Application of medicine prepared from irisin and hydrogel of irisin in diabetic skin wound repair

Abstract

The invention discloses an irisin hydrogel and an application of the irisin hydrogel in diabetic skin wound repair. The tectoridin hydrogel is prepared by combining the egg white matrix with tectoridin through a specific process, and has good slow release performance. Experiments prove that the tectoridin hydrogel can significantly accelerate healing of skin wounds of diabetic mice, and the action mechanism of the tectoridin hydrogel is to promote polarization of macrophages to M2 type and up-regulate expression of a vascular endothelial growth factor Vegf and an anti-inflammatory factor IL-10, thereby effectively promoting angiogenesis, collagen deposition and tissue reconstruction. The invention provides a novel treatment strategy for the refractory skin wound of diabetes mellitus.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field and discloses the application of irisin and its hydrogel preparation in the repair of diabetic wounds. Background Technology

[0002] Diabetic patients exposed to long-term high glucose levels experience chronic inflammation, impaired immune function, and impaired angiogenesis, leading to difficulties in skin wound repair. The core pathological mechanism lies in the disordered polarization of macrophages under high glucose conditions—the persistent presence of the pro-inflammatory M1 type, which fails to effectively convert to the reparative M2 type, resulting in persistent inflammation and hindered tissue regeneration. Existing treatments, such as growth factor and stem cell therapies, while showing some efficacy, suffer from issues such as unstable efficacy, high costs, or complex techniques.

[0003] Irisin is a small polypeptide secreted by muscle cells during exercise. It is an extracellular segment formed by the cleavage of protein 5, a component of type III fibronectin. Numerous studies have shown that irisin can improve metabolic disorders such as obesity and non-alcoholic fatty liver disease by regulating the browning of white adipose tissue. Furthermore, previous research by the authors of this invention has found that irisin treatment can significantly promote M2 polarization of macrophages and secrete large amounts of anti-inflammatory factors. However, whether irisin can promote the repair of diabetic wounds has not yet been investigated. Summary of the Invention

[0004] The present invention aims to provide an application of irisin and its hydrogel preparation in the repair of diabetic wounds, in order to solve the problems of unstable efficacy and high cost of existing diabetic wound treatment methods.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing an irisin hydrogel includes the following steps: S1. Take fresh egg white and add an equal amount of deionized water. Stir well at 4℃. Remove the white flocculent precipitate through a dialysis bag. Centrifuge at 10000rpm for 10min to remove the insoluble precipitate. Freeze-dry the supernatant for storage. S2. Prepare a 2.5 wt% NaOH aqueous solution and a 20 wt% egg white solution, and dissolve irisin in the egg white solution to obtain an irisin egg white solution with a concentration of 16 μg / mL. S3. Mix the irisin egg white solution and sodium hydroxide solution from step S2 at a ratio of 62.5%:37.5%, stir evenly, and let it stand in a mold with a thickness of 1.0 μm to form a gel; so that the concentration of irisin in the hydrogel is 10 μg / mL. S4. The hydrogels obtained in step S3 were placed in 1.5 g glycerol, glycerol containing 0.05 M CuSO4·5H2O, and glycerol containing 0.05 M ZnSO4·7H2O, respectively, and soaked for 10 min each. The excess liquid on the surface was removed with filter paper, and the reaction was promoted at 45℃ for 30 min. Finally, the hydrogels were cut to prepare irisin hydrogels with a diameter of 1 cm and a thickness of 1 μm. The irisin content in each hydrogel was 2.5 μg.

[0006] Furthermore, this hydrogel is used in the repair of diabetic refractory skin wounds.

[0007] Furthermore, the hydrogel uses endogenous irisin as its main component, and enhances the repair of diabetic skin wounds through targeted drug delivery.

[0008] The principle and beneficial effects of this technical solution: The drug of this invention uses irisin as the main component, and enhances the repair of diabetic skin wounds through targeted drug delivery via hydrogel, which is safe and highly effective. Subsequent experiments have demonstrated that the irisin hydrogel of this invention can effectively promote the secretion of large amounts of vascular endothelial growth factor (VEGF) and anti-inflammatory factor IL-10 by macrophages, and enhance the proliferation capacity of vascular endothelial cells and skin fibroblasts. After wound excision in diabetic mice, treatment with irisin significantly promotes angiogenesis and skin tissue reconstruction, and the wound healing speed is significantly accelerated. Attached Figure Description

[0009] Figure 1 This is a graph showing the cumulative release rate of irisin according to the present invention; Figure 2 This is a schematic diagram of the animal experiment protocol for an example. Figure 3 The following are the results of skin wound healing in diabetic mice in an animal experiment as an example. A shows representative pictures and traces of wound healing during the treatment process in mice, and BC are statistical charts of the wound healing rate in mice. Figure 4 The following are the pathological changes of wounds in diabetic mice in animal experiments as an example. A shows the H&E and Masson staining of mouse wound tissue on day 7, B is a statistical graph of wound width, and C is a statistical graph of collagen deposition. Figure 5 To illustrate angiogenesis in diabetic mice during an animal experiment, A shows an immunofluorescence staining image of CD31, and B shows a quantitative statistical graph of CD31 immunofluorescence staining. Figure 6 The image shows the macrophage phenotype in the wound area of ​​diabetic mice in an animal experiment. A and C represent the relative mRNA expression levels of CD206, Vegf, and IL-10 in the wound tissue, as determined by RT-PCR. Detailed Implementation

[0010] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments: Preparation of irisin hydrogel and determination of drug release efficiency The preparation process of irisin hydrogel is as follows: S1. Take fresh egg white and add an equal amount of deionized water. Stir well at 4℃. Remove the white flocculent precipitate through a dialysis bag. Centrifuge at 10000rpm for 10min to remove the insoluble precipitate. Freeze-dry the supernatant for storage. S2. Prepare a 2.5 wt% NaOH aqueous solution and a 20 wt% egg white solution, and dissolve irisin in the egg white solution to obtain an irisin egg white solution with a concentration of 16 μg / mL. S3. Mix the irisin egg white solution and sodium hydroxide solution from step S2 at a ratio of 62.5%:37.5%, stir evenly, and let it stand in a mold with a thickness of 1.0 μm to form a gel; so that the concentration of irisin in the hydrogel is 10 μg / mL. S4. The hydrogels obtained in step S3 were placed in 1.5 g glycerol, glycerol containing 0.05 M CuSO4·5H2O, and glycerol containing 0.05 M ZnSO4·7H2O, respectively, and soaked for 10 min each. The excess liquid on the surface was removed with filter paper, and the reaction was promoted at 45℃ for 30 min. Finally, the hydrogels were cut to prepare irisin hydrogels with a diameter of 1 cm and a thickness of 1 μm. The irisin content in each hydrogel was 2.5 μg.

[0011] The procedure for determining the drug release efficiency of irisin hydrogel is as follows: Irisin hydrogels were incubated in 5 mL PBS buffer with shaking, in triplicate. The gels were placed in a clean environment at 37 °C for 15 min, 30 min, 1 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h. 0.3 mL of the liquid was collected at each time point, and an equal volume of fresh PBS was added. The irisin concentration in the samples was measured using the Sigma-Aldrich Mouse irisin ELISA kit according to the manufacturer's instructions. The cumulative release of irisin from the gel at each time point was calculated, and a drug release curve was plotted.

[0012] like Figure 1 As shown, the cumulative release rate curve of irisin is obtained. ELISA analysis revealed that irisin was rapidly released from egg white hydrogel within 0-6 h, while the total amount of irisin gradually stabilized after 9 h, with a release rate of approximately (81±3)%.

[0013] Experimental verification To verify the therapeutic effect of the irisin hydrogel of the present invention, an animal diabetic skin trauma model was first constructed, and then the irisin hydrogel of the present invention was used for treatment to verify the therapeutic effect of the irisin hydrogel of the present invention.

[0014] Construction of a diabetic skin trauma model and intervention treatment plan: such as Figure 2 The diagram shows the experimental protocol for this animal study. Sixty male C57 mice (8 weeks old, weighing 20 ± 2 g) were selected and injected intraperitoneally with streptozotocin (0.05% STZ, Sigma) for 5 consecutive days. 72 hours later, random blood glucose levels were measured. A blood glucose level exceeding 16.7 mmol / L, along with symptoms of polydipsia, polyphagia, and polyuria, indicated a successful establishment of a diabetic model. A 1 cm diameter full-thickness skin wound was created on the back of each mouse using a biopsy punch. The mice were then randomly divided into three groups: a blank control group, a blank hydrogel group, and a drug-loaded hydrogel group, with 15 mice in each group. The diabetic model group was fed normally daily, while the other two groups received simple hydrogel and irisin hydrogel on days 0, 2, 4, and 6, respectively. The wound area was photographed and recorded on days 0, 3, 7, 10, and 14 post-surgery, and the wound area was measured using ImageJ software. The healing rate was calculated by comparing the wound area with the initial area. On the seventh day post-surgery, 12 animals from each of the three groups were sacrificed, and skin samples were collected for histopathological examination, angiogenesis, and changes in inflammatory signals.

[0015] Evaluation of the therapeutic effect of irisin hydrogel on diabetic wounds: (1) Wound healing rate. The dynamic changes of the wounds of each mouse were observed and photographed at 0, 3, 7 and 14 days after surgery. The wound area was measured by ImageJ and the wound healing rate was calculated by comparing it with the initial area.

[0016] (2) Pathological changes in the wound. After anesthetizing and euthanizing the animals, tissue blocks of the skin wound were excised, fixed with 4% paraformaldehyde, and prepared by paraffin embedding. Hematoxylin-eosin staining was used to detect pathological changes in the skin, and Masson's trichrome labeling was used to label collagen. After microscopic observation and photography, statistical analysis was performed.

[0017] (3) Angiogenesis. Skin tissue sections were dewaxed, and blood vessels were labeled with CD31 immunofluorescence staining and cell nuclei were labeled with DAPI staining. Angiogenesis in different treatment groups was analyzed.

[0018] (4) Macrophage phenotype. Skin tissue sections were dewaxed and incubated with fluorescent antibodies against F4 / 80-FITC, iNOS-Cy3, and CD206-Cy3. Cell nuclei were stained with DAPI, and macrophage phenotype changes were analyzed using immunofluorescence. Double positivity for F4 / 80 and iNOS indicated M1 type macrophages, while double positivity for F4 / 80 and CD206 indicated M2 type polarization.

[0019] (5) Cytokine secretion. ELISA was used to detect changes in the secretion of vascular endothelial growth factor (VEGF) and anti-inflammatory factor IL-10 in wound tissue.

[0020] Experimental results Wound healing rate like Figure 3 The figure shows the wound healing analysis of diabetic mice. In the figure, A represents representative images and trace analysis of wound healing during the treatment process. BC represents the statistical analysis of the wound healing rate of mice (N=6). Compared with the control group, *p<0.05, **p<0.01, ***p<0.001. As can be seen from the figure, on the 7th day of wound healing, the wounds in each group showed a certain degree of contraction. Compared with the control group, there was no significant change in wound size between the hydrogel group and the control group, while the iris extract hydrogel group showed a greater degree of wound contraction, with a wound healing rate of (68±3)% (P<0.05). On the 14th day, the wound healing rates of the disease model group and the hydrogel group were (72±3)% and (73±3)%, respectively, while the wound healing rate of the iris extract hydrogel group reached (92±3)% (P<0.05).

[0021] Pathological changes in the wound like Figure 4 The figure shows the pathological changes in the wounds of diabetic mice. In the figure, A represents H&E and Masson staining of the mouse wound tissue, B represents the statistical analysis of wound width, and C represents the statistical analysis of collagen deposition. The scale bar is 50 μm, n=6, and compared with the control group, ***p <0.001. The figure shows that on the 7th day after surgery, the wounds in the disease model group and the hydrogel-only group were longer, with extensive inflammatory cell infiltration, relatively immature granulation tissue, and incomplete epidermal bridges. In contrast, the iris extract hydrogel group had the shortest wound length, reduced inflammatory infiltration, densely arranged granulation tissue, and complete epidermal recovery. Masson staining results also showed that the collagen fibers in the iris extract hydrogel group were neatly and orderly arranged, suggesting that iris extract has the potential to reduce scar formation.

[0022] Angiogenesis like Figure 5The figure shows angiogenesis in diabetic mice. In the figure, AB represents the immunofluorescence staining and quantitative statistics of CD31. The scale bar is 50 μm, n=4, and compared with the control group, ***p <0.001. As can be seen from the figure, compared with the disease model group, the expression of CD31, a marker of angiogenesis, was not significantly changed in the hydrogel group alone, while the expression of CD31 in the irisin hydrogel group was significantly enhanced, showing obvious ring-shaped vascular distribution with large lumen and intact structure (p<0.001).

[0023] Macrophage phenotype like Figure 6 The figure shows the macrophage phenotype in the wound area of ​​diabetic mice. In the figure, A and C represent the relative mRNA expression levels of CD206, Vegf, and IL-10 in the wound tissue, as analyzed by RT-PCR (n=4). Compared with the control group, ***p <0.001. The figure shows that compared with the disease model group, the macrophage phenotype in the hydrogel group alone was not significantly changed, while the expression level of CD206 was increased in the irisin hydrogel group, indicating an increase in M2 polarized macrophages. Furthermore, RT-PCR analysis revealed increased levels of angiogenic factor Vegf and anti-inflammatory factor IL-10 in the mouse wound tissue. This suggests that irisin enhances the transformation of macrophages into a wound-healing phenotype.

[0024] The above experiments demonstrate that the irisin hydrogel of this invention can enhance M2 polarization of macrophages, effectively promote the secretion of large amounts of angiogenesis factor Vegf and anti-inflammatory factor IL-10 by macrophages, and enhance the proliferation capacity of vascular endothelial cells and skin fibroblasts. In diabetic mice, treatment with irisin after wound excision significantly promoted angiogenesis and skin tissue reconstruction, and significantly accelerated wound healing. Therefore, it can be applied to the repair of refractory skin wounds in diabetic patients.

[0025] The above descriptions are merely embodiments of the present invention, and common technical solutions or characteristics known in the schemes are not described in detail here. For those skilled in the art, various modifications and improvements can be made without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a tectorigenin hydrogel, characterized by, The method comprises the following steps: S1, fresh egg white is taken and an equal amount of deionized water is added, and the mixture is stirred uniformly at 4°C, white flocculent precipitate is removed by dialysis bag, and insoluble precipitate is removed by centrifugation at a speed of 10,000 rpm for 10 min, to obtain supernatant which is freeze-dried for preservation; S2, a 2.5 wt% NaOH aqueous solution and a 20 wt% egg white solution are prepared, and iridoid is dissolved in the egg white solution to obtain an egg white solution of iridoid with a concentration of 16 μg / mL; S3, the iridoid egg white solution of step S2 is mixed with the sodium hydroxide solution at a ratio of 62.5%:37.5%, and after being stirred uniformly, the mixture is placed in a mold with a thickness of 1.0 μm to form a gel; the concentration of iridoid in the hydrogel is 10 μg / mL; S4, the hydrogel prepared in step S3 is placed in 1.5 g of glycerol, glycerol containing 0.05 M CuSO4·5H2O, and glycerol containing 0.05 M ZnSO4·7H2O, respectively, and soaked for 10 min, the excess liquid on the surface is removed with filter paper, the reaction is promoted at 45°C for 30 min, and finally the hydrogel is cut into iridoid hydrogels with a diameter of 1 cm and a thickness of 1 μm, and the content of iridoid in each hydrogel is 2.5 μg.

2. The iridoid water gel prepared according to claim 1, characterized in that, The hydrogel is applied to repair of diabetic refractory skin wounds.

3. The use of a water gel of iridoid according to claim 2, characterized in that, The hydrogel uses endogenous iridoid in the body as the main component, and enhances the repair of diabetic skin wounds through targeted drug delivery of the hydrogel.

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