Application of NRP1 protein in preparation of medicine for promoting angiogenesis and / or healing of diabetic wound
By using NRP1 protein to regulate angiogenesis and inflammatory microenvironment, the angiogenesis disorder and inflammatory disorders of diabetic wounds in high-glycemic environments were solved, and the wound healing efficiency was significantly improved.
Patent Information
- Application Number
- CN202510461684.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-14
- Publication Date
- 2025-06-10
AI Technical Summary
The prior art is difficult to effectively improve the angiogenesis and inflammatory disorders of diabetic wounds in high-sugar environments.
By using NRP1 protein, the angiogenesis and inflammatory microenvironment are regulated, the migration, proliferation and neoangiogenesis of vascular endothelial cells are improved, and the overexpression of proinflammatory factors IL-6 and IL-8 are inhibited.
Significantly improves healing disorders in diabetic wounds, promotes angiogenesis and regulation of inflammatory microenvironment, shortens healing time and improves wound repair efficiency.
Smart Images
Figure CN120114569A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of NRP1 protein in the preparation of a drug for promoting angiogenesis and / or healing of diabetic wounds. Background Art
[0003] Neuropilin-1 (NRP1) is a multifunctional receptor that is widely expressed in vascular endothelial cells and other tissues and plays an important role in angiogenesis. NRP1 can promote vascular proliferation and remodeling by binding to growth factors such as VEGFR2 and TGF-β. Studies have shown that NRP1 plays an important role in the angiogenesis of tissues such as retinal lesions and tumors, but its role in wound angiogenesis and healing in a high-glucose environment remains unclear. In recent years, studies have further shown that NRP1 overexpression can significantly promote angiogenesis, especially in microvascular complications such as diabetic retinopathy, by regulating inflammatory and angiogenic signals. However, there are currently few studies on the specific role of NRP1 in diabetic wound angiogenesis and healing and its molecular mechanism, and existing technologies for diabetic wound healing solutions still have certain limitations, such as the inability to effectively improve the problem of angiogenesis disorders in a high-glucose environment.
[0004] Therefore, based on the molecular mechanism of NRP1 promoting angiogenesis in a high-sugar environment, the present invention proposes a potential drug for promoting angiogenesis and healing of high-sugar wounds, providing a new direction for improving wound healing in diabetes. Summary of the invention
[0005] In view of this, the purpose of the present invention is to provide the use of NRP1 protein in the preparation of drugs for promoting angiogenesis and / or healing of diabetic wounds. The present invention proposes for the first time the use of NRP1 protein in promoting angiogenesis and healing of diabetic wounds. NRP1 protein can effectively improve the healing disorder of diabetic wounds by regulating angiogenesis and inflammatory microenvironment.
[0006] In order to achieve the above object, the present invention provides the following technical solutions:
[0007] In a first aspect, the present invention provides the use of NRP1 protein in the preparation of a drug for promoting angiogenesis and / or healing of diabetic wounds.
[0008] Preferably, the NRP1 protein promotes angiogenesis and / or healing of diabetic wounds by: increasing the expression levels of endothelial markers CD31 and CD34 to enhance the migration, proliferation and angiogenesis ability of endothelial cells; inhibiting the overexpression of proinflammatory factors IL-6 and IL-8 to improve the inflammatory microenvironment of diabetic wounds.
[0009] In a second aspect, the present invention provides the use of NRP1 protein in the preparation of a drug for improving inflammation of diabetic wounds.
[0010] In a third aspect, the present invention provides a drug for promoting angiogenesis and / or healing of diabetic wounds, the drug comprising one or more of NRP1 protein, a recombinant vector comprising a nucleic acid molecule encoding NRP1 protein, a recombinant microorganism comprising a nucleic acid molecule encoding NRP1 protein, and a transgenic cell line comprising a nucleic acid molecule encoding NRP1 protein.
[0011] Preferably, the recombinant vector is an adeno-associated virus vector.
[0012] Preferably, the drug is a topical ointment, an injection, a nanocarrier preparation, or a hydrogel preparation.
[0013] Preferably, the drug is suitable for diabetic foot ulcers, diabetes-related chronic wounds, and diabetic wounds with limited angiogenesis.
[0014] Contains at least the following beneficial technical effects:
[0015] The present invention proposes for the first time the use of NRP1 protein in promoting angiogenesis and healing of diabetic wounds. The present invention finds that NRP1 protein can effectively improve the healing barrier of diabetic wounds by regulating angiogenesis and inflammatory microenvironment. Studies have shown that delivering the NRP1 gene to human endothelial cells in a high-sugar environment using adeno-associated virus (AAV) can significantly enhance the expression of angiogenesis-related factors (such as CD31 and CD34) and inhibit the overexpression of proinflammatory factors IL-6 and IL-8; further, in a diabetic mouse model, by upregulating the level of NRP1 protein, microangiogenesis can be significantly promoted, wound repair can be accelerated, and the inflammatory microenvironment can be improved. The preparation of related agents based on NRP1 protein can effectively solve the problems of limited angiogenesis and inflammatory disorders in diabetic wounds in the prior art, providing a new solution for the treatment of chronic diabetic wounds, with significant clinical value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 For wound healing.
[0017] Figure 2 It is a line graph of wound healing.
[0018] Figure 3 The results of immunohistochemical detection of CD31.
[0019] Figure 4 This is the result of immunohistochemical detection of CD34.
[0020] Figure 5Results of immunohistochemical detection of α-SMA
[0021] Figure 6 This is the result of WB detection of NRP1.
[0022] Figure 7 These are the results of wb detection of IL-6 and IL-8. DETAILED DESCRIPTION
[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.
[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0028] The "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C unless otherwise specified.
[0029] The raw materials or instruments used in the following examples of the present invention are all commercially available analytically pure or biological grade products unless otherwise specified.
[0030] Example 1
[0031] 1. Construction of AAV carrying NRP1
[0032] 1.1 Experimental methods
[0033] The linearized vector is obtained by restriction endonuclease digestion, and the target gene fragment is prepared by PCR amplification. The amplification primer used needs to add a homologous recombination sequence at its 5' end when it is designed. The 5' and 3' sequences of the amplified product of the target gene fragment amplified by this primer are completely consistent with the sequences at both ends of the linearized cloning vector. The reaction system is prepared with the linearized vector and the target gene amplification product, and the recombination reaction is carried out to achieve the in vitro circularization of the linearized vector and the target gene fragment. The recombinant product is directly transformed, and the single clone on the plate is picked for PCR identification, and the positive clone is sequenced and the results are analyzed. The correct clone bacterial solution is expanded and extracted to obtain a high-purity plasmid for downstream virus packaging.
[0034] Primers containing the NRP1 gene were designed and synthesized (see Table 1), and the full-length fragment of the NRP1 gene was amplified by q-PCR. The target plasmid vector (ENT) was digested with restriction endonucleases to linearize it for subsequent gene insertion. The amplified NRP1 gene fragment and the linearized vector were then integrated by homologous recombination to construct a recombinant plasmid.
[0035] The obtained recombinant plasmid was transformed into cells, and the accuracy of NRP1 sequence insertion was confirmed by colony PCR and sequencing. The colonies were identified, and if the result was positive, they were expanded and the plasmids were extracted to prepare for virus packaging. The recombinant plasmid containing the NRP1 gene was co-transfected into the cells with the auxiliary plasmid, and the cell supernatant and precipitate were collected after 72 hours of culture, and high-purity AAV (serological type ENT) virus fluid was obtained by ultracentrifugation and ultrafiltration purification.
[0036] Table 1
[0037]
[0038]
[0039] Primer description: Contains exchange pairing bases, restriction sites, and the 5' end sequence of the target gene for PCR fishing of the target gene
[0040] 1.2 Build Results
[0041] q-PCR is used to determine the virus titer. After obtaining the Ct value, a standard curve is drawn using the logarithmic value of the standard concentration and the average Ct value, and the concentration of other samples is calculated using this curve. The final concentration of the sample requires the measured value to be divided by the dilution factor and multiplied by 2 (because the standard is double-stranded and the AAV virus is single-stranded). The titers of samples with different dilution factors are averaged to obtain the final concentration of the virus. Ensure that the prepared virus concentration meets the standard, and perform sterility testing to confirm that the virus solution is free of bacterial and fungal contamination.
[0042] Example 2
[0043] 2. Construction of diabetic mouse wound model
[0044] 2.1 Experimental methods
[0045] Animals: A total of 20 C57BLKS / J-db / db mice were used, with 10 mice in each group. Mice were purchased from Saiye Biotechnology Co., Ltd. and bred in the Experimental Animal Center of the First Affiliated Hospital of Henan University of Science and Technology.
[0046] After one week of adaptive feeding, the blood sugar levels of 20 mice were tested. If the blood sugar levels were all >20mmol / L, the next step of wound preparation could be carried out.
[0047] Establishment of mouse full-thickness skin defect wound model: After the mice were anesthetized with isoflurane gas, the back hair was removed and the skin was prepared with iodine, and two circular full-thickness skin defect wounds with a diameter of 6 mm were prepared on the back skin with a skin biopsy device.
[0048] After two full-layer skin wounds were made, the mice were divided into an experimental group and a control group. The experimental group was injected with AAV virus (serotype ENT) carrying NRP1 vector (AAV-NRP1) (the amount per mouse was: 1.4E×10^11), and the control group was injected with blank virus (AAV-control) as a control.
[0049] Quantitative analysis of wound healing: After the wound model was established, the wound was photographed and recorded at time points such as 0, 2, 4, 6, and 10 days, the wound healing rate was calculated, and the time curve of the mouse wound healing was drawn.
[0050] 2.2 Experimental Results
[0051] After the wound was made with the skin biopsy device, the wound was photographed immediately to record its condition, and the wound healing condition was recorded every two days thereafter (e.g. Figure 1 On the tenth day, the healing rate was uniformly calculated to form a line graph (as shown in Figure 2 shown).
[0052] Figure 2The figure shows the changes in the relative area of the wound surface of mice under two different treatment conditions over time during the wound healing process. The ordinate represents the relative area of the wound surface, and the abscissa represents time (unit: day). The figure contains two sets of data, represented by blue and red, respectively, where blue represents the AAV-NRP1 treatment group and red represents the AAV-control control group. It can be observed that during the healing process, the relative area of the wound surface in the AAV-NRP1 treatment group was significantly lower than that in the AAV-control control group, especially on the 4th and 10th days, and the difference was highly statistically significant (marked with "**", indicating that the p value is less than 0.01). This shows that AAV-NRP1 treatment can significantly promote wound healing and shorten the healing time. The error bars in the figure represent the standard error of the data, which further illustrates the reliability of the experimental results. These results show that AAV-NRP1 treatment may be an effective method to promote wound healing, which deserves further research and development.
[0053] Example 3
[0054] Multiple intradermal injections of AAV-NRP1 on the wound surface promote the formation of blood vessels on the wound surface, inhibit inflammatory response, and promote wound healing
[0055] 3.1 Experimental methods
[0056] The wound surface model is the same as that in Example 2.
[0057] Multiple intradermal injections: After making two circular wounds with a diameter of 6 mm on the back of the mouse, 100 ul of AAV-NRP1 and AAV-control were injected intradermally at multiple points.
[0058] Immunohistochemistry: The full-thickness skin wound tissues of mice were collected on the 10th day, embedded in paraffin and stained to detect the expression of vascular markers such as CD31, CD34, and α-SMA.
[0059] Wb experiment: The full-thickness wound tissues of mice were collected on the tenth day, quickly frozen in liquid nitrogen, and then stored at -80°C for subsequent detection of IL-6 and IL-8 inflammatory indicators.
[0060] 3.2 Experimental Results
[0061] In order to evaluate the effect of NRP1 on wound microvascular formation, immunohistochemistry was used to detect the expression of vascular markers CD31, CD34 and α-SMA in wound tissue. The experimental results showed that in the AAV-NRP1 group, the number of CD31 and CD34 positive cells increased significantly, and the expression of α-SMA also increased significantly, indicating that NRP1 can effectively promote the formation and maturation of wound microvessels. In contrast, the expression of vascular markers in the AAV-control group was relatively low, the number of CD31 and CD34 positive cells was significantly less than that in the AAV-NRP1 group, and the expression of α-SMA was also at a low level (such as Figure 3-5 shown).
[0062] The expression of inflammatory factors IL-6 and IL-10 in the wound tissue of mice on the 10th day was detected by Western Blot experiment. The results showed that the expression of IL-6 and IL-8 in the AAV-NRP1 group was significantly lower than that in the AAV-control group. This change suggests that the expression of NRP1 not only promotes the formation of blood vessels in the wound, but also may further accelerate the wound healing process by regulating the inflammatory response (such as Figure 6-7 shown).
[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Application of NRP1 protein in the preparation of drugs for promoting angiogenesis and / or healing of diabetic wounds.
2. The use according to claim 1, characterized in that: The NRP1 protein promotes angiogenesis and / or healing of diabetic wounds by increasing the expression levels of endothelial markers CD31 and CD34 to enhance the migration, proliferation and angiogenesis of endothelial cells; and inhibiting the overexpression of proinflammatory factors IL-6 and IL-8 to improve the inflammatory microenvironment of diabetic wounds.
3. Application of NRP1 protein in the preparation of drugs to improve diabetic wound inflammation.
4. A drug for promoting angiogenesis and / or healing of diabetic wounds, characterized in that: The drug comprises one or more of NRP1 protein, a recombinant vector comprising a nucleic acid molecule encoding NRP1 protein, a recombinant microorganism comprising a nucleic acid molecule encoding NRP1 protein, and a transgenic cell line comprising a nucleic acid molecule encoding NRP1 protein.
5. The drug according to claim 4, characterized in that The recombinant vector is an adeno-associated virus vector.
6. The drug according to claim 4, characterized in that The medicine is one of a local smear, an injection, a nanocarrier preparation, and a hydrogel preparation.
7. The drug according to claim 6, characterized in that The drug is suitable for diabetic foot ulcers, diabetes-related chronic wounds, and diabetic wounds with limited angiogenesis.