Application of dipsacus asper saponin VI in regulating and controlling differentiation of surface stem cells

Abstract

According to the application of the dipsacus asper saponin VI in regulating and controlling the differentiation of the surface stem cells, the brand new pharmacological action of the dipsacus asper saponin VI in targeted promotion of the differentiation of the epidermal stem cells is found for the first time, the cognitive limitation of the prior art is broken through, a brand new target spot and direction are provided for research and development of innovative drugs for wound repair, and the application of the dipsacus asper saponin VI in regulating and controlling the differentiation of the epidermal stem cells is realized. A medicine containing teasel saponin VI is adopted, epidermal stem cells are taken as a core target, wound reepithelialization is accelerated by promoting directional differentiation of the epidermal stem cells, the mechanism is clear, the targeting property is high, a core link capable of directly hitting wound healing is adopted, epidermal stem cell differentiation is regulated in a targeted manner, and meanwhile, the medicine has the synergistic effects of resisting inflammation and promoting angiogenesis; the obvious application potential is realized on refractory wounds such as diabetic feet and pressure sores.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically providing the use of Dipsacus saponin VI in regulating the differentiation of surface stem cells. Background Technology

[0002] The mechanisms of action of existing wound healing agents are significantly limited, and their core targets are often misaligned. Current clinically used and investigational wound repair agents primarily focus on indirect auxiliary mechanisms such as anti-infection, anti-inflammation, and angiogenesis promotion, rarely targeting the core mechanism of action of "promoting the directed differentiation of epidermal stem cells and accelerating wound re-epithelialization." This fails to directly address the core aspects of wound healing, resulting in very limited therapeutic effects on refractory wounds such as diabetic foot ulcers and pressure ulcers.

[0003] Existing related technologies mainly fall into two categories: one is based on recombinant epidermal growth factor (EGF), keratinocyte growth factor (KGF), and other differentiation-promoting and wound-repairing agents. These agents can promote epidermal cell proliferation and accelerate wound healing to a certain extent, but they suffer from high production costs, short in vivo half-life, easy inactivation, and potential tumorigenic risks from inducing excessive cell proliferation, thus severely limiting their clinical application scenarios and target populations. The other category is based on traditional Chinese medicine compound wound-healing agents. These agents are mostly total extracts of medicinal materials and can play an auxiliary role in promoting healing through anti-inflammation and improving the wound microenvironment. However, their composition is complex and unclear, quality control is difficult, and there are significant differences in efficacy between batches. The core target and molecular mechanism cannot be clearly identified, making it difficult to pass drug regulatory approval and achieve standardized clinical translation and large-scale application. Furthermore, there is currently no single, clearly defined active ingredient from traditional Chinese medicine that can simultaneously achieve the dual core effects of targeting and promoting epidermal stem cell differentiation and accelerating skin wound healing. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems in the prior art. Therefore, one objective of this invention is to propose the use of Dipsacus saponin VI in regulating epidermal stem cells, thereby solving the problems of existing technologies that use growth factor compound preparations and multi-component traditional Chinese medicine compound preparations to promote epidermal stem cell differentiation, which suffer from complex composition, difficulty in quality control, poor stability, high potential risks, and high costs.

[0005] In a first aspect, the present invention provides the use of compounds in regulating the differentiation of epidermal stem cells, said compounds comprising bisacodyl VI or a pharmaceutically acceptable salt thereof.

[0006] In view of this, the inventors studied the molecular mechanism of the regulation of epidermal stem cell differentiation by Dipsacus saponin VI. The specific research ideas are as follows: (1) Screening of differentially expressed genes: The cells were treated with the optimal concentration of Dipsacus saponin VI and an equal volume of control solvent PBS, respectively. Total RNA was extracted and the transcriptome was sequenced. The differentially expressed genes of the two groups were analyzed. By GO and KEGG enrichment analysis, candidate core genes and signaling pathways related to the regulation of epidermal stem cell differentiation were screened. (2) Verification of candidate targets: The expression of the selected differentiation-related candidate genes was verified by RT-qPCR to confirm their expression changes after stimulation by Dipsacus saponin VI. (3) Confirmation of core regulatory targets: The activity of candidate genes / pathways was regulated by gene overexpression, siRNA knockdown, and intervention with signaling pathway-specific inhibitors. Combined with the expression changes of differentiation marker molecules, the core molecular targets and regulatory pathways mediating the differentiation-promoting effect of Dipsacus saponin VI were confirmed.

[0007] Based on current research findings, this invention addresses the following: 1. GPR30-mediated nongenomic estrogen signaling pathway: Dipsacus saponin VI, as a natural phytoestrogen, can activate the membrane-associated estrogen receptor GPR30, thereby rapidly initiating the downstream MAPK / ERK signaling cascade. This pathway differs from traditional nuclear estrogen receptor regulation pathways and also from the non-specific mechanisms by which existing traditional Chinese medicine components exert their effects through broad-spectrum anti-inflammatory effects. It may precisely regulate the transcriptional activation of epidermal stem cell differentiation marker genes (K10, Involucrin, etc.), achieving targeted regulation of epidermal stem cell differentiation. 2. De-inhibition regulation of the NF-κβ signaling pathway: Dipsacus saponin VI can inhibit the overactivation of the NF-κβ signaling pathway. Unlike the conventional role of this pathway in immune cells, which only regulates the release of inflammatory factors, in epidermal stem cells, this effect can relieve the inhibitory effect of excessive inflammation on epidermal stem cell differentiation in the chronic wound microenvironment. Simultaneously, it can directly regulate the transcription of differentiation-related genes, achieving a synergistic effect of "anti-inflammatory and differentiation-promoting."

[0008] In a second aspect, the present invention provides the use of compounds in the preparation of medicaments that promote the differentiation of epidermal stem cells, said compounds comprising bisacodyl VI or a pharmaceutically acceptable salt thereof.

[0009] In a third aspect, the present invention provides the use of compounds to promote wound healing by facilitating the differentiation of epidermal stem cells, said compounds comprising bisacodyl VI or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments of the present invention, the compound is used to achieve healing of chronic, refractory wounds.

[0011] Furthermore, the chronic, non-healing wounds include lower extremity vascular ulcers, post-traumatic chronic ulcers, diabetic foot ulcers, burns, or pressure ulcers, preferably diabetic foot ulcers or pressure ulcers.

[0012] In a fourth aspect, the present invention provides a pharmaceutical composition comprising dipsacus saponin VI or a pharmaceutically acceptable salt thereof, the pharmaceutical composition being used to promote the differentiation of epidermal stem cells.

[0013] In some embodiments of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable excipients.

[0014] In some embodiments of the present invention, the saponin VI or a pharmaceutically acceptable salt thereof is the sole active ingredient in the pharmaceutical composition.

[0015] Furthermore, the excipients include one or more pharmaceutically acceptable excipients or carriers.

[0016] In some embodiments of the present invention, the dosage form of the pharmaceutical composition includes an injection or a topical dosage form.

[0017] Furthermore, the topical dosage form includes ointments, granules, powders, or liquid preparations.

[0018] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: (1) Outstanding Innovation Value: This invention is the first to discover a novel pharmacological effect of Dipsacus saponin VI in promoting the differentiation of epidermal stem cells, breaking through the limitations of existing technologies and providing a new target and direction for the development of innovative drugs for wound repair. By proposing for the first time to use it in the preparation of a pharmaceutical formulation with both differentiation-promoting and wound-healing effects, it fills the technological gap in this application. This proposal breaks through the limitations of existing technologies in the application of Dipsacus saponin VI, and innovatively combines it with the development of epidermal stem cell-targeted formulations. Therefore, it not only expands the pharmaceutical boundaries of this component, but also provides a new active candidate for the development of wound repair formulations, breaking the raw material limitations of existing formulations. Secondly, this invention clarifies that its core mechanism of action is to target and regulate the differentiation of epidermal stem cells and accelerate wound re-epithelialization, while also having a synergistic effect of anti-inflammatory and angiogenesis-promoting effects. It clarifies the core target and pharmacological pathway of this component in promoting healing, rather than a non-specific broad-spectrum effect. Therefore, it provides clear theoretical guidance for the optimized development of formulations, and at the same time significantly improves the compliance and approval probability of drug application for formulations.

[0019] (2) Significant industrialization advantages: This invention uses a single active ingredient, Dipsacus saponin VI, which has a clear structure and controllable quality. The raw materials are major local medicinal materials in my country, which can be produced on a large scale and in a standardized manner. The raw materials are widely available, the extraction process is mature, and the pharmaceutical safety has been fully verified. The clinical transformation path is clear and the barriers are extremely low, which greatly reduces the research and development and production costs.

[0020] (3) Excellent therapeutic effect: The present invention uses a drug including Dipsacus saponin VI, with epidermal stem cells as the core target. It accelerates wound re-epithelialization by promoting their directional differentiation. The mechanism is clear and the targeting is strong. It can directly target the core link of wound healing, regulate the differentiation of epidermal stem cells, and at the same time has the synergistic effect of anti-inflammatory and angiogenesis promotion. It has significant application potential for difficult-to-heal wounds such as diabetic foot and pressure ulcers.

[0021] (4) Wide clinical applicability: The preparation containing Dipsacus saponin VI of this invention has high safety and stable physicochemical properties. It does not require cold chain storage and can be developed into a variety of topical dosage forms, covering various clinical treatment scenarios for acute and chronic skin wounds. It has broad market application prospects. In particular, the pharmaceutical preparation with Dipsacus saponin VI as the sole active ingredient has both the ability to promote epidermal stem cell differentiation and wound healing. It can be adapted to a variety of topical dosage forms and has the characteristics of clear composition, stable and controllable, safe and efficient. Because this preparation uses a single and clear small molecule active ingredient, it fundamentally solves the core pain points of existing preparations. Attached Figure Description

[0022] Figure 1 This is a diagram showing the identification results of mouse epidermal stem cells in Example 1 of the present invention; Figure 2 This is a graph showing the detection results of the proliferative activity of Dipsacus saponin VI on epidermal stem cells in Example 2 of the present invention; Figure 3 This is a graph showing the results of the activity of Dipsacus saponin VI on epidermal stem cell differentiation at different time points in Example 2 of the present invention. Figure 4 This is a graph showing the screening results of the optimal concentration of Dipsacus saponin VI for promoting epidermal stem cell differentiation in Example 2 of the present invention. Figure 5 This is a diagram showing the in vivo efficacy of Dipsacus saponin VI in promoting skin wound healing in mice, as described in Example 3 of this invention. Detailed Implementation

[0023] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way.

[0024] Example 1 Isolation, culture and purity identification of mouse epidermal stem cells (1) Using a two-step digestion method of neutral protease + trypsin, combined with the rapid adhesion method of type IV collagen, primary epidermal stem cells were isolated from the back skin tissue of SPF grade C57BL / 6 mice and placed in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin antibody. The cells were cultured in a 37°C, 5% CO2 incubator and the second generation cells were used for subsequent experiments.

[0025] (2) Cell purity identification: ① Cell immunofluorescence staining was used to detect the expression of epidermal stem cell characteristic markers β1 integrin and keratin 19 (CK19); ② Flow cytometry was used to double-label the expression of stemness marker α6 integrin and differentiation marker CD71 to verify cell stemness and purity and ensure that the experimental requirements were met.

[0026] To observe the effect of Dipsacus saponin VI on the differentiation of mouse epidermal stem cells, epidermal stem cells were isolated and cultured from mouse skin tissue, and the cells were identified by detecting epidermal stem cell marker molecules. The results showed that the primary cultured epidermal stem cells were arranged in a pebble-like pattern under a light microscope. Figure 1 (A, morphology of epidermal stem cells under a light microscope). Flow cytometry analysis of the expression of epidermal stem cell surface markers α6 integrin and CD71 revealed that 94.6% of cultured epidermal stem cells highly expressed α6 integrin while expressing low levels of CD71. Figure 1 B. Flow cytometry was used to detect the expression of α6 integrin and CD71 in epidermal stem cells. Immunofluorescence staining showed that 99% of these cells simultaneously expressed the epidermal stem cell markers CK19 and β1 integrin (B). Figure 1 The expression of epidermal stem cell markers CK19 (red) and β1 integrin (green) was detected by immunofluorescence staining, and DAPI was used to stain the cell nuclei. The figure shows representative images of the results from three experiments (scale bar = 50 μm). These results demonstrate that high-purity epidermal stem cells were obtained in this embodiment.

[0027] Example 2 In vitro efficacy verification of Dipsacus saponin VI on epidermal stem cell proliferation and differentiation (1) Proliferation activity detection: Dipsacin VI (solvent PBS) was set at a gradient concentration of 0~40μmol / L to stimulate epidermal stem cells for 24h, 48h and 72h respectively. Cell proliferation activity was detected by MTT assay to screen for safe concentration ranges that do not have cytotoxicity and do not affect normal cell proliferation.

[0028] See results Figure 2The results showed that: (1) ASAVI had no significant cytotoxicity to mouse epidermal stem cells within the concentration range of 0~40 μmol / L, and could be used for subsequent in vitro cell experiments; (2) Compared with the control group, different concentrations of ASAVI significantly inhibited the proliferation of mouse epidermal stem cells. (This also indirectly confirms the differentiation-promoting effect of ASAVI, because stem cells lose stemness and proliferative capacity after differentiation begins.) (2) Differentiation activity time gradient screening: Cells were stimulated with the above-mentioned safe concentration (40 μmol / L) of Dipsacus saponin VI, and samples were collected at 24h, 48h and 72h respectively. The mRNA expression of epidermal differentiation marker molecules K10, Involucrin, Loricrin and Filaggrin was detected by RT-qPCR to determine the optimal time for promoting differentiation.

[0029] For specific results, please refer to... Figure 3 ,from Figure 3 It can be seen that the mRNA expression of related genes changes over time, and the gene expression generally increases over time.

[0030] (3) Differentiation activity concentration gradient screening: According to the optimal action time, a gradient concentration of Dipsacus saponin VI was set to stimulate cells, and the expression of the above differentiation marker molecules was detected by RT-qPCR to determine the optimal concentration for promoting differentiation.

[0031] Epidermal stem cells (ESCs) were stimulated for 3 days with different concentrations of Dipsacus saponin VI, using 2 μM epigallocatechin gallate (EGCG) as a positive control. Immunofluorescence was used to detect the expression of filaggrin (FLG), a differentiation marker of ESCs. The results showed that both EGCG and 1-40 μM Dipsacus saponin VI promoted filaggrin expression, with the differentiation-promoting effect of Dipsacus saponin VI reaching its maximum at 20 μM (see [link to product description]). Figure 4 , Figure 4 In this study, mouse epidermal stem cells were stimulated for 3 days with different concentrations of Dipsacus saponin VI, an equal volume of solvent (PBS), or 2 mM epigallocatechin gallate (EGCG). Immunofluorescence staining was used to detect filaggrin (FLG), a cell differentiation marker. These results indicate that Dipsacus saponin VI can promote epidermal stem cell differentiation.

[0032] Example 3 The in vivo efficacy of Dipsacus saponin VI in promoting skin wound healing in mice can be found in [reference needed]. Figure 5 .

[0033] In vivo efficacy and optimal concentration screening for promoting healing: SPF-grade C57BL / 6 mice were used to establish a full-thickness skin defect model with a diameter of 1.2 cm on their backs. The mice were randomly divided into a PBS control group and three groups treated with different concentrations of Dipsacus saponin VI, with 6 mice in each group. The drug was applied to the wound immediately after modeling, once a day. Wound photos were taken every 2 days. The wound area was calculated using ImageJ, and the healing rate of each group was statistically analyzed using GraphPad to determine the optimal concentration for promoting healing in vivo.

[0034] Full-thickness skin lesions were prepared in mice, and different concentrations of Dipsacus saponin VI or a control solvent (PBS) were applied to the wounds. Wound healing was observed. Results showed that 10, 100, and 200 μg / mL of Dipsacus saponin VI significantly promoted skin wound healing, with 10 μg / mL showing the strongest effect. Figure 5 Representative images of mouse skin wounds treated with different concentrations of Dipsacus saponin VI (ASAVI) or an equal volume of PBS at different time points. Figure 5 Changes in A) and wound area ( Figure 5 (B), n=6 mice / group, data are mean ± standard deviation. **p<0.01, ***p<0.001, compared with control mice at the same time point).

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of the compound in regulating epidermal stem cell differentiation, characterized in that, The compounds include dipsaponin VI or a pharmaceutically acceptable salt thereof.

2. The use of the compound in the preparation of a drug that promotes the differentiation of epidermal stem cells, characterized in that, The compounds include dipsaponin VI or a pharmaceutically acceptable salt thereof.

3. The use of the compound to promote wound healing by facilitating the differentiation of epidermal stem cells, characterized in that, The compounds include dipsaponin VI or a pharmaceutically acceptable salt thereof.

4. The use according to claim 3, characterized in that, The compound is used to achieve healing of chronic, refractory wounds.

5. The use according to claim 4, characterized in that, The chronic, non-healing wounds include lower extremity vascular ulcers, post-traumatic chronic ulcers, diabetic foot ulcers, burns, or pressure ulcers, with diabetic foot ulcers or pressure ulcers being preferred.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises Dipsacin VI or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition is used to promote the differentiation of epidermal stem cells.

7. The pharmaceutical composition according to claim 6, characterized in that, Further includes: Pharmaceutically acceptable excipients; And / or, the saponin VI or a pharmaceutically acceptable salt thereof is the sole active ingredient in the pharmaceutical composition.

8. The pharmaceutical composition according to claim 7, characterized in that, The excipients include one or more pharmaceutically acceptable excipients or carriers.

9. The pharmaceutical composition according to any one of claims 6-8, characterized in that, The dosage form of the pharmaceutical composition includes injections or topical formulations.

10. The pharmaceutical composition according to claim 9, characterized in that, The external dosage forms include ointments, granules, powders, or liquid preparations.

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