Use of spina date seed saponin B in preparation of medicine for inhibiting skin scar hyperplasia

Abstract

The application discloses application of spina date seed saponin B in preparation of a medicine for inhibiting skin scar hyperplasia, and belongs to the technical field of medicines. The application finds that the spina date seed saponin B can directly combine and inhibit focal adhesion kinase (FAK), down-regulate the FAK / AKT / mTOR signal path, and then activate cell autophagy, reduce excessive deposition of extracellular matrix such as type I collagen, so as to effectively inhibit skin scar hyperplasia, reduce scar uplift and promote scar tissue remodeling. The spina date seed saponin B can be used alone or in combination with other active ingredients to prepare a medicine for treating skin scars such as hyperplastic scars, keloids, atrophic scars and the like, and provides a new, safe and effective medicine selection for treatment of skin scars.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of jujube seed saponin B in the preparation of drugs that inhibit the proliferation of skin scars. Background Technology

[0002] Hypertrophic scars are a common result of abnormal healing after skin injury. Hypertrophic scars and keloids are characterized by excessive proliferation of fibroblasts, differentiation of myofibroblasts, and excessive deposition of extracellular matrix (ECM) components such as type I collagen after damage to the dermal reticular layer. They often lead to functional impairment, itching, contractures, and significant psychological burden.

[0003] The pathogenesis of hypertrophic scars involves multiple cells, cytokines, and signaling pathways. Fibroblasts and their differentiated myofibroblasts play a central role in scar formation by driving excessive collagen synthesis and tissue contraction, manifested by the upregulation of fibrosis markers such as type I collagen (COL1A1, COL1A2) and α-smooth muscle actin (ACTA2). Studies have shown that focal adhesion kinase (FAK) mediates pro-fibrosis signals (such as the FAK-ERK-MCP-1 pathway) through mechanotransduction, and inhibiting FAK can alleviate fibrosis. Simultaneously, autophagy dysregulation is also closely related to scar progression, and activating autophagy can achieve effective scar treatment. Therefore, targeting FAK and regulating autophagy is a rational strategy for intervening in hypertrophic scars.

[0004] Jujuboside B (JUB) is a triterpenoid saponin derived from the dried, mature seeds of jujube (Ziziphus jujuba), with the molecular formula C. 52 H 84 O 21 With a molecular weight of 1045.21, jujuboside B has been reported to possess anti-inflammatory, antioxidant, anti-apoptotic, and anti-fibrotic pharmacological activities in disease models including liver injury, lung injury, and tumors. However, the use of jujuboside B for the treatment of skin scars, especially hypertrophic scars, is not disclosed in existing technologies. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings and defects of the prior art, the present invention provides a new use for jujube seed saponin B, for the preparation of a drug that inhibits the proliferation of skin scars.

[0006] Technical solution: The application of jujube seed saponin B of the present invention in the preparation of drugs that inhibit the proliferation of skin scars.

[0007] The scars mentioned are skin scars, including hypertrophic scars, keloids, atrophic scars, or flat scars.

[0008] The drug is a topical preparation, which may be an ointment, cream, gel, lotion, spray, patch, or transdermal drug delivery preparation.

[0009] The drug is an injectable preparation, which is a subcutaneous or intra-scar injection preparation.

[0010] The drug also contains a second active ingredient, which is at least one of glucocorticoids, 5-fluorouracil, or retinoic acid drugs.

[0011] The concentration of jujuboside B in the drug is below 1 nM. In in vitro experiments, jujuboside B inhibited the expression of COL1A1, COL1A2 and ACTA2 in human proliferative scar fibroblasts in a dose-dependent manner within the concentration range of 1 to 100 nM, with a significant inhibitory effect observed at a concentration of 1 nM.

[0012] The drug also contains a pharmaceutically acceptable carrier, which includes at least one of a thickener, emulsifier, penetration enhancer, humectant, preservative, and pH adjuster.

[0013] This invention utilizes a mechanism to inhibit collagen synthesis by targeting FAK: jujube seed saponin B directly binds to and inhibits focal adhesion kinase (FAK), reducing FAK protein levels and its phosphorylation at the Y397 site. This leads to downregulation of the FAK / AKT / mTOR signaling axis, activation of autophagy (manifested as an increased LC3-II / LC3-I ratio, p62 degradation, and upregulation of Beclin-1), and simultaneously increases intracellular reactive oxygen species (ROS) levels. Ultimately, this inhibits excessive deposition of extracellular matrix components such as type I collagen and reduces scar hyperplasia. These effects are not achieved by influencing fibroblast proliferation, apoptosis, or migration, suggesting specificity for the collagen synthesis pathway.

[0014] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention is the first to discover and confirm that jujube seed saponin B can inhibit scar hyperplasia by directly targeting FAK. 1) Clear and targeted mechanism: Cell thermal displacement analysis (CETSA) confirmed that jujube seed saponin B directly binds to FAK, and molecular docking further supports the strong binding potential between the two. FAK overexpression can reverse the anti-fibrotic effect of jujube seed saponin B, proving that it works by specifically targeting FAK. 2) Extremely low effective concentration and good safety: Jujube seed saponin B inhibits the expression of fibrosis markers in a dose-dependent manner at a nanomolar (nM) concentration, which is far lower than the micromolar (μM) concentration used in existing technologies, and has no significant effect on cell proliferation, apoptosis, and migration, suggesting its high activity, wide therapeutic window, and good safety. 3) Clear pathway: Jujube seed saponin B inhibits the FAK / AKT / mTOR signaling axis, activates autophagy, and moderately increases ROS, synergistically inhibiting excessive collagen deposition without disrupting mitochondrial membrane potential. The mechanism of action is self-consistent and controllable. 4) In vivo efficacy confirmation: In a rabbit ear hypertrophic scar model, subcutaneous administration of jujube seed saponin B can significantly reduce scar area, decrease collagen deposition and scar elevation index, confirming its definite in vivo anti-scar efficacy and practicality.

[0015] In vitro experiments showed that jujube seed saponin B, at nanomolar concentrations, dose-dependently inhibited the expression of type I collagen (COL1A1, COL1A2) and α-smooth muscle actin (ACTA2) in human hypertrophic scar fibroblasts without affecting cell proliferation, apoptosis, or migration. In a rabbit ear hypertrophic scar model, subcutaneous administration of jujube seed saponin B significantly reduced scar area, decreased collagen deposition, and lowered the scar elevation index. Therefore, jujube seed saponin B can be used to prepare drugs that inhibit scar hyperplasia, especially hypertrophic scars. It can be used alone or in combination with other active ingredients to formulate topical or injectable preparations, offering safety, efficacy, and convenient administration, providing a new drug option for the prevention and treatment of hypertrophic scars. Attached Figure Description

[0016] Figure 1 This is a schematic diagram showing the source and chemical structure of jujube seed saponin B in Example 1; Figure 2 This is a diagram showing the dose-dependent inhibitory effect of jujube seed saponin B on the expression of COL1A1, COL1A2 and ACTA2 mRNA in Example 1. Figure 3 This is a graph showing the effect of jujube seed saponin B on the expression of COL1A1, COL1A2, and ACTA2 proteins in Example 1; where... Figure 3 In the image, A represents the bands obtained from Western blot analysis of COL1A1, COL1A2, and ACTA2 protein expression. Figure 3B in the figure represents a quantitative analysis of the relative expression levels of COL1A1, COL1A2, and ACTA2 proteins. Figure 4 The graph shows the effect of different concentrations of jujube seed saponin B on cell viability as detected by the CCK-8 assay in Example 1. Figure 5 This is a flow cytometry image of apoptosis detected in Example 1; wherein, Figure 5 In the diagram, A represents the flow cytometry scatter plot of apoptosis in the control group. Figure 5 In the figure, B represents the flow cytometry scatter plot of apoptosis in the group treated with 100 nM jujuboside B. Figure 5 C in the figure represents a quantitative analysis of the apoptosis rate of the two groups of cells; Figure 6 This is a cell migration map detected by the scratch assay in Example 1; wherein, Figure 6 In the image, A represents a representative microscopic image of scratch healing at different time points; Figure 6 B in the figure represents a quantitative analysis of the scratch healing rate; Figure 7 This is a graph showing the effect of jujube seed saponin B treatment on the levels of FAK and pyruvate carboxylase (PC) proteins in Example 2; where, Figure 7 In the image, A represents the bands obtained from Western blot analysis of FAK and PC protein levels. Figure 7 B in the figure represents the quantitative analysis of the relative expression levels of FAK and PC proteins; Figure 8 This is a diagram illustrating the reversal effect of FAK overexpression on the downregulation of COL1A1 and ACTA2 mediated by jujuboside B in Example 2; where, Figure 8 In the image, A represents the bands representing the expression of COL1A1, ACTA2, and FAK proteins detected by Western blot. Figure 8 B in the graph represents the quantitative analysis of its relative expression level; Figure 9 This is a molecular docking model diagram of jujube seed saponin B and FAK in Example 2; Figure 10 This is a cellular thermal displacement analysis (CETSA) diagram illustrating the interaction between jujube seed saponin B and FAK in Example 2; where, Figure 10 In the image, A represents the Western blot bands of the FAK protein at different temperatures; Figure 10 B in the graph represents the relative expression level of FAK protein as a function of temperature. Figure 11 This is a graph showing the intracellular ROS levels after treatment with jujube seed saponin B in Example 3; where, Figure 11 In the diagram, A represents a bright-field observation of cells; Figure 11 In the image, B represents the fluorescence pattern of intracellular ROS detected by the DCFH-DA fluorescent probe. Figure 11C in the figure represents the overlay of bright field and fluorescence. Figure 11 D in the figure represents a quantitative analysis of the relative fluorescence intensity of intracellular ROS. Figure 12 This is a diagram of autophagy induced by jujube seed saponin B in Example 3; wherein, Figure 12 In the image, A represents the fluorescence of autolysosomes under the mCherry channel; Figure 12 In the image, B represents the fluorescence of autophagosomes under the EGFP channel; Figure 12 C in the image represents the overlay of two channels; Figure 12 In the figure, D represents a quantitative analysis of the number of autophagosomes and autolysosomes; Figure 13 This is a graph showing the effect of jujube seed saponin B on the levels of phosphorylated FAK and AKT proteins in Example 3; where, Figure 13 In the image, A represents the bands obtained by Western blot analysis of phosphorylated FAK (p-FAK) and AKT protein levels. Figure 13 B in the figure represents the quantitative analysis of the relative expression levels of p-FAK and AKT proteins; Figure 14 This is a graph showing the effect of jujube seed saponin B on the expression of autophagy-related proteins in Example 3; where, Figure 14 In the image, A represents the bands obtained by Western blot analysis of p-mTOR, mTOR, Beclin-1, LC3-II / I, and integrin β1 protein expression. Figure 14 B in the figure represents the quantitative analysis of the relative expression levels of the above proteins; Figure 15 This is a schematic diagram illustrating the animal model construction and drug administration time in Example 4; Figure 16 This is an observation diagram of the scar area after treatment with jujube seed saponin B in Example 4; Figure 17 This is a HE staining image from Example 4; where, Figure 17 In the image, A represents the low-power observation of scar tissue stained with HE in the control group and the jujube seed saponin B group; Figure 17 B in the image is a high-power view of the scar tissue stained with HE. Figure 18 This is a Masson trichrome staining image of the scar tissue in Example 4; Figure 19 This is a graph showing the scar elevation index and its quantitative analysis in Example 4; where, Figure 19 In the figure, A represents the quantitative analysis diagram of the relative intensity of Masson staining; Figure 19 B in the figure represents the quantitative analysis of the scar elevation index (SEI); Figure 20 This is a graph showing the change of relative scar area over time for the two groups in Example 4. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to embodiments. It should be understood that the following embodiments are only for illustrative purposes and are not intended to limit the scope of protection of the present invention. All equivalent substitutions or improvements made based on the essential content of the present invention should fall within the scope of protection claimed by the present invention.

[0018] The jujube seed saponin B used in this invention is an analytical grade standard (purity ≥98%), purchased from Aladdin (Shanghai), and can also be prepared according to conventional methods in the art. The cells used in this invention are human hypertrophic scar fibroblasts (HSF) isolated from surgically excised human hypertrophic scar tissue.

[0019] Example 1:

[0020] This embodiment was used to investigate the effect of jujube seed saponin B on the expression of fibrosis markers in proliferative scar fibroblasts in vitro.

[0021] Experimental Methods: Human hypertrophic scar tissue (donor number ≥ 3) was collected and digested with 0.25% (w / v) dispase at 4℃ for 16 h, followed by digestion with 0.1% (w / v) collagenase I at 37℃ for 4 h. After filtration and centrifugation, the tissue was cultured in fibroblast culture medium FM (ScienCell) with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin added. The culture was incubated at 37℃ in a 5% CO2 incubator, and cells from passages 3 to 6 were used for experiments. Jujube seed saponin B was dissolved in DMSO (final concentration ≤ 0.1% (v / v)). A DMSO solvent control group and treatment groups with different concentrations of jujube seed saponin B (1, 25, 50, 75, 100 nM) were set up. The mRNA expression of COL1A1, COL1A2, and ACTA2 was detected by real-time quantitative PCR (18S rRNA as internal control, calculated using the 2-ΔΔCt method), and the protein expression of COL1A1, COL1A2, and ACTA2 was detected by Western blot. Cell viability was detected by CCK-8 assay, apoptosis was detected by Annexin V-FITC / PI double staining flow cytometry, and cell migration was detected by scratch assay. Result: As Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 As shown, the results indicate that, compared with the DMSO control group, jujube seed saponin B significantly downregulated the mRNA expression of COL1A1, COL1A2, and ACTA2 at a concentration of 1 nM, and this downregulation was dose-dependent within the concentration ranges of 1, 25, 50, 75, and 100 nM. Figure 2Western blot results further confirmed that the protein levels of COL1A1, COL1A2, and ACTA2 decreased accordingly after 48 h of treatment with jujube seed saponin B. Figure 3 However, CCK-8 assays and Annexin V-FITC / PI flow cytometry showed that there was no significant difference in cell viability and apoptosis rate between the 100 nM jujuboside B treatment group and the control group. Figure 4 , Figure 5 Scratch assay showed that 100 nM jujuboside B had no inhibitory effect on HSF migration. Figure 6 The above results suggest that jujube seed saponin B can specifically inhibit the expression of fibrosis markers at nanomolar concentrations without affecting fibroblast proliferation, apoptosis, and migration, demonstrating specificity for the collagen synthesis pathway.

[0022] Example 2:

[0023] This embodiment is used to investigate the direct protein target of jujube seed saponin B and its binding and regulatory effect on FAK.

[0024] Experimental Methods: Based on previous research and literature, two key proteins in scar hyperplasia, FAK and pyruvate carboxylase (PC), were selected for investigation. HSF cells were treated with 100 nM jujuboside B for 48 h, and the levels of FAK and PC proteins were detected by Western blot. To further verify the functional relevance of FAK, HSF cells were divided into four groups: solvent control group, 100 nM jujuboside B group, pcDNA3.1 empty vector + jujuboside B group, and pcDNA3.1-PTK2 (FAK) overexpression plasmid + jujuboside B group. Cells were transfected with Lipofectamine 3000, and the medium was changed and 100 nM jujuboside B was added 6 h after transfection. After 48 h, cells were collected for Western blot detection of COL1A1 and ACTA2 expression. Molecular docking was used to analyze the binding potential of jujuboside B to FAK. Direct binding was verified using cell thermal shift analysis (CETSA): HSF was resuspended in PBS containing a mixture of 1% (v / v) protease inhibitors and incubated with 10 μM jujuboside B or an equal amount of DMSO at 37°C for 2 h. After aliquoting, the mixture was heated at 37–57°C (3°C intervals) for 3 min. After cooling, the mixture was lysed by three freeze-thaw cycles, centrifuged, and the supernatant was collected. The thermal stability of FAK protein was detected by Western blot.

[0025] Result: As Figure 7 , Figure 8 , Figure 9 , Figure 10 As shown, the results indicate that treatment with 100 nM jujube seed saponin B significantly reduced the levels of FAK and PC proteins. Figure 7FAK overexpression significantly reversed the downregulation of COL1A1 and ACTA2 mediated by jujuboside B. Figure 8 This demonstrates that FAK is a key target for the anti-fibrotic effect of jujuboside B. Molecular docking shows a strong binding potential between jujuboside B and FAK. Figure 9 CETSA results showed that treatment with 10 μM jujube seed saponin B caused a significant thermally stable shift in the FAK protein. Figure 10 Experiments have confirmed that jujube seed saponin B directly binds to FAK.

[0026] Example 3:

[0027] This embodiment was used to investigate the effects of jujube seed saponin B on intracellular ROS, autophagy, and the FAK / AKT / mTOR signaling pathway.

[0028] Experimental Methods: Intracellular reactive oxygen species (ROS) were detected using the DCFH-DA probe: HSF was treated with jujuboside B or a solvent control in 96-well plates (70% confluence) for 24 h, followed by incubation at 37℃ for 30 min in the dark with 10 μM DCFH-DA. Imaging was performed using a fluorescence microscope (488 nm excitation / 525 nm emission), and the average fluorescence intensity was quantified using ImageJ. Autophagic flux was detected using the mCherry-EGFP-LC3B dual fluorescent plasmid: HSF was transfected with pBobi-mCherry-EGFP-LC3B plasmid (0.5 μg / well) on coverslips of 24-well plates using Lipofectamine 3000. After 24 h, the plates were treated with 100 nM jujuboside B or a control for 24 h. Imaging was performed using a laser confocal microscope, and the autophagic flux was quantified using the autolysosome / autophagosome ratio. Western blot was used to detect phosphorylated FAK (p-FAK), AKT, phosphorylated mTOR (p-mTOR), mTOR, p62, Beclin-1, and the LC3-II / LC3-I ratio. Mitochondrial membrane potential (MMP) was detected using the JC-1 fluorescent probe.

[0029] Result: As Figure 11 , Figure 12 , Figure 13 , Figure 14 As shown, the results indicate that jujube seed saponin B can significantly increase intracellular ROS levels ( Figure 11 ), and induce autophagy ( Figure 12 Western blot further confirmed that jujube seed saponin B reduced the levels of phosphorylated FAK and AKT proteins. Figure 13 ), and lowered p-mTOR, mTOR and p62, and increased Beclin-1 and the LC3-II / LC3-I ratio ( Figure 14Flow cytometry and JC-1 fluorescence microscopy both showed that jujube seed saponin B did not alter mitochondrial membrane potential. These results suggest that jujube seed saponin B exerts its anti-fibrotic effect by inhibiting the FAK / AKT / mTOR pathway, activating autophagy, and moderately increasing ROS, without disrupting mitochondrial integrity.

[0030] Example 4:

[0031] This embodiment is used to investigate the therapeutic effect of jujube seed saponin B on a rabbit ear hypertrophic scar model in vivo.

[0032] Experimental Methods: Six male New Zealand White rabbits (2.5–3.0 kg, 8–10 weeks old) were acclimatized for one week. Under intravenous anesthesia with sodium pentobarbital (30 mg / kg), six full-thickness circular wounds (1 cm in diameter, preserving the perichondrium) were made on the ventral side of each rabbit's ear using a biopsy punch. The wounds were covered with Vaseline gauze, cleaned with saline, and changed until scabs formed (approximately 14 days). On days 14, 21, 28, and 35 after modeling, 100 μL of jujube seed saponin B (400 nM, dissolved in sterile PBS containing 0.1% (v / v) DMSO) or a solvent control was injected subcutaneously around the wounds. On day 42, the animals were sacrificed, and scar tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and prepared into 5 μm sections. Hematoxylin and eosin (HE) and Masson's trichrome staining were performed to evaluate scar area, collagen deposition, and scar elevation index (SEI).

[0033] Result: As Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 19 , Figure 20 As shown, the results indicated that, compared with the control group, the scar area in the jujube seed saponin B treatment group was significantly reduced on days 35 and 42 after modeling. Figure 16 On day 42, HE and Masson's trichrome staining showed that collagen deposition (blue) was significantly reduced in the jujube seed saponin B treatment group. Figure 17 , Figure 18 The scar elevation index was significantly reduced in the jujube seed saponin B group (). Figure 19 , Figure 20 The above results suggest that subcutaneous administration of jujube seed saponin B can significantly inhibit scar hyperplasia, reduce collagen deposition, and decrease scar elevation, demonstrating a definite in vivo anti-scarring effect.

[0034] Example 5:

[0035] This embodiment is an illustrative example. 0.001%~1% (recommended 0.01%~0.1%) of jujube seed saponin B is weighed by weight and mixed with a pharmaceutically acceptable carrier (including at least one of thickeners, emulsifiers, penetration enhancers, humectants, preservatives, and pH adjusters), and prepared into an ointment, cream, or gel according to conventional processes in the art. The resulting preparation is for external skin application to the scar site 1-2 times daily. Those skilled in the art can also, based on the essence of this invention, prepare lotions, sprays, patches, transdermal drug delivery preparations, or subcutaneous / intra-scar injection preparations, or use them in combination with other active ingredients (such as glucocorticoids, 5-fluorouracil, or retinoids).

[0036] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of jujube seed saponin B in the preparation of drugs that inhibit skin scar hyperplasia.

2. The application according to claim 1, characterized in that: The skin scars mentioned are hypertrophic scars, keloids, atrophic scars, or flat scars.

3. The application according to claim 1, characterized in that: The drug is a topical preparation, which may be an ointment, cream, gel, lotion, spray, patch, or transdermal drug delivery preparation.

4. The application according to claim 1, characterized in that: The drug is an injectable preparation, which is a preparation for subcutaneous or intra-scar injection.

5. The application according to claim 3 or 4, characterized in that: The drug also contains a second active ingredient, which is at least one of glucocorticoids, 5-fluorouracil, or retinoic acid drugs.

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