DNA tetrahedral framework nucleic acid and fg-4592 complex, and methods of making and uses thereof

Abstract

The application provides a DNA tetrahedral framework nucleic acid and FG-4592 complex, a preparation method and use thereof, and belongs to the field of biological medicines.The DNA tetrahedral framework nucleic acid and FG-4592 complex is a complex formed by mixing a DNA tetrahedral framework nucleic acid and FG-4592.The DNA tetrahedral framework nucleic acid and FG-4592 have the synergistic effect of reducing apoptosis of renal cortical proximal tubular epithelial cells, reducing kidney tissue necrosis, restoring kidney function, and preventing and treating renal fibrosis and preventing the development of acute kidney injury into chronic kidney disease, thereby providing a new option for clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to DNA tetrahedral framework nucleic acids and FG-4592 complexes, their preparation methods, and uses. Background Technology

[0002] Cisplatin-based chemotherapy has long been widely used in the treatment of head and neck cancer, ovarian cancer, and testicular cancer. Since its invention by Professor Rosenberg in 1978, this drug has saved millions of lives. It is hailed as "cancer penicillin" due to its unparalleled therapeutic effects compared to other treatments: it can achieve an overall cure rate of 90%, and even a near 100% cure rate in early-stage testicular cancer. However, there is currently no effective treatment for cisplatin-induced acute kidney injury (AKI). Up to 30% of patients immediately lose kidney function and may further develop life-threatening electrolyte imbalances. The mechanism of cisplatin's damage to the kidneys is similar to its anti-tumor effects caused by DNA damage. Studies have shown that the concentration of cisplatin DNA adducts is highest in the kidneys. After hydration, cisplatin forms an amine-water complex. This complex replaces the N7 atoms of guanine and adenine in the DNA double helix, forming a DNA adduct that blocks DNA and RNA polymerases, thus blocking DNA transcription. Studies on cisplatin metabolism show that its main metabolic pathways are glomerular filtration and renal tubular secretion. Cisplatin-induced glomerular and tubular damage can lead to acute kidney injury and chronic kidney disease (CKD). The impact of this clinical complication on patients' anti-tumor treatment and clinical prognosis cannot be overstated. Therefore, developing a chemoprotective agent to mitigate cisplatin-induced nephrotoxicity is a crucial issue in oncology.

[0003] FG-4592 is an HIF-1α inducer that mimics the hypoxic conditions of high-altitude regions, thereby increasing the production of endogenous erythropoietin (EPO), suggesting its potential application in tissue regeneration and wound healing. It is frequently administered orally for the treatment of renal anemia. However, recent studies have shown that it can also prevent kidney injury induced by chemotherapy or ischemia / reperfusion (I / R) treatments in animal models. In Chinese patients undergoing dialysis, oral FG-4592 was superior to beta-epofosine for treating anemia. These findings suggest that FG-4592 is a promising therapeutic agent for cisplatin-induced renal dysfunction. It was launched in China in 2018 due to its stability against HIF-1α and convenient oral administration. FG-4592 is a prolyl hydroxylase (PHD) inhibitor, an enzyme responsible for the hydroxylation of HIF-1α. This hydroxylation process leads to the ubiquitination and subsequent degradation of HIF-1α. Activation of HIF-1α can promote erythropoiesis and oxygen transport. The positive role of HIF-1α inducers in tissue regeneration has been increasingly supported by research. However, its poor water solubility and the potential cardiovascular risks associated with platelet activation introduce uncertainty for patients planning to use this small molecule drug in their treatment prescriptions. Therefore, developing a highly efficient drug delivery system with low toxicity and high renal aggregation holds promise for expanding the application of FG-4592. Summary of the Invention

[0004] The purpose of this invention is to provide a DNA tetrahedral framework nucleic acid and an FG-4592 complex, as well as their preparation methods and uses.

[0005] This invention provides a DNA tetrahedral framework nucleic acid and FG-4592 complex, which is a complex formed by mixing DNA tetrahedral framework nucleic acid and FG-4592.

[0006] Furthermore, the content of FG-4592 in the complex was determined to be 50 wt% using a UV spectrophotometer.

[0007] Furthermore, when the DNA tetrahedral framework nucleic acid and FG-4592 are mixed, the molar ratio of the DNA tetrahedral framework nucleic acid to FG-4592 is 1:80 to 200.

[0008] Furthermore, the molar ratio of the DNA tetrahedral framework nucleic acid to FG-4592 is 1:80.

[0009] Furthermore, the DNA tetrahedral framework nucleic acid is formed by base complementary pairing of four single-stranded DNA molecules with sequences as shown in SEQ ID NO. 1 to 4, respectively.

[0010] Furthermore, the method for synthesizing the DNA tetrahedral framework nucleic acid includes the following steps: adding four single-stranded DNAs to TM buffer, maintaining at 80-100℃ for 10 min, and maintaining at 2-4℃ for more than 20 min to obtain the product;

[0011] Preferably, four single-stranded DNA strands are added to TM buffer and maintained at 90°C for 10 minutes, then at 4°C for at least 20 minutes to obtain the final product.

[0012] The present invention also provides a method for preparing the aforementioned DNA tetrahedral framework nucleic acid and FG-4592 complex, which includes the following steps:

[0013] The DNA tetrahedral framework nucleic acid and FG-4592 solution were mixed, incubated, and then ultrafiltered to obtain the final product.

[0014] Preferably, the solvent of the FG-4592 solution is one or more of PBS and DMSO;

[0015] And / or, the incubation temperature is 20–30°C, and the incubation time is 5–10 hours.

[0016] The present invention also provides the use of the aforementioned DNA tetrahedral framework nucleic acid and FG-4592 complex in the preparation of medicaments for the prevention and / or treatment of acute kidney injury;

[0017] Preferably, the acute kidney injury is cisplatin-induced acute kidney injury.

[0018] Furthermore, the drug is a drug for preventing and alleviating renal tissue necrosis and / or restoring renal function;

[0019] And / or, the drug is a drug for the prevention and treatment of renal fibrosis;

[0020] And / or, the drug is a drug that reduces apoptosis of renal proximal tubular epithelial cells;

[0021] Preferably,

[0022] The drug in question is one that upregulates Hif-1α expression;

[0023] And / or, the drug is a drug that antagonizes the apoptosis signaling pathway.

[0024] Furthermore, the drug is a cisplatin-based chemotherapy enhancer and protectant;

[0025] Preferably, the drug is a chemotherapy protective drug that prevents cisplatin-induced acute kidney injury;

[0026] More preferably, the drug is a drug for preventing and treating cisplatin nephrotoxicity.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention provides a complex of DNA tetrahedral framework nucleic acid and FG-4592. The DNA tetrahedral framework nucleic acid and FG-4592 have a synergistic effect in reducing apoptosis of proximal tubular epithelial cells in the renal cortex, reducing renal tissue necrosis, restoring renal function, preventing renal fibrosis, and preventing the progression of acute kidney injury to chronic kidney disease, providing a new option for clinical practice.

[0029] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0030] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0031] Figure 1 For the preparation, characterization, and cell viability analysis of TFG: A shows the preparation route of TFG synthesis; B shows the AFM observation and semi-quantitative results of tFNAs, scale bar at 500 nm; C shows the TEM observation results of tFNAs, scale bar at 20 nm; D shows the PAGE results of tFNAs; E shows the particle size of tFNAs; F shows the PAGE results of TFG; G shows the UV spectra of tFNAs and TFG prepared with different concentrations of FG-4592; H shows the particle size of TFG; I shows the Zeta potential results of tFNAs and TFG; J shows the induction effect of FG-4592 (FG) alone on HIF-1α in HK-2 cells; K shows the semi-quantitative results of Figure J; L shows the cell viability analysis results of FG-4592 and TFG.

[0032] Figure 2 The results of cellular uptake and biodistribution analysis of tFNAs as a drug delivery system are as follows: A shows the confocal microscopy observation results of cellular uptake of Cy5-labeled tFNAs and S1 (ssDNA); B and C show the flow cytometry analysis and statistical analysis of mean fluorescence intensity of tFNAs and S1, respectively; D shows the frozen section results of mouse kidney ssDNA and tFNAs; E shows the schematic diagram of the in vivo experiment; F shows the in vivo images of the separated organs from mice 24 hours later and the statistical analysis of the signal intensity of the liver and kidney.

[0033] Figure 3Cellular uptake analysis results for tFNAs as a drug delivery system: A shows the cellular uptake results of Cy5-labeled tFNAs and TFG; B shows the cell entry efficiency of TFG and tFNAs by flow cytometry; C shows the qualitative analysis of how TFG and tFNAs are uptaken in each channel.

[0034] Figure 4 The following are in vitro analysis results of the anti-apoptotic effect of TFG in a cisplatin-induced cell model: A shows cisplatin-induced nephrotoxicity and its mechanism; B shows cell viability analysis of cisplatin-induced HK-2 cells with and without TFG treatment; C shows the quantitative analysis of the anti-apoptotic effect of TFG by annexin V (FITC) / PI staining; D shows the immunofluorescence analysis of HIF-1α and cleaved caspase 3 expression in cisplatin-pretreated HK-2 cells; E shows the analysis of Bax in the model cells; F shows the Western blot analysis of Bax expression and semi-quantitative analysis of three proteins: left: Bax, middle: HIF-1α; right: cleaved cysteine ​​protease 3.

[0035] Figure 5 Histological analysis results of cisplatin-induced AkI in animals after TFG treatment: A shows the method of inducing cisplatin AKI and treating with TFG to combat damage in C57 mice; B shows Mason staining and semi-quantitative analysis of kidney tissue; C shows PAS staining and semi-quantitative analysis of kidney tissue; D shows HE staining and semi-quantitative analysis of kidney tissue.

[0036] Figure 6 To explore the mechanism of TFG's anti-apoptotic effect and its induction of HIF-1α: A is the tunnel staining of kidney tissue; B is the Western blotting and semi-quantitative analysis of Hif-1α, Bcl-2, Bax and Kim-1 expression; C is the immunohistochemical qualitative analysis of Hif-1α, Bax and Cleaved Caspase expression in mouse kidney tissue; D is the statistical analysis of each factor. Detailed Implementation

[0037] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0038] The structure of FG-4592 is as follows: FG-4592 was purchased from Wuhan Chemical Stein Biotechnology Co., Ltd.

[0039] Example 1: Synthesis and characterization of the DNA tetrahedral framework nucleic acid and FG-4592 complex (TFG) of the present invention.

[0040] like Figure 1 As shown in the diagram, the synthesis of TFG involves two steps:

[0041] (1) tFNA synthesis: Four sequence-specific single-stranded DNAs (ssDNA) (Sangon, Shanghai, China) were added in equimolar amounts to a TM buffer composed of Tris-HCl and MgCl2 (10 mM Tris-HCl and 50 mM MgCl2 dissolved in water, and the pH was adjusted to 8.0 using hydrochloric acid). A one-pot annealing program was then performed in a PCR instrument (heated to 95°C and held for 10 minutes, then cooled to 4°C for 20 minutes), resulting in self-assembly of tFNAs. The specific sequences of the four sequence-specific single-stranded DNAs are shown in Table 1 below.

[0042] Table 1. Specific sequences of four sequence-specific single-stranded DNAs

[0043]

[0044] (2) Combination of FG-4592 and tFNAs: FG-4592 was prepared using cell culture grade DMSO (MP Biomedicals) as a stock solution. First, FG-4592 was dissolved in DMSO. Then, FG-4592 and tFNAs were mixed at molar ratios of 20:1, 40:1, 80:1, 160:1, and 200:1, and incubated at 4°C for 8 hours. Residual ssDNA and FG-4592 were then removed by ultrafiltration (30 kDa molecular weight membrane, Millipore, USA) to obtain TFG with different FG-4592 contents.

[0045] (3) Characterization: Polyacrylamide gel electrophoresis (PAGE) was used to verify the successful synthesis of tFNAs and TFG. Simultaneously, to verify the mechanism of action of tFNAs and FG-4592, FG-4592 was serially diluted with distilled water (ddH2O) (FG-4592 concentrations of 20 μM, 40 μM, 80 μM, and 160 μM). Then, FG-4592 was incubated with 1 μM tFNAs according to the method described in (2) above, followed by ultrafiltration to obtain TFG with different FG-4592 contents. The synthesis was verified using a UV spectrophotometer with an OD of 260 nm. The morphological characteristics of tFNAs were measured using TEM (Hitachi High Technology, Japan) and AFM (SPM-9600, Shimadzu Corporation, Japan). The particle distribution and charge of the complex and tFNAs were characterized using a ZETA potentiometer and particle size analyzer. The use of a UV spectrophotometer verified that FG-4592 and tFNAs are intercalated.

[0046] Figure 1 B represents the AFM characterization results of tFNAs. Figure 1 C represents the TEM characterization results of tFNAs. Figure 1 D represents the PAGE detection result of tFNAs. Figure 1B, 1C, and 1D indicate that the four DNA single strands can combine to form a tetrahedral structure, meaning that tFNAs were successfully synthesized.

[0047] Figure 1 F represents the PAGE test result of TFG, which indicates the successful synthesis of TFG.

[0048] Figure 1 G UV spectroscopy revealed a hypochromic reaction between FG-4992 and tFNAs. This result indicates that tFNAs bind to FG-4592 via intercalation and can form a TFG complex.

[0049] Figure 1 E represents the particle size of tFNAs. Figure 1 H represents the granularity of TFG. Figure 1 I represents the zeta potential results for tFNAs and TFG. Figure 1 E, 1H, and 1I indicate that the FG-4592 small molecule can bind to the grooves of tFNAs to form a small molecule-DNA complex.

[0050] Taking into account factors such as drug loading and encapsulation efficiency, the optimal molar ratio of tFNAs to FG-4592 was determined to be 1:80.

[0051] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0052] Cell culture: Human kidney-2 (HK-2) cell line was purchased from BNCC in Henan, China, and cultured in high-glucose DMEM medium supplemented with 10% (v / w) fetal bovine serum (ZETA, USA) and 5% (v / v) penicillin / streptomycin solution at 37°C and 5% CO2. The medium should be changed every 2 days. To maintain optimal growth, cells need to be cultured in fresh medium every two days and passaged at approximately 80% density with a passage ratio of 1:2.

[0053] Statistical Analysis: The researchers repeated the experiment three times to ensure reproducibility. Statistical significance was analyzed using GraphPad Prism 8 software, with *P<0.05, **P<0.01, ***P<0.001, or P>0.05 indicating no significance (ns). Appropriate statistical tests were performed on each dataset, and outliers or missing data were removed. The statistical analysis in this invention aims to ensure the validity and reliability of the results.

[0054] Experimental Example 1: Cellular Uptake

[0055] 1. Experimental Methods

[0056] (1) Observation of cellular uptake of tFNAs and TFG using confocal microscopy:

[0057] Cy5 was used to link S1, and then Cy5-tFNAs and Cy5-TFG (the molar ratio of FG-4592 to tFNAs was 80:1) were prepared according to the method described in Example 1. They were cultured in confocal culture dishes at a concentration of 1×10⁻⁶. 5 HK-2 cells were cultured at a density of 10 cells / mL. On the second day, the culture medium was replaced with serum-free medium containing 250 nmol / L Cy5-TFG, Cy5-tFNAs, or Cy5-S1 (1 mL per dish) for further incubation. After 24 hours of incubation, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 25 minutes. Finally, all cells were stained with DAPI and FITC (fiber oxidase inhibitor). Each staining step lasted 20–30 minutes, with three washes with PBS between each step. Finally, all cells were sealed with 10% glycerol before detecting fluorescence signals using a confocal microscope (Olympus Fluoview FV 1000; Tokyo, Japan). A control group was used.

[0058] (2) Flow cytometry was used to detect cellular uptake of tFNAs:

[0059] HK-2 cells were cultured in 12-well plates according to the method described in (1). After incubation with Cy5-TFG, Cy5-tFNAs, or Cy5-S1 for 48 hours, the culture medium was discarded, and the samples were washed twice with PBS. To harvest adherent cells, they were treated with 0.25% trypsin-EDTA (Gibco), separated, and then washed twice with PBS at 2000 rpm for 5 minutes each time before being transferred to 1.5 ml centrifuge tubes. Finally, the cells were resuspended in 200 μL of PBS and analyzed using a flow cytometer (CytolFLEX, Beckman Coulter, Brea, California, USA) at an excitation wavelength of approximately 650 nm using single-channel detection. Control was the cell control group.

[0060] 2. Experimental Results

[0061] Figure 2 A represents the confocal microscopy observation results of cellular uptake by Cy5-labeled tFNAs. Figure 2 B and 2C represent flow cytometry analysis of cellular uptake of tFNAs and statistical analysis of mean fluorescence intensity. Figure 2 The results of A-2C indicate that tFNAs can be well taken up by cells and thus enter the cell.

[0062] Figure 3Cellular uptake results for TFG, including cellular penetration analysis and flow cytometry quantification, showed that TFG exhibited better uptake efficiency than tFNAs. This demonstrates the successful synthesis of TFG and the synergistic effect of the small molecule with the DNA framework nucleic acids. Figure 3 A and 3C show that TFG can be better taken up by HK-2 cells; Figure 3 B showed that the uptake efficiency of TFG was significantly higher than that of tFNAs.

[0063] Experimental Example 2: In vivo distribution

[0064] 1. Experimental Methods

[0065] After one week of adaptive breeding, C57 mice were intravenously injected with 2 μM Cy5-S1 (… Figure 2 ssDNAs) and Cy5-tFNAs Figure 2 The tFNAs were used to ensure a Cy5 dose of 2 μM per mouse. Twenty-four hours after intravenous injection, mice were euthanized, and their hearts, livers, spleens, lungs, and kidneys were harvested for in vivo imaging using a Cy5 detection instrument. In strict darkness, the collected kidney tissue was stored in liquid nitrogen, sectioned using a cryostat (CM1950; Leica, Germany), and washed with PBS. DAPI was then added to the samples after 5 minutes of incubation. Samples were washed three times with PBST every 5 minutes and sealed before observation under a confocal microscope.

[0066] 2. Experimental Results

[0067] Figure 2 D is a frozen section of mouse kidney ssDNA and tFNAs. Figure 2 F represents in vivo images of isolated organs from mice 24 hours later, along with statistical analysis of signal intensity in the liver and kidneys. Figure 2 As shown in D and 2F, the fluorescence intensity in the kidneys was significantly higher than that in the liver after 24 hours. This indicates that tFNAs are targeted and distributed in the kidneys. The structure of TFNAs after complexation with FG-4592 did not change significantly, and TFG also exhibits in vivo targeted distribution capabilities.

[0068] Experimental Example 3: Cell Viability

[0069] 1. Experimental Methods

[0070] (1) Cell viability assay

[0071] The CCK8 assay kit (from MedChemExpress, Junction, Monmouth, NJ, USA) was used to evaluate the cytotoxicity of FG-4592 and TFG and to test their protective effect against cisplatin-treated cells. TFG was prepared according to the method described in Example 1 (molar ratio of FG-4592 to tFNAs was 80:1). HK-2 cells were cultured at a rate of 1 × 10⁻⁶ cells / year. 5 After incubating the cells at high density in 12-well plates for 24 hours, add medium containing FG-4592 (25 μM) or TFG (25 μM) and incubate for another 24 hours. Then, plant the cells at a density of 9 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. The next day, the cells were incubated for 0.5 hours with CCK8 solution diluted in serum-free medium instead of the medium. The samples were then tested at 450 nm using an enzyme-labeled immunosorbent assay (Variskan LUX, Thermo Fisher Scientific) to obtain cell viability analysis.

[0072] The cisplatin-treated cell culture protocol is as follows: After incubating cells with FG-4592 and TFG according to the above protocol, remove the culture medium, wash three times with PBS, add 5 μg / mL cisplatin (Aladdin, Shanghai), and culture for another 24 hours. Then, plant the cells at 9 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. The next day, the cells were incubated for 0.5 hours with CCK8 solution diluted in serum-free medium instead of the medium. The samples were then tested at 450 nm using an enzyme-labeled immunosorbent assay (Variskan LUX, Thermo Fisher Scientific) to obtain cell viability analysis.

[0073] (2) Quantitative analysis of apoptosis using flow cytometry

[0074] To perform Annexin V (FITC) / PI dual staining analysis, this invention uses the staining kit KeyGenBiotech Co., Ltd. HK-2 cells were stained at 1.2 × 10⁻⁶. 5 Cells were seeded in 12-well plates and treated for 24 hours with serum-free medium containing 25 μM FG-4592 and 25 μM TFG, respectively. The next day, the medium was removed, cells were washed three times with PBS, and then 5 μg / mL cisplatin (Aladdin, Shanghai) was added. After another 24 hours of incubation, serum-free medium was added to each well. Cells were collected, washed with PBS, resuspended in 400 μL of 1× binding buffer, and stained with both dyes in complete darkness according to the product description. Flow cytometry was used for analysis.

[0075] (3) Western blot analysis of Bax expression

[0076] HK-2 cells were fed at 2.5 × 10⁻⁶ 5 The cells were seeded at a density of 1 / 2 well in 6-well plates and incubated for 24 hours. Afterward, they were treated with serum-free medium containing 25 μM MFG-4592 and 25 μM TFG for 24 hours each. The next day, the medium was removed, and the cells were washed three times with PBS. Then, 5 μg / mL cisplatin (Aladdin, Shanghai) was added, and the cells were incubated for another 24 hours. The samples were then collected and washed twice with PBS. A total protein extraction kit was used for cell protein extraction. The samples were then incubated at a density of 1 × 10⁻⁶. 4 After centrifugation at rpm for 15 minutes, the sample was mixed with 5× loading buffer, and the supernatant was collected. The protein sample was then heated at 100°C for 15 minutes and subjected to SDS-PAGE gel electrophoresis. The gel was then transferred to a PVDF membrane and sealed with 5% skim milk. After rinsing with TBSTzai, the membrane was incubated with a 1:1000 diluted anti-Bax antibody at 4°C for 10 hours. The membrane was then incubated with a second antibody diluted 1:5000 at room temperature for 1 hour. The samples were imaged using an exposure machine (Bio-Rad, California, USA), and semi-quantitative analysis was performed using ImageJ software.

[0077] (4) Immunofluorescence detection of protein expression

[0078] With 1×10 5 Cells were seeded at a density of [number] cells / dish, and the cell treatment protocol was the same as described above for flow cytometry analysis of apoptosis. The cells were then incubated with the antibody at a concentration of 1:200 at 4°C for 10 hours. The next day, the cells were washed and incubated with a suitable secondary antibody excited at 594 nm and DAPI staining. The next day, after washing and fixation, the cells were blocked with goat serum and rinsed with PBS. The samples were observed under a confocal microscope (Olympus Fluoview FV 1000, Tokyo, Japan).

[0079] 2. Experimental Results

[0080] Figure 1 J and 1K are validation results of the Hif-1α stabilizing effect of FG-4592 on HK-2 cells, derived from... Figure 1 J and 1K indicate that after incubation at 25 μM for 24 h, the abundance of Hif-1α protein in HK-2 cells was significantly upregulated, verifying the pharmacodynamic effects of FG-4592.

[0081] Figure 1 L represents the effect of FG-4592 and TFG on HK-2 cell viability, derived from... Figure 1L indicates that TFG has almost no toxicity to HK-2 cells, demonstrating the biosafety of this nanoparticle.

[0082] Figure 4 B represents the cell viability analysis of cisplatin-induced HK-2 cells with or without TFG treatment, by... Figure 4 B shows that HK-2 cells treated with TFG survived better in the cisplatin-induced injury environment than those without TFG treatment, indicating that TFG can reduce the occurrence of damage at the cellular level by preserving cells.

[0083] Figure 4 C represents the quantitative analysis results of annexin V (FITC) / PI staining on the anti-apoptotic effect of TFG, derived from... Figure 4 C indicates that TFG can significantly reduce both early and late apoptosis in cells.

[0084] Figure 4 D represents the immunofluorescence analysis results of HIF-1α and cleaved Caspase 3 expression in cisplatin-pretreated HK-2 cells, derived from... Figure 4 D indicates that TFG can significantly reduce the expression of the apoptosis marker Cleaved-Caspase 3.

[0085] Figure 4 E represents the results of Western blot analysis of Bax expression, from... Figure 4 E indicates that TFG can significantly downregulate Bax protein in renal cells, which is upregulated by cisplatin.

[0086] Figure 4 F represents the semi-quantitative results for three proteins: the left graph shows Bax, the middle graph shows HIF-1α, and the right graph shows cleaved cysteine ​​protease 3. Figure 4 F indicates that TFG inhibits cisplatin-induced kidney damage and related cell apoptosis by upregulating Hif-1α and then downregulating apoptosis-related pathways.

[0087] Experiment 4, Animal Experiment

[0088] 1. Experimental Methods

[0089] (1) Animal reproduction and establishment of cisplatin-induced AKI model

[0090] All animal experiments were conducted in accordance with the Ethics Committee of the West China School of Stomatology, Sichuan University. Twenty-four C57 mice (12 males and 12 females), aged 6–8 weeks and weighing 18–22 g, were housed under standard animal room conditions. To establish cisplatin (20 mg / kg)-induced AKI, mice were divided into four experimental groups: (1) control group, (2) cisplatin (10 mg / kg), (3) cisplatin (10 mg / kg) + FG-4592 (25 μM), and (4) cisplatin (20 mg / kg) + TFG (25 μM). In the control group, mice were intravenously injected with 0.9% sodium chloride for two days, followed by intraperitoneal injection of the same solution for the last two days. In the second group, mice were intravenously injected with 0.9% NaCl and intraperitoneally injected with a 10 mg / kg cisplatin dilution for two days. The proportion of DMF in the dilution should not exceed 5%, as this may affect the mice's tolerance. In the FG-4592 and TFG groups, all solutions were administered intravenously for two days at a dose of 12.5 mg / kg. Mice were then intraperitoneally treated with cisplatin for the next two days to induce a mouse model.

[0091] (2) Western blot analysis of kidney tissue

[0092] The prepared cryolysis buffer (from Kaigen Biotech Co., Ltd., China) was placed in a grinding tube, and fresh kidney tissue was added. The mixture was then thoroughly ground using a grinder at 4°C. Protein samples were prepared as described in the manufacturer's instructions. After sodium dodecyl sulfate gel electrophoresis, the membrane was sealed with skim milk and washed with TBST. The PVDF membrane was incubated with 1:1000 antibodies (HIF-1α, anti-BCL-2, anti-Kim-1, anti-β-actin, anti-β-actin, anti-Bax, and anti-BCL-2 were purchased from HUABIO (Hangzhou, Zhejiang); anti-Kim-1 was purchased from Abcam (Cambridge, UK); and HIF-1α was purchased from Cell Signaling Technology (Massachusetts, USA), followed by incubation with a secondary antibody, as described above. Finally, the protein expression signal of the membrane was obtained using an exposure unit, and semi-quantitative analysis was performed using ImageJ software.

[0093] (3) Immunohistochemical histological analysis of AKI mouse tissues

[0094] All organs were collected from mice. Mouse kidneys were fixed in 4% polyoxymethylene and then stained with HE, Masson, PAS, and HIF-1α.

[0095] 2. Experimental Results

[0096] Figure 5 B represents Masson staining and semi-quantitative analysis of kidney tissue, by... Figure 5B showed that renal tubular damage was significantly reduced in the TFG group, and neovascularization was observed in some tissues.

[0097] Figure 5 C represents PAS staining and semi-quantitative analysis of kidney tissue, derived from... Figure 5 C indicates a significant decrease in collagen near the renal tubular basement membrane.

[0098] Figure 5 D represents HE staining and semi-quantitative analysis of kidney tissue, from... Figure 5 D indicates that kidney damage was significantly alleviated in the TFG group.

[0099] Figure 6 A is PAS staining of kidney tissue, by Figure 6 A shows that in the experimental group that received TFG pretreatment, the polysaccharide deposits in and around the renal tubules of the mice were significantly reduced, and the thickening of the glomerular mesangium was significantly alleviated.

[0100] Figure 6 B represents the results of Western blotting and semi-quantitative analysis of Hif-1α, Bcl-2, Bax, and Kim-1 expression, derived from... Figure 6 B indicates that TFG can alleviate cisplatin-induced acute kidney injury by inhibiting the apoptosis pathway, and this process is achieved through the upregulation of Hif-1α.

[0101] Figure 6 C represents immunohistochemical staining for Hif-1α, Bax, and Cleaved Caspase 3, by... Figure 6 As can be seen from C, the trends of all indicators are consistent with those of Western blotting.

[0102] Figure 6 D is, by Figure 6 D indicates that TFG can significantly downregulate the apoptosis signaling pathway associated with Hif-1α. TUNEL staining showed a significant downregulation of apoptosis in renal tubular epithelial cells within the kidney tissue. Furthermore, Western blot analysis of kidney tissue homogenates containing Hif-1α, Bax, and Kim-1 yielded the same conclusion, demonstrating that TFG can restore kidney function by antagonizing the apoptosis signaling pathway.

[0103] Using 1 μM tFNAs alone did not have any repair effect on kidney damage in mice. The results of this invention show that tFNAs and FG-4592, when combined, have a synergistic effect in repairing kidney damage.

[0104] In summary, this invention provides a complex of DNA tetrahedral framework nucleic acid and FG-4592. The DNA tetrahedral framework nucleic acid and FG-4592 have a synergistic effect in reducing apoptosis of proximal tubular epithelial cells in the renal cortex, reducing renal tissue necrosis, restoring renal function, preventing renal fibrosis, and preventing the progression of acute kidney injury to chronic kidney disease, thus providing a new option for clinical practice.

Claims

1. A DNA tetrahedral framework nucleic acid and FG-4592 complex for preventing and / or treating acute kidney injury, characterized by: It is a complex formed by mixing DNA tetrahedral framework nucleic acid and FG-4592; when DNA tetrahedral framework nucleic acid and FG-4592 are mixed, the molar ratio of DNA tetrahedral framework nucleic acid and FG-4592 is 1:80~200; the DNA tetrahedral framework nucleic acid is formed by base complementary pairing of four single-stranded DNA molecules with sequences as shown in SEQ ID NO.1~4 respectively; The structure of FG-4592 is .

2. The DNA tetrahedral framework nucleic acid and FG-4592 complex of claim 1, wherein: The content of FG-4592 in the complex was measured to be 50 wt% using a UV spectrophotometer.

3. The DNA tetrahedral framework nucleic acid and FG-4592 complex according to claim 1, characterized in that: The molar ratio of the DNA tetrahedral framework nucleic acid to FG-4592 is 1:

80.

4. The DNA tetrahedral framework nucleic acid and FG-4592 complex of claim 1, wherein: The method for synthesizing the DNA tetrahedral framework nucleic acid includes the following steps: adding four single-stranded DNA strands to TM buffer, maintaining at 80~100℃ for 10 min, and maintaining at 2~4℃ for more than 20 min to obtain the final product.

5. The DNA tetrahedral framework nucleic acid and FG-4592 complex of claim 4, wherein: Add four single-stranded DNA strands to TM buffer, maintain at 90°C for 10 min, and then at 4°C for at least 20 min to obtain the final product.

6. A method of preparing the DNA tetrahedral framework nucleic acid and FG-4592 complex of any one of claims 1-5, characterized by: It includes the following steps: The DNA tetrahedral framework nucleic acid and FG-4592 solution are mixed, incubated, and ultrafiltered to obtain the final product.

7. The method of claim 6, wherein: The solvent for the FG-4592 solution is one or more of PBS and DMSO; And / or, the incubation temperature is 20~30℃, and the incubation time is 5~10 hours.

8. Use of the DNA tetrahedral framework nucleic acid and FG-4592 complex according to any one of claims 1 to 5 in the preparation of a medicament for the prevention and / or treatment of acute kidney injury.

9. Use according to claim 8, characterized in that: The acute kidney injury mentioned was cisplatin-induced acute kidney injury.

10. Use according to claim 8, characterized in that: The drug is a chemotherapy protective agent against cisplatin-induced acute kidney injury.

11. Use according to claim 10, characterized in that: The drug in question is for the prevention and treatment of cisplatin nephrotoxicity.

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