Application of hederagenin in preparation of medicine for maintaining dryness of ATII

Abstract

The invention discloses a novel application of hederagenin in preparation of a medicine for maintaining the dryness of alveolar II epithelial cells, the hederagenin is definitely used for preparing the medicine for maintaining the dryness of the alveolar II epithelial cells for the first time, and the repairing effect of the hederagenin on ATII dry damage is verified through in-vivo and in-vitro experiments. A brand new treatment strategy is provided for lung diseases such as acute lung injury, pulmonary fibrosis and the like which are characterized by ATII dry failure, the technical blank of intervention of natural active ingredients in ATII dry is filled up, a new way which is efficient, low in cost and easy to obtain is provided for ATII dry maintenance, the concentration of a medicine prepared from the natural active ingredients is screened, and the clinical application prospect is wide. An important theoretical basis is provided for further medication and medicine preparation, meanwhile, the pharmacological application range of the hederagenin is expanded, and a new target and a new strategy are provided for subsequent intervention treatment of lung diseases such as acute lung injury and pulmonary fibrosis needing injury repair.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology, specifically relating to the application of ivy saponin in the preparation of drugs that maintain the dryness of ATII. Background Technology

[0002] Alveolar type II (ATII) cells, as progenitor cells of the lungs, possess a certain stem potential and are capable of self-renewal and differentiation into type I alveolar epithelial cells. This is fundamental to maintaining the integrity of alveolar structure and the stability of lung function. With continuous advancements in research techniques, researchers have gradually discovered that intervening in the stem potential of ATII cells may be an important pathway for lung injury repair.

[0003] Currently, research on the dryness regulation of ATII lacks low-cost, readily available, and large-scale application intervention programs using natural active ingredients from traditional Chinese medicine, resulting in technical bottlenecks in research related to lung damage repair and intervention for pulmonary diseases.

[0004] Currently, the following patents are related to ATⅡ dry proliferation: 1. Patent No. 202311088227.1, Cell culture medium for maintaining the phenotype and function of type II alveolar epithelial cells in vitro; 2. Patent No. 202310439572.9, An immortalized human type II alveolar epithelial cell line, its construction method, and its uses; 3. Patent No. JP2011196269, TYPE II ALVEOLAR EPITHELIAL CELL ACTIVATOR; 4. Patent No. 202310439572.9, An immortalized human type II alveolar epithelial cell line, its construction method, and its uses; 5. Patent No. 202211599247.0, A method for enhancing the expansion capacity of in vitro cultured type II alveolar epithelial cells and its drug combination. The technical content of the above patents does not include any content related to the maintenance of ATII stemness through hederone saponins.

[0005] As a natural triterpenoid saponin, the existing pharmacological activity studies of ivy saponins have mostly focused on anti-inflammatory, anti-tumor, liver protection and respiratory mucus regulation. Relevant literature has clarified its application potential in some diseases, but no related functions of ivy saponins in the maintenance of ATII dryness have been found. Summary of the Invention

[0006] The purpose of this invention is to provide a new application of ivy saponins in the preparation of drugs for maintaining the stemness of alveolar II epithelial cells, and to provide a theoretical basis for verifying the use of ivy saponins in the regulation of specific cell stemness, which has broad application prospects.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the application of ivy saponins in the preparation of drugs for maintaining ATII dryness.

[0008] This invention also provides its application in the preparation of drugs that improve ATⅡ tissue morphological abnormalities.

[0009] This invention also provides its application in the preparation of drugs that restore the self-renewal potential of ATII.

[0010] This invention also provides its application in the preparation of drugs that improve the proliferation and differentiation capacity of ATⅡ.

[0011] To further realize the present invention, the present invention provides a pharmaceutical concentration of hederogenin of 12.5 μM.

[0012] This invention also provides its application in the preparation of drugs that protect ATII mitochondria and lamellar bodies.

[0013] This invention also provides its application in the preparation of drugs that protect the dry potential of ATII.

[0014] This invention also provides its application in the preparation of drugs that improve ATII function and secretion.

[0015] This invention also provides its application in the preparation of drugs for maintaining ATII function and promoting lung tissue repair.

[0016] The advantages of this invention compared to the prior art are as follows: The core innovation of this invention lies in the first-time use of ivy saponins in the preparation of drugs that maintain the stemness of type II alveolar epithelial cells. Through in vitro and in vivo experiments, its repair effect on ATII stemness damage is verified, providing a novel treatment strategy for lung diseases characterized by ATII stemness failure, such as acute lung injury and pulmonary fibrosis.

[0017] Cellular and animal experiments have fully demonstrated its therapeutic effects. 1. Cellular level: After TNF-α intervention caused significant damage to the stemness of ATII cells, hederonein at concentrations of 6.25, 12.5, 25, and 50 μM could improve the proliferation capacity of ATII cells. 12.5 μM hederonein intervention for 12 h could completely or partially improve abnormal cell morphology (such as disordered colony arrangement and cytoplasmic vacuolation). The number of cell colony clones formed recovered from (70.67±5.13) to (123.00±8.89), which is close to the normal level. In the 3D spheroidization experiment, the maximum diameter of cell clusters increased from (50.72±14.89) μm to (113.68±31.27) μm, and the area increased by about 3.5 times, which proved that it can restore the self-renewal potential of ATII.

[0018] 2. Molecular level: Hederogen can reverse the inhibition of TNF-α on ATII specific markers, and restore the expression of SP-A and SP-C genes from 0.65±0.01 and 0.14±0.00 to 0.86±0.01 and 0.85±0.01, respectively, and the protein expression level is completely restored to the level of the normal control group; at the same time, it upregulates the abnormal expression of HOPX, a marker of alveolar type I epithelial cells, and maintains the proliferation-differentiation balance of ATII.

[0019] 3. Animal level: After oral administration of 25 mg / kg ivy saponin to a rat model of LPS-induced acute lung injury for 7 days, the pathological changes such as alveolar septal widening and inflammatory infiltration were significantly reduced. The expression of SP-A in lung tissue recovered from 0.94±0.29 to 6.15±0.24, close to the normal level, which proves that it can maintain ATII function and promote lung tissue repair in vivo.

[0020] This invention does not follow the conventional approach of current lung disease treatments, which often target the pathogenesis of alveolar epithelial cell inflammation or epithelial-mesenchymal transition, abnormal fibroblast proliferation, and interstitial collagen deposition. Instead, it focuses on a novel treatment strategy that promotes lung tissue repair by maintaining the stemness of alveolar epithelial cells. Its core advantages include: 1. Reparative treatment: By protecting the self-renewal and differentiation potential of ATII, it can delay or reverse the progression of lung injury, rather than simply treating the symptoms. It is especially suitable for refractory diseases such as idiopathic pulmonary fibrosis and acute respiratory distress syndrome (ARDS).

[0021] 2. Synergistic effect of multiple mechanisms: It works together to maintain the stemness of alveolar epithelial cells (upregulation of SP-A and SP-C expression), inhibit the inflammatory response (downregulation of TNF-α and IL-6 expression levels), and protect mitochondria (improving mitochondrial morphology and structure and enhancing mitochondrial function), which has a more comprehensive therapeutic advantage than a single mechanism.

[0022] 3. High safety due to natural origin: As a plant-derived compound, its toxicity is significantly lower than that of chemically synthesized drugs, making long-term use possible (such as maintenance therapy for pulmonary fibrosis).

[0023] This invention demonstrates the application and verification of hederonein in maintaining the dryness of ATII, confirming its regulatory effect on the dryness potential of ATII. It fills a technological gap in the intervention of natural active ingredients in ATII dryness, providing a new, efficient, low-cost, and readily available approach for maintaining ATII dryness. Furthermore, the concentration of drugs prepared using hederonein was screened, providing important theoretical basis for further drug use and preparation. Simultaneously, it expands the pharmacological application scope of hederonein, providing new technical support for subsequent research on interventions requiring damage repair in lung diseases such as acute lung injury and pulmonary fibrosis.

[0024] This invention proposes and confirms the role of ivy saponins in maintaining ATII dryness, which can be used to prepare drugs that maintain ATII dryness. This not only provides a new target and strategy for the treatment of lung diseases, but also has the potential for dosage form optimization and industrial production feasibility, and has important scientific value and clinical application prospects. Attached Figure Description

[0025] Figure 1 The morphology of primary alveolar type II epithelial cells with excellent morphology in Experimental Example 1 of the present invention (×40). Figure 2 Cell morphology (×40) 12 h after TNF-α cytokine intervention in Experiment Example 1 of this invention. Figure 3 Cell morphology (×40) 12 h after immediate administration of ivy saponin in Experiment Example 1 of this invention. Figure 4 The cell colony count in the cell cloning experiment of Experiment Example 1 of this invention is represented by A (blank group), B (model group), and C (hederone group). Figure 5 Ultrastructure of type II alveolar epithelial cells in the blank group in Experiment Example 1 of this invention (×7000). Figure 6 Ultrastructure of type II alveolar epithelial cells in the model group of Experiment Example 1 of this invention (×7000). Figure 7 Ultrastructure of type II alveolar epithelial cells after 12 hours of intervention with ivy saponin in Experiment Example 1 of this invention (×7000). Figure 8 Images of ATⅡ cell clusters (×40) from each group in Experiment Example 1 of this invention, where A is the blank group, B is the model group, and C is the hederaponin group; Figure 9 This shows the cell viability trend after hedyotis diffusa intervention in cells in Experiment Example 1 of this invention. Figure 10 The immunofluorescence expression of SP-A in ATII cells of each group in Experiment Example 1 of this invention (×40). Figure 11 The immunofluorescence expression of SP-C in ATII cells of each group in Experiment Example 1 of this invention (×40). Figure 12 The HOPX immunofluorescence expression of ATII cells in each group of Experiment Example 1 of this invention (×40). Figure 13 The expression of SP-A and SP-C proteins in each group after gene knockout in Experiment Example 1 of this invention; Figure 14The expression of SP-A in the lung tissue of rats in Experiment Example 2 of this invention (×40), where A is the blank group, B is the model group, and C is the hederaponin group; Figure 15 The changes in the expression of proliferative differentiation proteins SP-A and SP-C in the lung tissue of rats in Experiment Example 2 of this invention are shown. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0027] Experiment Example 1: Cell Experiment 1. Cell culture and drug intervention protocol: On days 5-6 after primary extraction, the culture dish was observed under a microscope, and well-formed cell colonies could be observed (see...). Figure 1 The cells exhibited square, rhomboid, and other polygonal morphologies, growing in a regular, cobblestone-like clonal pattern. They were cultured for another 4-5 days until the cell confluence reached 80-90% before further processing. Cells in the logarithmic growth phase were digested with trypsin, centrifuged, resuspended in 20% complete culture medium, and re-seeded. Culture dishes that had regrow to 60-70% confluence were then subjected to drug intervention. Following TNF-α model intervention, 12.5 μM hedyotis diffusa was immediately administered. (This is repeated in the original text.)

[0028] 2. Observation of cell morphology and cell number: After TNF-α intervention, ATII tissue morphology showed significant abnormalities (see...). Figure 2 The abnormal phenomena observed under the microscope include: (1) cell colonies lose their regular arrangement; (2) cell edges are irregular or have gaps; (3) a large number of small vesicle-like structures appear in the cytoplasm or nucleus; (4) the three-dimensionality of the cells is reduced, the cytoplasm is flattened, and some cells show floating or semi-adherent phenomena; (5) most cells show filamentous structures; (6) the edges of normal cells are bright, but the brightness of their edges is significantly reduced, or the boundaries are not obvious; (7) the number of cells counted in the culture dish after intervention is significantly reduced by counting with a counter.

[0029] After 12 hours of intervention with hederaponin, all of the above-mentioned abnormalities improved (see...). Figure 3 ),and Figure 2 In comparison, the cells after intervention regained a regular and three-dimensional morphology, were arranged relatively densely, had a compact cytoplasm that was relatively transparent, and had fewer impurities or vacuolation. The cell edges became clear and bright, and the cell count in the culture dish increased.

[0030] 3. Cloning experiment: The colony formation assay is a classic experimental technique for evaluating the long-term proliferative capacity, self-renewal potential, and survival ability of cells in vitro. By detecting the colony formation, the proliferative capacity of cells can be evaluated.

[0031] Experimental Methods: When the ATII cells in the control group reached approximately 75% confluence with the culture dish, they were digested with trypsin, centrifuged, and the resulting precipitate was resuspended in 1 mL of 20% complete culture medium to form a cell suspension. Cells were then counted using a cell counter. After counting, 800 cells / well were seeded into each experimental group in a 6-well plate. Culture was continued until day 3, with medium changes every 2 days. Cell status was observed daily. Cell culture was stopped when the number of cloned cells exceeded approximately 50. After cloning, cells were washed twice with PBS, fixed with 1 mL of 75% ethanol for 20 minutes in each well, the ethanol was discarded, and 1 mL of crystal violet staining solution was added to each well for 20 minutes. Cells were washed several times with water, air-dried, and photographed using a professional camera.

[0032] The results are as follows Figure 4 As shown in Table 1, the number of colonies formed in the blank group was (141.00±1.73), the number of colonies formed after TNF-α intervention was (70.67±5.13), and the number of colonies formed after hedyotis diffusa intervention was (123.00±8.89), indicating that hedyotis diffusa has an improving effect on the proliferation ability of ATII.

[0033] 4. Electron microscopy observation of ultrastructure: The ultrastructure of ATII was observed using transmission electron microscopy, including mitochondria and lamellar bodies. Figure 5 As shown, the mitochondria in the blank group cells have complete and clear morphological structure, with neatly arranged cristae and good regularity.

[0034] Compared to the control group, TNF-α intervention resulted in swollen or vacuolated mitochondria, reduced or absent mitochondrial cristae, shortened or broken mitochondrial cristae, or a decrease in the number of mitochondria. Furthermore, a decrease in the number of lamellar body precursors and extracellular secretory granules was observed (see...). Figure 6 ).

[0035] After 12 hours of intervention with hederaponin, all of the above-mentioned abnormalities improved (see...). Figure 7 ).and Figure 6 compared to, Figure 7 The number of mitochondria in the cells increased after the intervention, the mitochondrial cristae were more complete and arranged relatively neatly; the number of lamellar bodies was restored, and the number of extracellular secretory granules was also restored.

[0036] 5. 3D sphere formation test: The role of hederasein in maintaining the stemness of AECII was indirectly assessed by measuring and analyzing the maximum diameter and area of ​​AECII cell clusters under an optical microscope. The 3D spheroidization ability of AECII is one of the important external manifestations of its stemness characteristics; changes in cell cluster diameter and area can reflect its state of stemness maintenance or impairment.

[0037] Experimental Methods: The hanging drop method was used. Cells were digested with trypsin, centrifuged, resuspended in 20% complete culture medium, and counted. TNF-α or ivy saponin was added to a 15 mL centrifuge tube. The cell suspension in the same tube was repeatedly mixed before inoculation, and simultaneously seeded by dropping the cells into the bottom of a 60 mm culture dish. The droplet size was strictly controlled at 25 μL, ensuring sufficient fluid tension and minimizing deformation or dripping due to slight movements. The cell density was 750 cells / drop, and the droplet count was 40 drops / dish. The dish was then capped, and the entire dish was carefully and evenly inverted. 1-2 mL of PBS was added to the lid to reduce evaporation. The dish was then horizontally moved back to the incubator for incubation. At 12 hours, cell clusters were photographed using an optical microscope, and the size of the cell clusters was measured using ImageView software. The diameter and area of ​​the largest cell cluster were recorded.

[0038] The results are as follows Figure 8 As shown in Table 2, the average maximum cell cluster diameter in the blank group was (88.26±15.08) μm, and the average maximum area was (5286.21±1631.26) μm². After TNF-α intervention, the changes were (50.72±14.89) μm and (2074.53±1299.06) μm², respectively. After hedyotis diffusa intervention, the changes were restored to (113.68±31.27) μm and (7298.14±2978.66) μm², respectively. This demonstrates that hedyotis diffusa has a protective effect on the stem potential of ATII.

[0039] 6. CCK8 Experiment: The CCK8 assay is a classic cell proliferation validation assay that can verify cell proliferation results by evaluating cell viability.

[0040] Experimental methods: Cells were digested with trypsin at 80-90% confluence, centrifuged, resuspended in 20% complete culture medium, and then counted. The total cell and liquid volumes were estimated, and the mixture was thoroughly mixed in 50 mL centrifuge tubes and seeded into 96-well plates with a total liquid volume of 100 μL per well and a cell count of 3 × 10⁶ cells / well. 3After seeding, the cells were shaken well and incubated for 24-36 hours. Intervention was initiated when cell confluence reached 60%-70%. An ALI cell model was established using TNF-α, and hederaponin at concentrations of 6.25, 12.5, 25, and 50 μM were administered at 0, 2, 4, and 6 hours post-modeling. The cells were then returned to the incubator for 12 hours. At 12 hours, the culture medium was removed in the dark, and 100 μM of CCK8 dilution buffer (CCK8:DMEM = 1:9) was added to each well. After incubation for another hour, OD values ​​were measured using a microplate reader.

[0041] The results are as follows Figure 9 As shown, the activity of ATII was increased after intervention with the same concentration of hederaponin at different time points, indicating that hederaponin can enhance the proliferation ability of ATII. Among them, the intervention with a concentration of 12.5 μM hederaponin was the most suitable.

[0042] 7. Specific gene and protein expression 7.1 Immunofluorescence observation of protein expression Immunofluorescence was used to detect SP-A, SP-C, and HOPX proteins in lung tissue. SP-A and SP-C are ATII-specific expression proteins, while SOPX is a type I alveolar epithelial cell-specific expression protein, representing the degree of "proliferation" and "differentiation" of ATII, respectively.

[0043] Experimental methods: (1) Cell slides: Soak round cell slides in anhydrous ethanol, carefully hold them with forceps and ignite them over an alcohol lamp for 3-6 seconds. After temperature control, place them into 6-well plates. Digest and centrifuge well-grown cells according to the method described above, resuspend them in 20% complete culture medium, and count the cells. Estimate the total cell count and total liquid volume, mix them repeatedly in 50mL centrifuge tubes, and seed them into 6-well plates. The total liquid volume per well is 2mL, and the cell count is 5×10⁶ cells / well. 3 Cell density per well; after seeding, shake well and incubate in an incubator for 36-48 hours; when cell confluence reaches 60%-70%, use different drugs for intervention, and collect samples after 12 hours.

[0044] (2) Cell fixation: Remove the 6-well plate, discard the culture medium, wash twice with PBS on a shaker and aspirate, add 4% paraformaldehyde for fixation for 20 min, wash twice again with PBS and discard the liquid. Add methanol and dehydrate for 5 min.

[0045] (3) Serum blocking: Add BSA (bovine serum albumin + Tritium) and block for 20 min, then recover the blocking solution; (4) Incubation in the dark: Add primary antibody and incubate overnight in a 4°C refrigerator in the dark, then incubate secondary antibody at room temperature in the dark for 1 hour. Add DAPI anti-fluorescence quenching agent to stain the nucleus for 10 minutes, and then mount the sample with a glass slide. Immediately acquire images of the processed sample using laser confocal microscopy and store it in a 4°C refrigerator in the dark for up to one week.

[0046] The results are as follows Figure 10-12 As shown in Table 3, ImageJ statistical analysis of SP-A and SP-C protein expression revealed the following in immunofluorescence images: In the blank control group, the average fluorescence signal intensities were: SP-A (1016094.67±247326.85), SP-C (332067.40±177582.95), and HOPX (197632.00±103627.50); ​​after TNF-α intervention, the fluorescence signal intensity decreased (SP-A 130304.67±...). The fluorescence signal intensity of hedyotis diffusive agents was restored after intervention with SP-A (781958.67±103792.73), SP-C (368074.00±203146.38), and HOPX (1181089.33±235086.33). These results demonstrate that hedyotis diffusive agents have an ameliorative effect on ATII function and secretion, and indirectly contribute to improved proliferation and differentiation capabilities.

[0047] 7.2 Gene and Protein Expression The expression of SP-A and SP-C genes and proteins of ATⅡ was verified using Rt-qPCR and WB detection techniques, which can represent the changes in ATⅡ proliferation level to a certain extent.

[0048] The gene expression results are shown in Table 4. The expression levels of ATⅡ gene in the blank group were SP-A (1.00±0.04) and SP-C (1.00±0.00), respectively. After TNF-α intervention, SP-A (0.65±0.01) and SP-C (0.14±0.00) were observed. After hedyotis diffusa intervention, SP-A (0.86±0.01) and SP-C (0.85±0.01) were observed.

[0049] Protein expression results as follows Figure 13As shown in Table 5, the expression levels of ATⅡ protein in the blank group were SP-A (21288.17±2959.92) and SP-C (20677.87±469.14), respectively; after TNF-α intervention, SP-A (10068.39±3849.73) and SP-C (16807.37±2406.69), respectively; and after ivy saponin intervention, SP-A (21276.38±1224.50) and SP-C (23348.22±4533.61), respectively.

[0050] The above results demonstrate, at the gene and protein level, the effect of ivy saponins on improving ATII function.

[0051] Experiment Example 2, Animal Experiment: After endotracheal intubation and infusion of LPS to replicate the acute lung injury model, 25 mg / kg of ivy saponin was administered by gavage in physiological saline solution on the same day, with a dosing cycle of 7 days.

[0052] 1. Immunohistochemical expression in lung tissue ATII is one of the fundamental components of alveolar structure; therefore, observing the integrity of lung tissue structure can indirectly reflect the function of ATII. Simultaneously, immunohistochemical detection of SP-A secretion can directly demonstrate the relationship between ATII dry maintenance and disease during lung injury repair.

[0053] See results Figure 14 As shown in Table 6, in the control group, the alveolar septa of rats were narrowed, the alveolar tissue structure was clear and intact, and there were no abnormal pathological manifestations. Compared with the control group, the alveolar septa of rats in the model group were significantly widened, the alveolar spaces were narrowed, and there was a large amount of inflammatory cell infiltration, erythrocyte leakage, and hyaline membrane formation in some cases. Compared with the model group, the alveolar septa of rats in the ivy saponin group were restored to narrowing, the alveolar spaces were enlarged, the number of inflammatory cells and erythrocyte leakage was reduced, and hyaline membranes were rare.

[0054] SP-A expression in lung tissue was detected, with the values ​​being (8.38±1.96) in the blank group, (0.94±0.29) in the model group, and (6.15±0.24) in the hederaponin group. These results demonstrate that hederaponin plays a protective role in lung tissue structure and ATII proliferation.

[0055] 2. Detection of SP-A and SP-C expression in lung tissue Protein expression reflects cellular function to some extent. Western blotting was used to detect the expression of SP-A and SP-C proteins in lung tissue. We also intervened in animals using the active ingredients of Astragalus membranaceus, gentiocarbazone, hederaponin, quercetin, and the positive control drug dexamethasone. Therefore, during Western blotting, these groups were simultaneously detected to compare their intervention effects on ATII function.

[0056] The protein expression results are shown in Figure 15 According to Table 7, the protein expression levels in lung tissue of the blank group were SP-A (22235.22±1478.03) and SP-C (24202.20±2071.99), respectively; after TNF-α intervention, SP-A (16942.82±1623.11) and SP-C (16839.37±1286.04), respectively; and after ivy saponin intervention, SP-A (19553.05±1671.98) and SP-C (21200.33±2701.03), respectively.

[0057] .

Claims

1. The use of a saponin aglycone in the preparation of a medicament for maintaining the stemness of AT II.

2. Use according to claim 1, characterized in that: in the preparation of a medicament for improving the morphological abnormalities of AT II.

3. Use according to claim 1, characterized in that: in the preparation of a medicament for restoring the self-renewal potential of AT II.

4. Use according to claim 1, characterized in that: in the preparation of a medicament for improving the proliferation and differentiation capacity of AT II.

5. The use according to claim 1, characterized in that: in the preparation of a medicament for protecting the mitochondria and lamellar bodies of AT II.

6. Use according to claim 1, characterized in that: in the preparation of a medicament for protecting the stemness potential of AT II.

7. Use according to claim 1, characterized in that: in the preparation of a medicament for improving the function and secretion of AT II.

8. The use according to claim 1, characterized in that: in the preparation of a medicament for maintaining the function of AT II and promoting lung tissue repair.

9. Use according to claim 4, characterized in that: The concentration of the saponin aglycone is 12.5 μM.

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