Application of notoginseng saponins FT1 in preparation of U2AF1 protein stabilizer

Abstract

The present application belongs to the field of medicine biotechnology, and relates to application of panax notoginseng saponin FT1 in preparation of a U2AF1 protein stabilizer. The present application finds that panax notoginseng saponin FT1 has a significant stability regulation effect on U2AF1 protein and has good safety. Meanwhile, the present application verifies that panax notoginseng saponin FT1 has a good curative effect on improving sustained damage of renal tubular epithelial cells and delaying renal interstitial fibrosis. Therefore, panax notoginseng saponin FT1 has a broad application prospect in relieving and treating AKI and blocking AKI-CKD transition.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology and relates to the application of Panax notoginseng saponin FT1 in the preparation of U2AF1 protein stabilizers. Background Technology

[0002] Acute kidney injury (AKI) is a common critical illness in clinical practice. Epidemiological data show that some patients with severe AKI may still have persistent renal tubular damage after the acute phase, increasing the risk of transitioning from AKI to chronic kidney disease (AKI-to-CKD). This pathological process often presents with subacute progressive kidney injury characteristics, and its core components involve the persistent activation of DNA damage responses in renal tubular epithelial cells, mitochondrial dysfunction, and cell cycle arrest (such as G2 / M arrest), ultimately leading to renal tubular atrophy and abnormal repair. Although clinical attention to this pathological process is increasing, specific intervention drugs for the AKI-to-CKD transition phase are still relatively scarce, and the long-term prognosis of patients urgently needs improvement.

[0003] Notoginsenoside FT1 is a dammarane-type triterpenoid saponin unique to Panax notoginseng and enriched in its leaves and fruits. Its most distinctive pharmacological characteristic is its bidirectional regulation: on the one hand, it promotes platelet aggregation and shortens bleeding time to achieve hemostasis; on the other hand, it dilates blood vessels and promotes endothelial regeneration to improve blood circulation, reflecting the material basis of Panax notoginseng's "hemostasis without blood stasis." Furthermore, notoginsenoside FT1 can improve obesity and insulin resistance, inhibit tumor cell proliferation, and induce tumor cell death. The target map of notoginsenoside FT1 in the field of organ protection is not yet fully elucidated, especially in the area of ​​RNA splicing regulation related to maintaining renal tubular epithelial homeostasis; its potential role remains to be explored.

[0004] U2AF1 (U2 small nuclear RNA auxiliary factor 1, also known as U2AF35) is a key cofactor of the RNA spliceosome. It plays a crucial role in constitutive and alternative splicing by recognizing the 3′ splice site of pre-mRNA and mediating the assembly of the U2 snRNP complex. Dysregulation of U2AF1 expression affects key biological processes such as DNA damage repair and cell cycle transitions (e.g., G2 / M phase), and is associated with fibrosis in various organs. However, no definitive research has yet been published on whether natural products or small molecule compounds can exert nephroprotective effects by specifically targeting U2AF1.

[0005] Current strategies for protecting against kidney injury primarily focus on anti-oxidation, anti-inflammation, or broad-spectrum cell protection. Precise interventions targeting the renal tubular epithelial splicing regulatory network remain to be explored. Given the potential activity of FT1 at the splicing regulatory level and the crucial role of U2AF1 in kidney injury, exploring their potential applications in renal protection, especially the development of novel renal protective lead compounds based on splicing factor regulation, has significant clinical implications and broad application prospects. Summary of the Invention

[0006] This invention discovered that Notoginsenoside FT1 (CAS No. 155683-00-4) can specifically bind to U2AF1 and significantly prolong the half-life of the U2AF1 protein, thereby maintaining the stability of U2AF1 in renal tubular epithelial cells and exhibiting good safety. Based on this research, this invention provides the application of Notoginsenoside FT1 in the preparation of U2AF1 protein stabilizers.

[0007] This invention provides the following technical solution: The first aspect is the application of a Panax notoginseng saponin FT1 in the preparation of a U2AF1 protein stabilizer.

[0008] Secondly, the application of Panax notoginseng saponin FT1 in the preparation of drugs to alleviate or treat diseases related to abnormal U2AF1 expression.

[0009] In some implementations, diseases associated with abnormal U2AF1 expression include breast cancer, ovarian cancer, or acute kidney injury.

[0010] In some embodiments, the drug is a drug for relieving or treating subacute progressive kidney injury caused by chemical toxicity or persistent damage.

[0011] In some embodiments, the drug is a drug for relieving or treating chemotherapy-induced kidney damage. Further, the chemotherapy drug is cisplatin, etc.

[0012] In some implementations, the drug is a drug for relieving or treating the progression from acute kidney injury (AKI) to chronic kidney disease (CKD).

[0013] In some implementations, the drug has the effect of improving persistent damage to the renal tubular epithelium.

[0014] In some implementations, the drug has the effect of delaying renal interstitial fibrosis.

[0015] In some implementation schemes, the dosage of Panax notoginsenoside FT1 is 4.5~5.5 mg / kg for mice.

[0016] In some implementations, the drug includes pharmaceutical excipients. Specifically, the pharmaceutical excipients may be physiological saline, etc.

[0017] In some implementations, the drug is administered via intraperitoneal injection.

[0018] In some embodiments, the dosage form of the drug is tablets, capsules, granules, drops, lyophilized products, granules, or injections.

[0019] The beneficial effects of this invention are as follows: This invention discovered that notoginsenoside FT1 has a significant stability-regulating effect on U2AF1 protein and exhibits good safety. Furthermore, this invention verified that notoginsenoside FT1 has good efficacy in improving persistent renal tubular epithelial damage and delaying renal interstitial fibrosis. Therefore, notoginsenoside FT1 has broad application prospects in alleviating and treating AKI and blocking the progression of AKI-CKD. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 This is a schematic diagram illustrating the interaction between U2AF1 protein and Panax notoginseng saponin FT1 analyzed by molecular docking in Example 1 of the present invention.

[0022] Figure 2 This is a graph showing the results of the interaction between FT1 and U2AF1 observed by the Cellular Thermal Shift Assay (CETSA) in Example 2 of the present invention; where A is the effect of the CETSA on the interaction between FT1 and U2AF1 and the thermal stability of U2AF1 protein observed; and B is a grayscale analysis graph.

[0023] Figure 3 The image shows the cell viability detection results of cells co-cultured with different concentrations of Panax notoginseng saponin FT1 (5, 10, 15, and 20 μM) and cisplatin (DDP) in Example 2 of this invention.

[0024] Figure 4 The image shows the HE staining results of paraffin sections of heart, liver, spleen, lung, and kidney tissues from mice injected intraperitoneally with physiological saline and 5 mg / kg Panax notoginseng saponin FT1 in Example 3 of this invention.

[0025] Figure 5 This is an ELISA statistical chart of ALT (A), AST (B), urea nitrogen (C), and creatinine (D) in mice after intraperitoneal injection of physiological saline and 5 mg / kg Panax notoginseng saponin FT1 in Example 3 of the present invention.

[0026] Figure 6 This is an ELISA statistical graph of blood urea nitrogen (B) and creatinine (A) in paraffin sections of the kidneys induced by 2 mg / kg cisplatin in Example 4 of the present invention, showing the improvement of kidney injury by using 5 mg / kg Panax notoginseng saponin FT1.

[0027] Figure 7 This is an HE staining result of paraffin sections used in Example 4 of the present invention to improve kidney injury induced by 2 mg / kg cisplatin using 5 mg / kg Panax notoginseng saponin FT1.

[0028] Figure 8 This is a PAS staining result of using 5 mg / kg Panax notoginseng saponin FT1 to improve 2 mg / kg cisplatin-induced kidney injury in Example 4 of the present invention.

[0029] Figure 9 This is a Collagen 3 staining result of using 5 mg / kg Panax notoginseng saponin FT1 to improve 2 mg / kg cisplatin-induced kidney injury in Example 4 of the present invention.

[0030] Figure 10 This is a diagram showing the α-SMA staining results of 5 mg / kg Panax notoginseng saponin FT1 used to improve 2 mg / kg cisplatin-induced kidney injury in Example 4 of the present invention. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1: Molecular docking analysis This embodiment uses the RBS method to screen potential binding drugs targeting U2AF1 (UniProt ID: Q9D883). The protein structure model is derived from the AlphaFold Protein Structure Database (https: / / alphafold.ebi.ac.uk / ). Using 3191 natural products from the BioBioPha database (BBP Natural Product Library) as the ligand set, 3D modeling and energy minimization were performed using Chemdraw 20.0. High-throughput docking was driven by AutoDock Vina 1.1.2, with batch computation implemented using the Python script SailVina. The novel pocket detection module built into RBS can automatically identify highly active binding cavities and calculate docking box parameters based on pre-docking results using AutoDockTools. In the initial screening stage, the box size was set to 98 Å × 74 Å × 65 Å, the grid spacing to 1.00 Å, and the center coordinates to be x : y : z = -6.801 : -5.059 : -4.426. After global docking and energy optimization using SailVina, the samples were sorted by binding affinity; subsequently, samples with binding energy ≤ The compound with a concentration of 9.0 kcal / mol was precisely verified using AutoDockVina 1.1.2 within a reduced-size box (30 Å × 30 Å × 30 Å, center x : y : z = 3.155 : -1.702 : 1.758, grid spacing 1.00 Å).

[0033] Molecular docking analysis revealed that notoginsenoside FT1 can bind to specific amino acid residues of U2AF1 via hydrogen bonds and van der Waals forces, such as... Figure 1 As shown.

[0034] Example 2: In vitro experiment CETSA (Cellular Thermal Migration Assay) can be used to assess the binding of drugs to their target proteins within cells. When the target protein binds to an active small molecule, it often exhibits higher thermal stability. In this example, human renal tubular epithelial cells HK-2 were cultured with DMSO as a negative control or with notoginseng saponin FT1. After 24 hours of treatment, the cells were collected and lysed to prepare a complete protein extract. The protein sample was aliquoted into multiple PCR tubes and heated sequentially at gradient temperatures for 10 minutes each to induce thermal denaturation. Each tube was then centrifuged, and the supernatant was analyzed by Western blot to determine the residual amount of soluble protein at different temperature points. The CETSA assay showed that notoginseng saponin FT1 can bind to U2AF1, increasing the stability of the U2AF1 protein. Figure 2As shown in A and B in the diagram.

[0035] HK-2 cells were treated with different concentrations of notoginsenoside FT1 (5, 10, 15, 20 μM) and 20 μM DDP in normal culture medium for 24 hours. Cell viability was detected by CCK-8 assay. HK-2 cells treated with notoginsenoside FT1 showed a concentration-dependent increase in cell viability, suggesting that this compound may protect cells by targeting U2AF1. Figure 3 As shown.

[0036] Example 3: Biosafety of Panax notoginseng saponin FT1 Toxicity assessment was conducted by intraperitoneal injection of 5 mg / kg of Panax notoginseng saponin FT1 into mice. Panax notoginseng saponin FT1 was dissolved in physiological saline, and 5 mg / kg of Panax notoginseng saponin FT1 was injected intraperitoneally into mice for 14 consecutive days. Sections of major organs such as the heart, liver, spleen, lungs, and kidneys were collected for pathological examination, and serological tests were performed using microvenous blood. HE staining and serological results showed that, compared with the control group, intraperitoneal injection of Panax notoginseng saponin FT1 did not produce pathological effects on the major internal organs of mice. Figure 4 As shown, it does not affect liver or kidney function, such as Figure 5 As shown in A, B, C, and D, these results indicate that Panax notoginseng saponin FT1 has good biocompatibility.

[0037] Example 4: In vivo animal experiments This invention evaluates the therapeutic effect of notoginsenoside FT1 on acute kidney injury (AKI) by intraperitoneal injection of notoginsenoside FT1 (concentration 5 mg / kg) into a mouse model of subacute progressive kidney injury.

[0038] To establish a subacute progressive kidney injury model, experimental animals received intraperitoneal injections of cisplatin (2 mg / kg) for 14 consecutive days. Simultaneously, the treatment group received intraperitoneal injections of Panax notoginseng saponin FT1 (5 mg / kg).

[0039] 1. On day 14, tail vein blood was collected from mice in each group, and renal function indicators were assessed using blood urea nitrogen and creatinine assay kits. Results showed that serum BUN and Cr levels in the model group were significantly higher than those in the control group. However, after FT1 intervention, these indicators significantly decreased, suggesting that Panax notoginseng saponin FT1 has a good protective and therapeutic effect on cisplatin-induced kidney injury. Figure 6 As shown in A and B in the diagram.

[0040] 2. Hematoxylin and eosin (HE) staining: Paraffin sections of kidney tissue were prepared and baked at 65°C for 30 minutes to fix the tissue.

[0041] (2) After the slices were treated with xylene for 15 min, they were hydrated by sequentially passing through a gradient of ethanol from 100% to 70% for 3 min at each stage, and finally rinsed with distilled water for 5 min.

[0042] (3) Gently shake off the moisture from the surface of the slices.

[0043] (4) Hematoxylin staining: stain the sections for 2 minutes.

[0044] (5) Briefly differentiated for a few seconds by 1% hydrochloric acid alcohol to enhance nucleocytoplasmic contrast.

[0045] (6) Rinse with tap water for 15 minutes to turn blue, and confirm under a microscope that the cell nuclei are clearly blue.

[0046] (7) Re-dye with eosin solution for 30 s, then wash away excess dye with a gradient of 95% and anhydrous ethanol.

[0047] (8) Clear xylene for 10 min, let it dry, and then seal it with a coverslip.

[0048] (9) Evaluation of HE staining effect and image acquisition under microscope.

[0049] The results showed that in the HE-stained control group, the renal tubular structure was clear, the epithelial arrangement was regular, and the lumen morphology was relatively uniform; in the DDP group, the renal tubular arrangement was disordered, the lumen was dilated / irregular, the interstitial cell density was increased, and inflammatory cell infiltration and tissue damage were visible; the DDP+FT1 group showed improvement compared to the DDP group, with more regular renal tubular structure and reduced interstitial infiltration. Figure 7 As shown.

[0050] 3. Glycogen (PAS) staining: Staining was performed using the PAS kit (Solarbio, G1281), as briefly described below: (1) The specimen was dehydrated, embedded in paraffin, and then slided. The slide was dewaxed and hydrated in distilled water.

[0051] (2) The slices were oxidized with PAS oxidant at room temperature for 10 min.

[0052] (3) Rinse twice with distilled water, 2 minutes each time.

[0053] (4) Stain with Schiff solution for 20 min under dark conditions.

[0054] (5) Rinse twice with distilled water, 2 minutes each time.

[0055] (6) Stain cell nuclei with hematoxylin for 1 min.

[0056] (7) Rinse with tap water for 15 minutes to promote blueing.

[0057] (8) Dehydrated stepwise with 75%~100% ethanol (2 min per step), cleared with xylene (3 min × 2 times), and sealed with resin.

[0058] (9) Evaluate the PAS staining effect under a microscope and take photos for record.

[0059] The results showed that in the PAS-stained control group, the renal tubular basement membrane and brush border structure were clear; in the DDP group, the renal tubules were disordered, the lumen was dilated, the brush border was lost and interstitial cell infiltration was present; in the DDP+FT1 group, the renal tubular structure was more regular, the lumen morphology was improved, and the tissue integrity was significantly restored compared with the DDP group. Figure 8 As shown.

[0060] 4. Immunofluorescence staining of fibrosis markers α-SMA and Collagen 3: (1) Paraffin sections of kidney tissue were baked at 65°C for 30 min.

[0061] (2) Routine dewaxing to hydration: The sections were dewaxed with xylene for 15 min, then hydrated with 100%, 90%, 80% and 70% ethanol for 3 min each, and finally rinsed with ddH2O for 3 min.

[0062] (3) High-temperature and high-pressure antigen retrieval was performed using sodium citrate buffer for 20 min, followed by natural cooling to room temperature and washing with PBS for 5 min.

[0063] (4) Block non-specific binding sites in the slices at room temperature for 60 min.

[0064] (5) Preparation of primary antibody working solution: α-SMA and Collagen 3 antibodies were diluted in antibody dilution solution at a ratio of 1:400.

[0065] (6) Place the slide in a humidified chamber, add the primary antibody working solution, and incubate overnight at 4°C.

[0066] (7) The next day, the slices were removed and allowed to equilibrate at room temperature for 1 hour.

[0067] (8) Wash with PBS 5 times, 3 min each time; subsequent operations should be performed in the dark.

[0068] (9) Preparation of secondary antibody working solution: Dilute 488 and 561 anti-rabbit fluorescent secondary antibodies at a ratio of 1:300 in a dark room and add an appropriate amount of DAPI dye solution.

[0069] (10) Each slice was incubated with secondary antibody for 1 h (37℃) in the dark, and then washed with PBS for 30 min.

[0070] (11) Add anti-fluorescence quenching mounting medium to mount the slide and acquire images under a confocal microscope.

[0071] The results are as follows Figure 9 and Figure 10 As shown, during the progression from AKI to CKD, FT1 intervention significantly reduced cisplatin-induced collagen deposition and downregulated the abnormal expression of fibrosis markers α-SMA and Collagen III, confirming that it can effectively block the process of renal fibrosis and delay the onset of CKD.

[0072] In summary, the results indicate that Panax notoginseng saponin FT1 can significantly improve the transformation of cisplatin-induced acute kidney injury into chronic kidney disease (AKI-to-CKD).

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of Panax notoginseng saponin FT1 in the preparation of U2AF1 protein stabilizer.

2. The application of Panax notoginseng saponin FT1 in the preparation of drugs to alleviate or treat diseases related to abnormal expression of U2AF1.

3. The application as described in claim 2, characterized in that, Diseases associated with abnormal U2AF1 expression include breast cancer, ovarian cancer, or acute kidney injury.

4. The application as described in claim 2, characterized in that, The drug is used to relieve or treat subacute progressive kidney injury caused by chemical toxicity or persistent damage.

5. The application as described in claim 2, characterized in that, The drug is used to alleviate or treat kidney damage induced by chemotherapy drugs.

6. The application as described in claim 2, characterized in that, The drug is used to alleviate or treat the progression from acute kidney injury to chronic kidney disease.

7. The application as described in claim 2, characterized in that, The dosage of Panax notoginseng saponin FT1 is 4.5~5.5 mg / kg for mice.

8. The application as described in claim 2, characterized in that, The drug includes pharmaceutical excipients.

9. The application as described in claim 2, characterized in that, The drug is administered via intraperitoneal injection.

10. The application as described in claim 2, characterized in that, The dosage form of the drug is tablets, capsules, granules, drops, lyophilized products, granules, or injections.

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