Use of stem cell exosomes in the preparation of a medicament for the treatment of uranium poisoning

Abstract

The application provides application of stem cell exosomes in preparation of a medicine for treating uranium poisoning. The application utilizes human umbilical cord mesenchymal stem cell-derived exosomes (MSC-EXS) to pre-administer to protect human renal tubular epithelial HK-2 cells from uranium damage, reduce intracellular ROS and MDA levels, increase GSH levels, enhance SOD activity, and reduce cell apoptosis.

Description

Technical Field

[0001] This invention relates to the field of chemical pharmaceutical technology, specifically to the application of stem cell exosomes in the preparation of drugs for treating uranium poisoning. Background Technology

[0002] Uranium (U) is a naturally occurring radioactive heavy metal that releases alpha and beta particles with high linear energy transfer, primarily used in nuclear energy production. Depleted uranium (DU) is mainly composed of... 238 U and 0.2%-0.3% 235 U is composed of uranium (U₂), a residual product from the enrichment of natural uranium used in nuclear fuel. Once ingested, it exhibits both heavy metal toxicity and radiotoxicity. After being absorbed into the bloodstream, uranium primarily forms the most stable form, U(VI), which then forms uranyl ions (UO₂). 2 + ), uranyl ion [UO2 2+ Uranyl ions (U(VI)) complex with biomolecules (mainly transferrin (50%), albumin (fetoglobulin A, 30%), and osteopontin (20%)) or small molecule ligands (such as bicarbonate, citrate, and phosphate) to form stable complexes at physiological pH. Approximately 35% of U(VI) binds to proteins, and 65% binds to carbonates. Transferrin (TF) is a key protein with an affinity for uranium. The complex formed by uranyl ions and proteins enters the cell, primarily controlled by protein coordination chemistry. In the early stages of injury, the kidneys are the key target organ for uranyl ion poisoning (DU). A characteristic feature of DU nephrotoxicity is that uranyl ions are mainly deposited in renal tubular epithelial cells, entering mitochondria and lysosomes, leading to cell death and renal tubular damage. In the later stages of injury, the skeleton becomes the primary target organ.

[0003] Studies have shown that high doses of uranium (DU) contamination can seriously harm human health, even leading to kidney failure, and has the potential to cause cancer, birth defects, and mutagenesis. However, an ideal uranium poisoning eliminator has not yet been found. Most DU chelating agents have certain side effects. For example, sodium bicarbonate (SB), recommended by the EU and the US Department of Energy, may cause acid-base imbalance, hypokalemia, and alkalosis; diethylenetriaminepentaacetic acid (DTPA) has side effects such as poor cell membrane penetration, short half-life in vivo, and hepatotoxicity and nephrotoxicity. Studies have shown that most uranium ions in the blood are excreted from the body within 3 days through the kidneys and intestines. Depleted uranium remaining in the body is a major cause of severe damage to target organs such as the kidneys.

[0004] Mesenchymal stem cells (MSCs) are pluripotent stem cells that differentiate into multiple lineages. They can adhere to and recognize damaged tissues via molecular adhesion, possessing important immunomodulatory capabilities and are widely used in tissue repair, transplant immunomodulation, and other fields. In particular, they have shown significant repair effects in cisplatin-, glycerol-, and ischemia-perfusion-induced acute kidney injury in rodents, enhancing functional and morphological recovery. However, the risk of immune rejection of MSCs remains unresolved. Studies have found that MSCs are mainly located in the renal tubules, only transiently accumulating in renal vessels, and play a role in maintaining renal tubular cell differentiation and renal tubular regeneration. Simultaneously, they can improve the survival rate of injured animals through secretory mechanisms. In addition to the repair and therapeutic effects of cytokines secreted by MSCs, MSC exosomes (EXs) have a significant repair effect on acute kidney injury, and their effect is stronger than that of MSCs alone.

[0005] EXs are monolayered vesicles with a diameter between 30 and 130 nanometers, secreted by cells into the extracellular space. EXs can transfer substances such as proteins, DNA, RNA, and microRNA from parent cells to other cells. They can act effectively on corresponding tissues over long distances, for extended periods, and at multiple targets. Due to the nanoscale size and lipid outer layer of exosomes, they can easily reach the site of injury through blood circulation after intravenous injection. There are three mechanisms of action between exosomes and somatic cells: (1) Exosomes connect with target cells through adhesion molecules and receptors, activating downstream signals. For example, there are transferrin receptors (TFR), osteopontin (OPN) receptors, integrins, and CD44 on the membrane of mesenchymal stem cells and EXs, and albumin receptor gp60. Therefore, it is speculated that transferrin, OPN, and albumin bind to uranyl ions in the blood and then bind to the corresponding receptors on the MSCEXs membrane, which can significantly reduce the chance of uranyl ion complexes binding to renal tubular cells and reduce renal uranium deposition. The number of TF receptors expressed by MSCs is 10772±6626 per cell, with many binding sites. Therefore, the number of receptors on the MSC-EXs membrane should also be relatively large, which should have a significant therapeutic effect on uranium poisoning, and can be squeezed, deformed and excreted through the urinary system. (2) In addition, the direct fusion of exosomes with target cells promotes the transfer of exosome signaling molecules; (3) Exosomes transfer their contents into cells through endocytosis. In this process, the lipid bilayer structure of EXs can prevent the degradation of contents, maintain the activity of enzymes and genetic material, and simulate the function of parent cells without causing rejection by the recipient cells, and can even replace the parent cells in performing functions. Multiple studies have found that exosomes play an important role in cell apoptosis, inflammatory response, immune response, angiogenesis and other processes. Exosomes not only have a restorative effect on kidney damage, but also on various organ damage, such as myocardial infarction and reperfusion injury, limb ischemia, liver injury, hypoxic lung injury and so on. Although, as mentioned above, MSC-EXs have significant repair and therapeutic effects on various types of kidney injury, their mechanisms are not yet fully understood. Summary of the Invention

[0006] This invention provides the application of stem cell exosomes in the preparation of drugs for treating uranium poisoning.

[0007] In one embodiment of the invention, it contains 10-50 μg / mL of stem cell exosomes.

[0008] The present invention also provides a drug for treating uranium poisoning, which contains an effective amount of stem cell exosomes.

[0009] In one embodiment of the present invention, the content of the stem cell exosomes is 10-50 μg / mL.

[0010] In one embodiment of the invention, it also contains an effective amount of mesenchymal stem cells and / or sodium bicarbonate.

[0011] In one embodiment of the invention, sodium bicarbonate is used in an aqueous solution with a concentration of 5% (110 μL).

[0012] In one embodiment of the present invention, the amount of MSC cells used is 1 × 10⁻⁶. 6 .

[0013] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0014] In this invention, stem cell exosomes can effectively reduce uranium deposition in the kidneys and enhance repair. They can protect human renal tubular epithelial HK-2 cells from uranium damage, reduce intracellular ROS and MDA levels, increase GSH levels, enhance SOD activity, reduce cell apoptosis, improve the pathological morphology of the kidneys and bone marrow, promote uranium excretion in urine, and reduce uranium retention in the body. Attached Figure Description

[0015] Figure 1 The image shows the characterization patterns of EXs, where A: NTA size map of EXs; B: Transmission electron microscope image of EXs; C: Western blot identification map of EXs.

[0016] Figure 2 The study aimed to investigate the protective effect of EXs against DU damage in HK-2 cells, including: A: the effect of different concentrations of DU on the proliferation rate of HK-2 cells; B: the effect of EXs on the proliferation rate of HK-2 cells; C: intracellular ROS fluorescence intensity; and D: the average intracellular ROS fluorescence intensity.

[0017] Figure 3 To assess the antioxidant effects of exogenous antioxidants (EXs), the study included: A: the effect of EXs on intracellular GSH levels; B: the effect of EXs on intracellular SOD activity; and C: the effect of EXs on intracellular MDA levels. *P<0.05, **P<0.01

[0018] Figure 4 The effects of excimerolenic acid (EXS) on apoptosis were analyzed. A: Effect of EXS on intracellular mitochondrial membrane potential; B: Effect of EXS on the content of Caspase 3 / 7 protein in cells; C: Effect of EXS on apoptosis as detected by flow cytometry. *P<0.05, **P<0.01

[0019] Figure 5The effect of EXs on improving biochemical indicators of depleted uranium injury; where A: survival of mice under different uranium doses; BC: renal function index, **P<0.01; DF: liver function index; GI: blood routine index;

[0020] Figure 6 EXs improve the morphology of DU damage;

[0021] Figure 7 The excretion-promoting effect of EXs on uranium excretion in mice; where A: urine volume on days 1-4; B: uranium content in urine on days 1-4; C: uranium content in femur on day 20; D: uranium content in kidney on day 20; E: uranium content in blood on day 20.

[0022] Figure 8 To detect the expression of apoptosis-related proteins and uranyl ion-binding proteins in mouse kidneys using Western blot and immunohistochemistry, the following images were used: A: Electrophoretic bar graph of apoptosis-related proteins in mouse kidneys detected by Western blot; B: Electrophoretic bar graph of uranyl ion-binding proteins in mouse kidneys detected by Western blot; C: Immunohistochemistry was used to observe the expression of uranyl ion-binding proteins in the kidneys. Detailed Implementation

[0023] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0024] Unless otherwise specified, all reagents used in this embodiment are of analytical grade, and the progress of all chemical reactions is detected by thin-layer chromatography.

[0025] Example 1: Materials and Methods

[0026] 1.1 Isolation, Extraction and Identification of EXs

[0027] Human umbilical cord mesenchymal stem cells (hUC-MSCs) were isolated and cultured by Formosa Stem Cell Biotechnology Co., Ltd. (Chongqing, China). The MSCs used in the experiments were all derived from passages 5–10, thoroughly characterized by flow cytometry.

[0028] hUC-MSCs were cultured to passage P6 using serum-free mesenchymal stem cell culture medium provided by Fumei Biotechnology. After collecting the culture medium, exosomes were extracted by differential centrifugation. The extracted exosomes were lysed with protein lysis buffer, sonicated for 10 min, incubated on ice for 30 min, precipitated with acetone overnight, centrifuged at 12000g for 20 min, and reconstituted with 5M urea. The protein concentration of exosomes was then determined using a Bradford protein assay kit (TAKARA, AIG1851A).

[0029] The particle size of exosomes was determined using a NanoSight nanoparticle size analyzer (Malvern Instruments, UK, NS300), with specific parameters set according to the user manual (NanoSight NS300 User Manual, MAN0541-01-EN-00, 2017). To measure the size distribution of exosomes, NanoSight Software NTA3.3.301 (Malvern, UK) from Malvern Panalytical Ltd. was used. Transmission electron microscopy (HITACHI, HT7700) was used to observe the morphology of exosomes. Western blotting detected the exosome marker proteins Alix, TSG101, and CD9.

[0030] 1.2 Cell Culture

[0031] Human proximal renal tubule (HK-2) cell line (ATCC CRL-2190) was purchased from the Chinese Academy of Sciences (Beijing, China). HK-2 cells were grown in RPMI 1640 (Gibco, USA) containing 10% (vol / vol) FBS (Hyclone, USA), 100 units / mL penicillin G, and 100 μg / mL streptomycin sulfate. All cells were cultured in a humidified incubator at 37°C with 5% (vol / vol) CO2 in air.

[0032] 1.3 EXs' effects on cell proliferation and antioxidant activity

[0033] To determine the optimal concentration of DU poisoning, HK-2 cell viability was measured using the CCK-8 assay kit after culturing for 24 h at final DU concentrations of 62.5 μM, 125 μM, 250 μM, 500 μM, and 1000 μM.

[0034] To evaluate the effect of EXs on the proliferation activity of DU-poisoned cells, HK-2 cells were cultured with 10 μg / mL and 50 μg / mL EXs for 24 h, respectively, and then contaminated with 250 μM and 500 μM DU solutions for 24 h, respectively. Cell proliferation activity in each group was detected using a CCK-8 assay. Serum ROS, SOD, GSH, and MDA levels in each group were measured according to the instructions of the following kits: Reactive Oxygen Species (ROS) Assay Kit (Beyotime, S0033S), Total Superoxide Dismutase (WST-8) Assay Kit (Beyotime, S0101S), Reduced Glutathione (GSH) Assay Kit (Nanjing Jiancheng Bioengineering Institute, A-006-2-1), and Lipid Peroxidation MDA Assay Kit (Beyotime, S0131S).

[0035] 1.4 Anti-apoptotic effects of EXs

[0036] The treatment of HK-2 cells in each group was the same as in the previous experiment. Flow cytometry was used to detect cell apoptosis after Annexin V-FITC and PI double staining (BD, 556547). The mitochondrial membrane potential and Caspase-3 activity of HK-2 cells were detected using the JC-1 assay kit (Beyotime, C2006) and the Caspase-3 / 7 activity assay kit (Beyotime, C1116).

[0037] 1.5 Animals and Treatment

[0038] In the experiment, AREVA-NC (France) provided urea nitrate hexahydrate (UO2(NO3)2-6H2O), which had a purity of 99.74%. 238 U, 0.255% 235 U and 0.0055% 234 The composition is U. The specific activity of DU is 2.4 × 10⁻⁶. 4 Bq / g. All reagents used were of trace element analytical grade, and the water was glass distilled water.

[0039] Male BALB / c mice (6 weeks old, 18±0.37g) were purchased from the Animal Center of Army Medical University (Chongqing, China) in accordance with the protocol approved by the Animal Ethics Committee of Army Medical University. The study design and all animal experimental procedures were conducted in accordance with the Laboratory Animal Guidelines and Usage (Institute of Laboratory Animal Resources), and the study design was specifically approved by the Institutional Animal Care and Use Committee (IACUC) of Army Medical University.

[0040] Sixty mice were randomly divided into six groups. Five groups received intraperitoneal injections of 0.5 mg DU / kg, 1.0 mg DU / kg, 2.0 mg DU / kg, 4.0 mg DU / kg, and 6.0 mg DU / kg, respectively. The normal control group received an intraperitoneal injection of the solvent. Mice survival rates were observed daily, and the optimal DU exposure dose for biological effect was determined to be 2 mg / kg.

[0041] Thirty mice were randomly divided into 5 groups. All four groups received an intraperitoneal injection of depleted uranium solution at 2.0 mg / kg (DU group). Immediately after depleted uranium exposure, 100 μg of MSC-EXs (EXs group) and 1×10⁻⁶ mg / kg of MSC-EXs were injected via the tail vein. 6MSC cells (MSC group) were used, and one group received an intraperitoneal injection of 110 μL of 5% SB (SB group). The control group received no treatment. Urine output and urinary DU content were measured and recorded daily. Blood routine tests and liver and kidney function tests were performed on day 5. Liver, kidney, spleen, and bone marrow of mice were collected for HE staining. On day 20, uranium content in blood, kidney, and bone was measured using ICP-MS 7800 (Agilent, USA) according to the literature (Hao Y, Ren J, Liu J, Yang Z, Liu C, Li R, Su Y. Immunological changes of chronicoral exposure to depleted uranium in mice. Toxicology. 2013 Jul 5; 309:81-90.).

[0042] 1.6 Western blot analysis

[0043] Total protein was extracted from exosomes and kidney tissue according to standard procedures. All protein samples were mixed with SDS-PAGE loading buffer (Beyotime), heated to 95°C for 10 minutes, separated on 8-12% SDS-PAGE gels (Beyotime), electrotransferred to polyvinylidene fluoride membranes (Beyotime), and blocked with QuickBlock. TM Blocking buffer (Beyotime) for 1 hour. The following antibodies were then used: anti-Alix (1:2000, 3A9, abcam), TSG101 (1:2000, 3A9, abcam), CD9 (1:2000, EPR2949, abcam), β-Actin (1:2000, Beyotime), Caspase-3 (1:2000, 5A1E, Cell Signaling Technology), Bax (1:2000, D2E11, Cell Signaling Technology), and Caspase-9 (1:2000, Cell Signaling Technology).

[0044] 1.7 Immunohistochemistry

[0045] Kidney tissue samples were fixed in formalin, embedded in paraffin, and sectioned. Slides were dewaxed and pretreated in a microwave oven with 1 mmol / L EDTA and heat-mediated antigen recovery solution. Further steps were performed in a hydration chamber at room temperature. Slides were pre-cultured in goat serum for 15 minutes. They were then incubated overnight with albumin (1:200, 66051-1-1g, Proteintech), osteopontin (1:200, 12952-1-AP, Proteintech), and transferrin (1:200, 17435-11AP, Proteintech). Slides were then washed in PBS and detected with HRP-conjugated anti-mouse IgG polymer (PV-6002, ZSGB Bio) or HRP-conjugated anti-rabbit IgG copolymer (PV-6002, ZSGB Bio). All slides were counterstained with hematoxylin.

[0046] 1.8 Statistical Analysis

[0047] All data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using SPSS 24.0, GraphPad Prism 8.0, and ImageJ software. One-way ANOVA was used, followed by Duncan's multiple range analysis. A p-value < 0.05 was considered statistically significant.

[0048] 2.1 Separated EXs features

[0049] The concentration of EXs protein determined by BCA was 0.943 mg / mL. Transmission electron microscopy revealed that the EXs were of uneven size and exhibited a typical disc-shaped bilayer membrane structure. Figure 1 B). Particle size results show that the main peak is around 134 nm, and the two side peaks are at 199 nm and 297 nm, respectively. Figure 1 A). Since exosomes can be taken up by cells via endocytosis or pinocytosis, the increase in exosome size may be due to aggregation and fusion. Western blot analysis showed expression of the signature proteins Alix, TSG101, and CD9 in huc-MSCs. Figure 1 C). Combining particle size analysis and appearance analysis results, it was confirmed that the sample contained the EXs component.

[0050] Example 2: Protective effect of MSC-EXs on renal tubular epithelial cells poisoned by DU

[0051] HK-2 cells were treated with DU at concentrations ranging from 62.5 μM to 1000 μM, and cell viability was observed. The median lethal dose (LD50) of DU for HK-2 cells was determined to be 500 μM using a CCK-8 assay. Figure 2Therefore, this study selected uranium doses of 250 μM and 500 μM. HK-2 cells were pretreated with EXs at concentrations of 10 μg / mL and 50 μg / mL, and 250 μM and 500 μM uranium solutions were added after 24 h. Compared with the control group, in the low-concentration (250 μM) DU group, EXs increased cell viability by 80% to 100%. In the 500 μM DU group, cell viability decreased to 50%, while the high-concentration EXs effectively increased cell viability from 50% to 90%, an increase of 40%. Figure 2 B).

[0052] DU exposure induces a cellular stress response, leading to a significant increase in ROS levels. Conversely, 10 μg / ml and 50 μg / ml EXs significantly reduced intracellular ROS levels. Figure 2 The results (C and 2D)(p < 0.05) indicate that EXs can reduce ROS generation caused by DU.

[0053] Example 3: Antioxidant effect of MSC-EXs on DU poisoned cells

[0054] Exosyltransferases (EXs) exhibited antioxidant activity against DU-induced oxidation in HK-2 cells. HK-2 cells were pretreated with exosomes for 24 h, followed by treatment with 250 μM DU. Compared to the DU group, the 50 μg / mL EXs group showed a 50% significant increase in the endogenous antioxidant GSH content. Figure 3 A), SOD activity was significantly increased ( Figure 3 B)(p<0.05), MDA content was significantly reduced to that of the control group ( Figure 3 C), and it was superior to the 10 μg / mL EXs group (p < 0.01).

[0055] Further examination of mitochondrial membrane potential (MMP), which is closely related to apoptosis, revealed that 250 μM DU decreased MMP, and significant loss of MMP may lead to activation of the mitochondrial apoptosis pathway. However, exogenous mitochondria (EXs) maintained normal mitochondrial membrane potential. Figure 4 A) The 50 μg / mL group was more effective than the 10 μg / mL group (p < 0.01).

[0056] Cell apoptosis and Caspase 3 / 7 levels were detected by flow cytometry. 250 μM DU significantly induced apoptosis by up to 10% and increased Caspase 3 / 7 expression levels, while EXs significantly reduced apoptosis by up to 5% and decreased Caspase 3 / 7 expression levels. Figure 4(B and 4C), the 50 μg / mL EXs group was superior to the 10 μg / mL group (p < 0.01). The results indicate that EXs can protect cells from DU by enhancing the antioxidant capacity of cells, maintaining normal mitochondrial membrane potential, and reducing cell apoptosis by decreasing the activity of Caspase3 / 7.

[0057] Example 4: The therapeutic effect of EXs on DU-poisoned mice

[0058] To determine the optimal DU exposure dose, male BALB / c mice were intraperitoneally injected with different doses of DU, and their survival rates were observed. The results showed that the 6 mg / kg DU group and the 4 mg / kg DU group began to die on day 5, with survival rates of only 30% and 40% on day 9, respectively. Mice in the 0.5 mg / kg and 1 mg / kg groups showed no mortality; therefore, the optimal dose of depleted uranium exposure was determined to be 2 mg / kg. Figure 5 A)

[0059] The results showed that serum urea levels in mice exposed to 2 mg / kg DU increased to 86 mmol / L, and the levels in the Exs, MSC, and SB groups ( Figure 5 The levels of B and 5C decreased to 40, 43, and 19 mmol / L, respectively; creatinine levels increased to 220 μmmol / L in the DU group, while they decreased to 90 μmol / L, 60 μmol / L, and 50 μmol / L in the Exs, MSC, and SB groups, respectively, effectively alleviating renal function damage caused by DU. The levels of ALT and ALP, reflecting liver function indices, did not differ significantly among the groups. Figure 5 D and 5F), AST was slightly elevated in the DU group ( Figure 5 E), estimated to be related to the relatively small dose of uranium used, which has not yet damaged liver function. Hematological changes in WBC, RBC, and PLT were not significantly different between the groups and the control group. Figure 5 G, 5H and 5I).

[0060] HE staining of kidneys, bone marrow, liver, and spleen from mice treated with DU, Exs, MSC, and SB is shown. Figure 7 In summary, in the DU group, renal congestion, proximal tubular necrosis, protein-rich tubules, and glomerular enlargement were observed. After treatment with EXs, MSCs, and SB, congestion significantly improved, glomerular morphology returned to normal, and renal tubular necrosis decreased. Uranium poisoning led to angiogenesis in the bone marrow, with dispersed and loosely packed cells, and a reduced number of nucleated cells. EXs treatment significantly reduced angiogenesis, resulted in more compact cell packing, and increased nucleated cells. Compared with the control group, 2 mg / kg of depleted uranium had no significant effect on the morphology of the liver and spleen in mice. Figure 6 ), and also not in the EXs, MSC and SB groups.

[0061] Example 5: EXs promotes the elimination of DU in mice

[0062] Total urine output in mice was measured, and it was found that from day 2 of DU exposure, both the DU group and the treatment group had higher urine output than the control group. After treatment with EXs and SB, urine output decreased significantly on day 3. Figure 7 A). On days 1-3, Exs, MSC, and SB significantly increased uranium excretion in the urine of DU-poisoned mice (P<0.01). Figure 7 B), reducing uranium retention in blood, kidneys, and bones. On day 20, uranium levels in bones, kidneys, and blood were significantly higher in the DU group than in the control group, but uranium levels in all organs were significantly lower in the EXs, MSC, and SB groups, with similar levels in the EXs and SB groups. Figure 7 (C, 7D, and 7E). The results showed that EXs effectively promoted the excretion of uranium through urine and reduced the retention of uranium in the blood, kidneys, and bones.

[0063] Example 6: Western blotting and immunohistochemistry verification of the expression of EXs in kidney apoptosis-related proteins and uranyl ion-binding proteins in DU-poisoned mice.

[0064] Western blotting verified the effect of exogenous methylphenidate (EXs) on the expression of kidney apoptosis-related proteins in DU-poisoned mice. The expression levels of Caspase3, Caspase9, and Bax were significantly increased after DU poisoning (p<0.01). However, treatment with MSCs and EXs could reverse this phenomenon. In particular, the inhibitory effect of EXs treatment was more pronounced compared to MSCs and SBs. Figure 8 A).

[0065] Western blotting verified the effects of exogenous esters (EXs) on the expression of proteins related to uranyl ion binding in the kidneys of mice poisoned with DU. It was found that the expression levels of transferrin, ostipontin, and albumin were significantly increased after DU poisoning (p<0.01). However, treatment with MSCs and EXs could reverse this phenomenon. In particular, the inhibitory effect of EXs treatment was more pronounced compared to the MSC and SB groups. Figure 8 B). The expression of adhesion molecule CADM is similar to that of transferrin. Figure 8 B).

[0066] Example 7 Immunohistochemical Validation

[0067] Immunohistochemical analysis was conducted to verify the effect of EXs on the expression of uranyl ion-binding proteins in the kidneys of DU-poisoned mice. Compared with the control group, the expression levels of Transferrin, Osteopontin, and albumin were significantly increased in the DU group, presumably due to increased uranyl ion levels and the resulting increase in the number of binding proteins entering the kidneys. However, treatment with MSCs, EXs, and SB reduced the expression of related proteins in the renal tubular cell membrane, cytoplasm, or surrounding interstitium. Compared with the MSC and SB groups, the inhibitory effect of EXs treatment was more pronounced. Figure 8 C).

[0068] Transferrin enters the renal tubular cell after binding to its receptor TFR on the cell membrane. Transferrin stains dark brown in renal tubular cells; the brown substance is distributed in the cell membrane and cytoplasm, while immunostaining in the surrounding interstitium is negative.

[0069] Osteopontin is mainly expressed in renal tubules, interstitium, and juxtaglomerular apparatus. It binds to OPN receptors, integrins, and CD44 on the renal tubular cell membrane, and can also bind to integrin αVβ1 in the surrounding interstitium, such as fibronectin or porphyrin. Phosphorylated OPN binds to cell surface integrin receptors, while dephosphorylated OPN binds to CD44 receptors, thus evoking different effects. OPN stains dark brown in renal tubular cells, with the brown substance distributed in the cell membrane and cytoplasm; it is positive for immunostaining in the surrounding interstitium.

[0070] Renal tubular epithelial cells are extensions of glomerular vascular endothelial cells and also express the albumin receptor gp60. Therefore, the overall structure of albumin shows four potential metal-binding sites. After binding with uranyl ions, albumin enters renal tubular epithelial cells via blood circulation, through a specific pathway of binding to albumin receptors, or through non-specific pathways such as endocytosis.

[0071] The reduced expression of renal tubular cell-related proteins in all treatment groups is presumably due to the expression of TF receptors and OPN receptor integrins or CD44 on the membranes of MSCs and MSC-EXs. These receptors competitively bind to uranyl ion-TF and uranyl ion-OPN complexes, reducing the binding of uranium to protein receptors on the renal tubular cell membrane and thus reducing uranium deposition. Some EXs may be excreted through the urinary system via compression or deformation, reducing the body's uranium content. MSCs do not express albumin receptors, but the albumin-uranyl ion complex can be degraded into small molecular fragments within renal tubular cells and reused. In the SB group, the increased levels of bicarbonate ions in the blood led to an increase in the levels of more stable uranyl tricarbonate in both blood and urine, generating stable uranyl ion complexes, which reduced the interaction between uranyl ions and renal tubular cells.

[0072] This invention investigates the therapeutic effect of MSC-EXS on uranium poisoning in mice, finding that it significantly promotes uranium excretion in urine, reduces uranium content in the kidneys and bones, and lowers serum urea and creatinine levels, indicating that EXS can alleviate kidney damage and improve the pathological morphology of the kidneys and bone marrow. The main mechanism of this protective effect is that the protein receptors on MSC-EXS competitively bind to receptors on renal tubular cells, significantly reducing the deposition of uranyl ions bound to transferrin, osteopontin, and albumin in the kidneys. Some MSC-EXS are expelled through extrusion and deformation. Simultaneously, reduced expression of adhesion molecules helps reduce the entry of protein-uranyl ion complexes into tissue cells, thereby reducing uranium retention in the blood, kidneys, and bones. Furthermore, MSC-EXS reduces ROS levels, increases SOD and GSH expression, and reduces membrane lipid peroxidation. An increased bcl-2 / Bax ratio inhibits apoptosis. These results suggest that MSC-EXS may be a novel treatment for DU poisoning.

[0073] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of stem cell exosomes in the preparation of drugs for treating uranium poisoning, wherein the stem cell exosomes are derived from human umbilical cord mesenchymal stem cells; The method for preparing stem cell exosomes is as follows: mesenchymal stem cells are cultured to passage P6 in serum-free culture medium, and the culture medium is collected and extracted by differential centrifugation.

2. The application as described in claim 1, characterized in that, It contains 10-50 μg / mL of stem cell exosomes.

3. The application of an effective amount of stem cell exosomes, an effective amount of human umbilical cord mesenchymal stem cells, and sodium bicarbonate in the preparation of a drug for treating uranium poisoning, wherein the stem cell exosomes are derived from human umbilical cord mesenchymal stem cells, the content of the stem cell exosomes is 10-50 μg / mL, the amount of sodium bicarbonate is 110 μL of a 5% aqueous solution, and the amount of human umbilical cord mesenchymal stem cells is 1×10⁻⁶. 6 indivual; The method for preparing stem cell exosomes is as follows: mesenchymal stem cells are cultured to passage P6 in serum-free culture medium, and the culture medium is collected and extracted by differential centrifugation.

Images