates were incubated in a constant temperature incubator for 48 hours.

[0054] Cell processing: Aspirate the liquid from each culture dish, recover the uranium nitrate solution, and wash twice with PBS. After trypsin digestion, resuspend the cells. Ensure the cell density in the cell suspension is 1 × 10⁻⁶. 6 per mL.

[0055] Gel preparation: Both normal melting point agarose (NMA) and low melting point agarose (LMA) were prepared using PBS.

[0056] First layer of gel plating: Set the water bath temperature to 100℃, dissolve 1% NMA, and preheat the ground glass slide. Add 100μL of NMA to the ground surface, cover with a coverslip, and incubate at 4℃ for 10 minutes.

[0057] Second gel layer plating: Set the water bath temperature to 70℃ and dissolve 0.7% LMA. Take 10 μL of cell suspension and mix it with 75 μL of LMA. After mixing, remove the coverslip from the first gel layer plate, drop 85 μL of the liquid onto the first layer, cover with a coverslip, and incubate at 4℃ for 10 minutes.

[0058] Third gel layer: Remove the coverslip, add 100 μL of 0.7% LMA on top of the gel, and cover with the coverslip again. Incubate at 4°C in a humid environment for 40 minutes.

[0059] Lysis: The lysis buffer was prepared by mixing 45 mL of Lysis Bufffer and 5 mL of DMSO. Lysis was carried out at 4°C for two hours. The lysed slides were then removed and rinsed with PBS for 10 minutes, repeated twice.

[0060] Electrophoresis: The alkaline electrophoresis buffer has a concentration of 1 mM EDTA and 300 mM NaOH.

[0061] Unwinding: Place the glass slide in a flat electrophoresis pool, add freshly prepared alkaline electrophoresis buffer solution, ensuring the solution level is 0.25 cm above the gel layer on the slide. After adding the solution, let it stand for one hour.

[0062] Electrophoresis: 25V, 30 minutes.

[0063] Staining: After electrophoresis, wash three times with PBS for 10 minutes each time. After washing, add 20 μL of PI staining solution, cover with a coverslip, and stain for 10 minutes in the dark.

[0064] Microscopic photography: Under excitation light of 515~560nm, observe and photograph using an inverted microscope.

[0065] 2. Experimental Results HK-2 cells were exposed to different concentrations of uranium nitrate (0 μM, 200 μM, 400 μM, and 800 μM) for 48 h. The degree of DNA damage in HK-2 cells was then detected using single-cell gel electrophoresis (comet assay). The experimental results are as follows: Figure 5 As shown, the comet tail length of HK-2 cells gradually increased with increasing uranium nitrate concentration. After the addition of NAC, the tail length decreased, but remained slightly higher than that of the control group. These results indicate that uranium nitrate exposure for 48 hours leads to DNA damage in HK-2 cells, and NAC can significantly alleviate this damage.

[0066] V. RNA synthesis detection.

[0067] 1. Experimental Methods Seeding plate: Seed 5×10⁻⁶ holes in a six-hole plate. 5 Each cell was placed in a constant temperature incubator and allowed to adhere to the culture vessel.

[0068] Sample preparation: Discard the culture medium, wash with PBS, add only 2 mL of DMEM to the control group, add the corresponding concentration of uranium nitrate solution (diluted with DMEM) to the experimental group, and pretreat the 800 μM + NAC group with 10 mmol of NAC for 15 minutes before co-incubation with 800 μM uranium nitrate. After all reagents have been added, incubate the plates in a constant temperature incubator for 48 hours.

[0069] EU doping: Aspirate the liquid from each well, recover the uranium nitrate solution, and wash twice with PBS.

[0070] Preparation of working solution: Prepare a 2mM EU working solution using 4.9mL of DMEM and 100μl of EU. After mixing, heat in a 37°C water bath.

[0071] Add 1 mL each of EU working solution and DMEM to a six-well plate and incubate in a constant temperature incubator for two hours.

[0072] After EU labeling, remove the working solution, add 1 mL of immunostaining fixative to each well, fix for 15 minutes at room temperature.

[0073] Remove the fixative and wash the cells three times with 1 mL of PBS for 5 minutes each time.

[0074] Add 1 mL of PBS containing 0.3% Triton X-100 to each well and incubate at room temperature for 15 minutes.

[0075] Wash three more times with PBS, each time for 5 minutes.

[0076] Preparation of working solution: Dissolve Click Additive in 1.3 mL of ultrapure water and mix well. Pipette 250 μL of Click Reaction Buffer, 2.15 mL of CuSO4, and 5 μL of Azide 594, and mix thoroughly. Add 0.5 mL of the working solution to each well of a six-well plate. Incubate in the dark for 30 minutes at room temperature.

[0077] Test: Aspirate the working solution and wash three times with PBS, five minutes each time. After washing, observe under an inverted microscope.

[0078] 2. Experimental Results HK-2 cells were exposed to different treatment groups for 48 hours, and the synthesis level of newly generated RNA in HK-2 cells was detected. The experimental results are as follows: Figure 6 As shown, compared with the control group, the intracellular EU fluorescence intensity decreased significantly in a dose-dependent manner after 48 h of uranium nitrate exposure. The addition of NAC slightly increased the EU fluorescence intensity, but it remained lower than the control group. These results indicate that uranium nitrate exposure for 48 h significantly inhibited RNA synthesis in HK-2 cells, while NAC partially alleviated the inhibitory effect.

[0079] VI. Protein synthesis detection.

[0080] 1. Experimental Methods This experiment was divided into a control group and an experimental group. The uranium nitrate concentrations in the experimental groups were 800 μM and 800 μM + NAC, respectively. The treatment time for uranium nitrate was 48 h.

[0081] (1) Seed plate: Plant 5×10 seeds in a petri dish with a diameter of 10 cm. 6 Each cell was placed in a constant temperature incubator and allowed to adhere to the culture vessel.

[0082] (2) Sample preparation: Discard the culture medium, wash with PBS, add only 10 mL of DMEM to the control group, and add the corresponding concentration of uranium nitrate solution (diluted with DMEM) to the experimental group. The 800 μM + NAC group was pretreated with 10 mmol of NAC for 15 minutes, and then co-incubated with 800 μM uranium nitrate. After all reagents were added, the plates were incubated in a constant temperature incubator for 48 hours. 10 minutes before sample extraction, add 10 μg / mL puromycin to each plate and incubate for 10 minutes before proceeding to the next step.

[0083] (3) Protein extraction: Aspirate the liquid from each culture dish, recover the uranium nitrate solution, and wash twice with PBS. Add 1 mL of lysis buffer to the culture dish and scrape off the cells using a cell scraper. After repeated freeze-thaw cycles at -80°C three times, centrifuge at 12000 g and 4°C for 15 minutes, and retain only the supernatant.

[0084] (4) Protein quantification: a. Preparation of working solution: Take a certain amount of 25 mg / ml protein standard solution and dilute it to a final concentration of 0.5 mg / mL. The diluent is PBS. Add 5 mL of BCA reagent A to 100 μL of BCA reagent B and mix well to prepare the BCA working solution.

[0085] b. Sample loading: Add standards to the 96-well plates at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, respectively. Use PBS to bring the liquid level in each well to 20 μL. These concentrations correspond to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively. Add 20 μL to each sample well.

[0086] c. Measurement: Add 200 μL of BCA working solution to each well and incubate at room temperature for 2 hours. Measure A562 using a microplate reader, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0087] (5) Protein sample preparation: a. Use 5x protein loading buffer and add it to the supernatant at a protein:buffer ratio of 5:1.

[0088] b. After uniform mixing, place in a 100°C metal bath for 10 minutes to fully denature the protein.

[0089] c. After brief centrifugation, dispense into portions and store at -80℃.

[0090] (6) SDS-PAGE gel electrophoresis: Install the glass plates on the gel casting rack, ensuring the glass plates are completely sealed. Prepare a 10% separating gel using the kit and add it to the glass plate interlayer, filling approximately three-quarters of the space. Flatten the liquid surface with ultrapure water and let it stand for 30 minutes to solidify. After solidification, pour off the water layer, prepare a stacking gel, fill the plate completely, and insert a comb to prevent air bubbles from forming.

[0091] (7) Preparation of electrophoresis solution: After the gel is completely solidified, place it in the electrophoresis tank and add 1X electrophoresis solution. The electrophoresis solution formula is as follows: 3.03g of Tris base, 14.4g of glycine and 1g of SDS dissolved in 1L of ultrapure water.

[0092] (8) Electrophoresis: a. Gently and vertically remove the comb, and add the marker and protein to the sample wells. The marker loading volume is 2 μL, and the protein loading volume is 30 μg / well. Turn on the power. For the stacking gel stage, use a constant voltage of 80V for 30 minutes. For the separating gel stage, use a constant voltage of 120V for 1 hour. Stop electrophoresis when the bromophenol blue indicator line approaches the bottom of the gel.

[0093] b. After electrophoresis, carefully remove the gel. Immerse the PVDF membrane in ethanol for at least 30 seconds. During transfer, the structure from negative to positive electrode is as follows: sponge pad, filter paper, gel, PVDF membrane, filter paper, sponge pad. When making the transfer clamp, use a roller to remove all air bubbles.

[0094] c. Place the transfer clamp into the transfer tank, which is in an ice-water mixture environment. The transfer buffer formula is as follows: pour the rapid transfer buffer (powder) into 800 mL of ultrapure water and 200 mL of ethanol and mix well. Turn on the power and use a constant current of 250 mA for 30 minutes for transfer.

[0095] d. After the transfer is complete, remove the PVDF membrane and wash it three times with TBST. Immerse the membrane in a TBST blocking solution containing 5% skim milk and seal it on a shaker at room temperature for 2 hours.

[0096] e. After blocking, wash the membrane three times with TBST, and dilute the anti-puromycin antibody according to the instructions. The diluent is TBST. Place the membrane in the primary antibody (recombinant anti-puromycin antibody, abcam) and incubate overnight at 4°C.

[0097] f. After primary antibody incubation, wash three times with TBST for 10 minutes each time. Dilute the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG (H+L), Beyotime) according to the instructions. The diluent is TBST. Place the membrane in the secondary antibody dilution buffer and incubate on a shaker at room temperature for 1 hour.

[0098] g. After the secondary antibody incubation is complete, wash three times with TBST, 10 minutes each time.

[0099] h. Mix solutions A and B of the chemiluminescence (ECL) reagent in a 1:1 ratio, preparing fresh each time. Use tweezers to remove the membrane, blot away excess liquid with filter paper (do not allow the membrane to dry completely). With the protein side of the membrane facing up, evenly add the ECL working solution and incubate at room temperature for 2 minutes. Place the membrane in the dark chamber of the chemiluminescence imaging system. Acquire and save the image.

[0100] 2. Experimental Results HK-2 cells were exposed to 800 μM uranium nitrate for 48 h, and the synthesis level of newly synthesized proteins in HK-2 cells was detected. The experimental results are as follows: Figure 7As shown, uranium nitrate treatment significantly reduced the amount of newly synthesized proteins in cells, but the addition of NAC could restore some protein synthesis.

[0101] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0102] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0103] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of N-acetylcysteine ​​in the preparation of drugs for treating uranium poisoning.

2. The application according to claim 1, characterized in that, The drug is used to reduce the amount of ROS generated in cells after uranium poisoning.

3. The application according to claim 1, characterized in that, The drug is used to increase intracellular GSH levels after uranium poisoning.

4. The application according to claim 1, characterized in that, The drug is used to increase intracellular CAT levels after uranium poisoning.

5. The application according to claim 1, characterized in that, The drug is used to reduce intracellular MDA levels after uranium poisoning.

6. The application according to claim 1, characterized in that, The drug is used to alleviate cellular DNA damage following uranium poisoning.

7. The application according to claim 1, characterized in that, The drug is used to enhance cellular RNA synthesis after uranium poisoning.

8. The application according to claim 1, characterized in that, The drug is used to restore cellular protein synthesis levels after uranium poisoning.

9. The application according to claim 1, characterized in that, The drug is made from the N-acetylcysteine ​​with a pharmaceutically acceptable carrier and / or excipient.

Images