Use of an agent that enhances the interaction of HSP90 with CD93 in the preparation of a medicament for promoting healing of diabetic wounds

Abstract

This invention relates to the field of angiogenesis and healing technology for diabetic wounds, and discloses a method for promoting angiogenesis and healing of diabetic wounds based on HSP90-CD93 interaction, comprising the following steps: applying an HSP90 agonist, delivering a CD93 overexpression vector, or applying a CD93 glycosylation agonist, thereby enhancing the HSP90-CD93 interaction locally at the wound site and / or maintaining the O-linked N-acetylglucosamine (O-GlcNAc) modification level of CD93 to activate the downstream focal adhesion kinase (FAK) signaling pathway, thereby effectively promoting endothelial cell migration, adhesion, and angiogenesis, and ultimately accelerating wound healing under diabetic conditions.

Description

Technical Field

[0001] This invention relates to the field of diabetic wound healing technology, specifically to a method for promoting diabetic wound healing based on the interaction between HSP90 and CD93. Background Technology

[0002] Wound healing is a complex process involving the synergistic effects of cell migration, proliferation, matrix remodeling, and angiogenesis, with angiogenesis being the core component—new blood vessels deliver oxygen and nutrients to the wound, regulate inflammation resolution, and restore microenvironmental homeostasis. Under normal physiological conditions, vascular endothelial cells complete angiogenesis through migration, adhesion, and tubular structure formation, supporting wound healing. However, the persistently high glucose microenvironment in diabetic patients disrupts angiogenesis mechanisms: on the one hand, it induces endothelial cells to produce reactive oxygen species, causing oxidative stress damage and reducing their migration and adhesion abilities; on the other hand, it interferes with intercellular signal transduction, inhibiting tubular structure formation and stabilization, ultimately leading to impaired angiogenesis. This directly results in delayed wound healing, chronic ulcers, severely impacting patients' quality of life and increasing the medical burden. Current wound treatments for diabetes focus on improving the wound environment and promoting repair, including wound debridement, functional dressing changes, negative pressure wound therapy (NPWT), and exogenous growth factor (VEGF) supplementation. Debridement and dressing changes remove necrotic tissue and reduce infection; NPWT promotes blood circulation and drains exudate; and growth factor therapy attempts to activate angiogenesis by supplementing pro-angiogenic factors. However, existing treatments have significant limitations: First, debridement, dressing changes, and NPWT are only "symptomatic supportive treatments" and cannot fundamentally repair the angiogenesis impairment induced by high glucose levels, thus having limited effectiveness for deep chronic ulcers. Second, exogenous growth factors (such as VEGF) are unstable, easily degraded, require repeated administration, and are costly and difficult to widely implement. Furthermore, the high glucose levels in diabetic patients disrupt the overall homeostasis of the endothelial cell angiogenesis signaling network; supplementing with a single factor cannot restore this balance, making it difficult to initiate and maintain the repair process. In fact, supplementing with a single factor may disrupt the signal balance, inducing abnormal angiogenesis, tissue fibrosis, and other side effects.

[0003] Therefore, developing a stable and low-cost method to promote the healing of diabetic wounds is of great significance for rapidly healing diabetic wounds and improving the safety of diabetic patients. This will not only effectively compensate for the shortcomings of existing technologies, but also effectively promote the healing of diabetic wounds. Summary of the Invention

[0004] The present invention aims to provide a method for promoting wound healing in diabetic patients based on the interaction of HSP90-CD93, in order to solve the technical problem of poor stability and unstable efficacy of existing methods for promoting wound angiogenesis in diabetic patients.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a method for promoting diabetic wound healing based on HSP90-CD93 interaction, comprising the following steps: applying an HSP90 agonist, delivering a CD93 overexpression vector, or applying a CD93 glycosylation agonist, thereby promoting diabetic wound healing by enhancing the HSP90-CD93 interaction at the wound site.

[0006] Preferably, as an improvement, the HSP90 agonist is AMP-PCP and the CD93 glycosylation agonist is tiotropium bromide G (TMG).

[0007] Preferably, as an improvement, the protein sequence of the recombinant protein of CD93 is shown in SEQ_ID_NO.1.

[0008] Preferably, as an improvement, the application of the CD93 glycosylation agonist is the delivery of a CD93 overexpression vector after the use of tiotropium bromide G. Tiotropium bromide G inhibits the activity of O-GlcNAc hydrolase, thereby glycosylating the overexpressed CD93.

[0009] Preferably, as an improvement, this solution also provides the application of AMP-PCP as an HSP90 agonist.

[0010] The technical advantages of this solution are as follows: 1. Novel Target, Directly Addressing the Root Cause: This application reveals for the first time that the HSP90-CD93 interaction is a core element in maintaining the stability and function of the CD93 protein, and elucidates its key mechanism for impaired angiogenesis under high glucose conditions. The intervention strategy targeting this point (enhancing HSP90-CD93 binding) stabilizes the entire pro-angiogenic signaling hub (CD93) upstream, rather than simply supplementing a single factor downstream, thus solving the problem of existing growth factor therapies that only treat the symptoms and not the root cause.

[0011] 2. Synergistic Effect, Significant Results: The key step in this protocol—"enhancing the binding of HSP90 to CD93"—directly prevents the ubiquitination and degradation of CD93. A high-glucose environment disrupts the HSP90-CD93 binding, leading to accelerated CD93 degradation, which is the core reason for the technical problem (downregulation of CD93 expression, impaired angiogenesis). Reversing this process directly solves the root cause of the problem. Using agonists that promote the binding of HSP90 to its client proteins can maximize the stability of CD93 protein without affecting basic cellular activity.

[0012] 3. Clear Mechanism, Promising Effects: Another key step – “Maintaining the O-GlcNAc Modification Level of CD93” – O-GlcNAc modification is a necessary biochemical switch for CD93 to perform its pro-angiogenic function. High glucose levels not only lead to CD93 degradation but also disrupt its glycosylation modification, rendering it functional even if present. The stabilizing effect of HSP90 precisely preserves both the “quantity” and “quality” of CD93. By supplementing substrates such as GlcNAc to increase the overall intracellular O-GlcNAc level, sufficient substrates can be ensured for the functional modification of CD93.

[0013] 4. Clear downstream pathways: The stability and functional integrity of the CD93 protein enable it to effectively activate the downstream FAK signaling pathway. FAK is a core molecule regulating cell migration and adhesion, and its activation directly leads to improvements in endothelial cell function, such as enhanced migration ability in Transwell assays and enhanced tube formation ability in tube formation assays. This perfectly explains how molecular-level interventions can be translated into tissue-level angiogenesis-promoting effects.

[0014] 5. Sufficient in vitro and in vivo validation: This protocol fully demonstrates the necessity of this signaling axis through gene knockout (CD93-KO) and pharmacological inhibition (HSP90 inhibitor) experiments as comparative examples; at the same time, the adequacy and therapeutic potential of this strategy are demonstrated through experiments on CD93 overexpression rescuing angiogenesis function in a high glucose environment.

[0015] In summary, this application provides a method for promoting diabetic wound healing by stabilizing CD93 protein. The essence of this method lies in discovering and utilizing the interaction between the molecular chaperone HSP90 and CD93, thereby maintaining the stability and function of CD93 in a high-glycemic environment. The specific principle is as follows: HSP90, as an important molecular chaperone, can specifically recognize and bind to the extracellular domain of CD93. This binding acts like a "protective shield" for CD93, stabilizing its O-GlcNAc glycosylation modification on the one hand, and directly shielding the ubiquitination recognition site or altering the protein conformation on the other, thus effectively blocking the degradation process of CD93 through the ubiquitin-proteasome pathway. The stable and accumulated level of CD93 protein enables it to continuously activate the downstream FAK signaling pathway, thereby efficiently performing its pro-angiogenic function. Specifically, by enhancing the interaction between HSP90 and CD93, and / or maintaining the O-GlcNAc (O-GlcNAcylation) modification level of CD93, the downstream focal adhesion kinase (FAK) signaling pathway is activated, thereby effectively promoting endothelial cell migration, adhesion and angiogenesis, and ultimately accelerating wound healing under diabetic conditions.

[0016] Specifically, this approach enhances the function of the HSP90-CD93 signaling axis at the wound site by one of the following methods: application of an HSP90 agonist (such as AMP-PCP), gene manipulation (such as delivery of an overexpression vector for HSP90 or CD93), or provision of an O-GlcNAc modified substrate, thereby promoting the healing of diabetic wounds. Attached Figure Description

[0017] Figure 1 The image shows the Co-IP verification of CD93-HSP90 interaction (left) and HSP27-CD93 no interaction (right) in Embodiment 1 of the present invention.

[0018] Figure 2 This refers to the inhibition of CD93 protein expression after HSP90 by 50 nM 17-AAG in Example 1 of the present invention.

[0019] Figure 3 For the analysis of the relative amount of CD93 protein in Example 1 of the present invention ( p <0.05; p <0.01; p <0.001, ns = no significant difference; N=3).

[0020] Figure 4 This is a schematic diagram of the full-length CD93 plasmid and the truncated plasmid (CD93△) lacking the intracellular domain in Example 2 of the present invention (TM, transmembrane domain; Cy, intracellular domain; RFP, red fluorescent protein).

[0021] Figure 5 This is the full-length plasmid map of CD93 in Example 2 of the present invention.

[0022] Figure 6 This is the CD93△ spectrum in Embodiment 2 of the present invention.

[0023] Figure 7 The results of CD93 and CD93△ plasmid transfection observed under a fluorescence microscope in Example 2 of this invention (scale bar = 200 μm).

[0024] Figure 8 This is the Co-IP detection of the interaction between HSP90-CD93 or HSP90-CD93Δ in Embodiment 2 of the present invention.

[0025] Figure 9 The effect of CD93 on cell budding after 17-AAG inhibits HSP90 in Example 3 of the present invention (scale bar = 200 μm).

[0026] Figure 10 The Transwell assay in Example 3 of this invention shows the effect of CD93 on cell migration after HSP90 inhibition (scale bar = 200 μm).

[0027] Figure 11 Analysis of cell budding length (left) and analysis of the number of migrating cells (right) in Example 3 of the present invention. p <0.05; p <0.01; p <0.001, ns = no significant difference; N=3).

[0028] Figure 12 The expression of CD93 protein after treatment with 17-AAG and / or bortezomib in Example 4 of this invention is shown in the left image and the ubiquitination level of CD93 protein is shown in the right image.

[0029] Figure 13 This is an analysis of the relative amount of CD93 protein in Example 4 of the present invention.

[0030] Figure 14 This is a schematic diagram of embodiment 4 of the present invention.

[0031] Figure 15 This is a GO analysis chromatogram of the mass spectrometry results in Example 5 of the present invention.

[0032] Figure 16 The effect of CD93 overexpression on FAK phosphorylation in Example 5 of this invention (left) and the relative amount of FAK protein (right). p <0.001, ns = no significant difference; N=3).

[0033] Figure 17 The effect of siCD93 on FAK phosphorylation in Example 5 of this invention (left) and the relative amount of FAK protein (right). p <0.05; p <0.01; p <0.001, ns = no significant difference; N=3).

[0034] Figure 18 The effect of 17-AAG and / or ad.CD93 on FAK phosphorylation in Example 5 of this invention (left) and the relative amount of FAK protein (right). p <0.05; p <0.01; p <0.001, ns = no significant difference; N=3).

[0035] Figure 19 The image above shows the expression of CD93 protein in endothelial cells after treatment with 5mM and 30mM glucose and / or ad.CD93, as detected by Western blot in Example 6 of this invention, along with the relative expression level analysis. p <0.05; p <0.01; p <0.001, ns = no significant difference; N=3).

[0036] Figure 20 The Transwell assay used in Example 7 of this invention to detect the migration of endothelial cells after treatment with 5mM, 30mM glucose and / or ad.CD93 (scale bar = 200μm); p <0.05; p <0.01; p <0.001, ns = no significant difference; N=3).

[0037] Figure 21 This is an analysis of the number of endothelial cells that migrated after treatment with 5mM glucose, 30mM glucose and / or ad.CD93 as detected by Transwell in Example 7 of the present invention (scale bar = 200 μm). p <0.05; p <0.01; p <0.001, ns = no significant difference; N=3).

[0038] Figure 22 This is an experiment on endothelial cell budding after treatment with 5mM, 30mM glucose and / or ad.CD93 in Example 8 of the present invention (scale bar = 200μm).

[0039] Figure 33 This is a magnified view of the upper right quarter of the cell sphere in Embodiment 8 of the present invention.

[0040] Figure 24 Statistical analysis of bud length in Example 8 of the present invention ( p <0.05; p <0.01; p <0.001, ns = no significant difference; N=3).

[0041] Figure 25 The left image shows the effect of 5mM and 30mM glucose on HSP90 protein expression in Example 9 of this invention, and the right image shows the relative expression levels of HSP90 protein after treatment with 5mM and 30mM glucose. p<0.001, ns=no significant difference; N=3).

[0042] Figure 26 This is from Example 9 of the present invention, which uses Co-IP to detect the effect of 5mM and 30mM glucose on the interaction between HSP90 and CD93.

[0043] Figure 27 This is a flowchart of the animal experiment in Embodiment 10 of the present invention.

[0044] Figure 28 The blood glucose level of the mice in Example 10 of this invention.

[0045] Figure 29 The body weight level of the mice in Example 10 of this invention ( p<0.05; p<0.01; p<0.001, ns=no significant difference; N=5).

[0046] Figure 30 The healing process of the wounds of mice D0, D5, and D10 in Example 10 of this invention is shown (scale bar = 5mm).

[0047] Figure 31 This is a schematic diagram simulating the healing process of wounds D0-D10 in Embodiment 10 of the present invention.

[0048] Figure 32 The left image shows the variation of wound area from D0 to D10 in Example 10 of the present invention, and the right image shows the statistical analysis of the wound area of ​​D0. p<0.05; p<0.01; p<0.001, ns=no significant difference; N=5).

[0049] Figure 33 Statistical analysis of wound areas D5 and D10 in Embodiment 10 of the present invention (left figure is D5, right figure is D10); p<0.05; p<0.01; p<0.001, ns=no significant difference; N=5).

[0050] Figure 34 H&E staining of the wound at D0, D5, and D10 in Example 10 of this invention (black dashed lines in the magnified image indicate the boundary between the epidermis and dermis, and red arrows indicate discontinuous epidermal layers; scale bar = 1 mm).

[0051] Figure 35 Masson staining of the wound at times D0, D5, and D10 in Example 10 of this invention (blue represents collagen fibers; scale bar = 1 mm).

[0052] Figure 36 This refers to the double immunofluorescence staining of neovascularization in the wound at D5 in Example 10 of this invention (A: red represents CD31 (neovascularization marker), green represents CD93, and blue represents DAPI; scale bar = 100 μm) and the percentage of CD31-positive areas in the total area (B: p<0.05; p<0.01; p<0.001, ns=no significant difference; N=5).

[0053] Figure 37This refers to the double immunofluorescence staining of neovascularization in the skin wound at D10 in Example 10 of this invention (A: red represents CD31 (neovascularization marker), green represents CD93, and blue represents DAPI; scale bar = 100 μm) and the percentage of CD31-positive areas in the total area (B: p<0.001, ns=no significant difference; N=5). Detailed Implementation

[0054] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0055] Example 1: Verification of CD93-HSP90 interaction To verify the interaction between CD93 and HSP90, this study conducted a systematic analysis using Co-IP experiments and HSP90 inhibitor experiments.

[0056] Analysis of Co-IP experimental results: such as Figure 1 As shown, there is a significant interaction between CD93 and HSP90. Figure 1 (Middle left image, marked with a red asterisk), while no interaction was detected with another molecular chaperone protein, HSP27. Figure 1 (Right image in the middle). In this mass spectrometry analysis, no other molecular chaperone proteins were detected besides HSP90, suggesting that CD93 may be one of the specific client proteins of HSP90.

[0057] Analysis of HSP90 inhibitor experimental results: 17-AAG is a specific inhibitor of HSP90. It binds to the ATP-binding pocket at the N-terminus of HSP90, inhibiting its function. The inventors used 17-AAG to detect the effect of HSP90 on CD93 expression. Western blot results showed that compared with the ad.CD93 group, the expression of both glycosylated and non-glycosylated CD93 was significantly reduced in the 17-AAG + ad.CD93 group (p<0.05). Figure 2 and Figure 3 In summary, the interaction between HSP90 and CD93 stabilizes CD93 glycosylation, and inhibiting HSP90 can significantly reduce the expression of CD93 glycosylation, suggesting that HSP90 plays a key role in maintaining CD93 glycosylation homeostasis.

[0058] Example 2: Site analysis of HSP90-CD93 interaction CD93 is a single-pass transmembrane protein whose protein structure includes an extracellular domain, a transmembrane domain, and an intracellular domain. To determine whether the interaction site between HSP90 and CD93 is located intracellularly or extracellularly on CD93, the inventors constructed a full-length CD93 plasmid (1-652 amino acids) and a truncated CD93 plasmid lacking the intracellular domain (CD93Δ, 1-604 amino acids), both of which are tagged with a red fluorescent protein. Figure 4 The sequence maps of CD93 plasmid and CD93Δ plasmid are shown below. Figure 5 and Figure 6 Since 293T cells do not express the endogenous CD93 gene, this study transfected two plasmids ectopically into 293T cells to investigate the interaction between exogenous CD93 and HSP90. Red fluorescence was observed in the transfected cells under a fluorescence microscope, indicating successful plasmid transfection. Figure 7 Co-IP results showed that HSP90 interacted with both the full-length CD93 plasmid and the CD93Δ plasmid in cells transfected with it. Figure 8 The left-middle figure (marked with a red asterisk) shows that the interaction site between HSP90 and CD93 is located in the extracellular domain of CD93 rather than the intracellular domain.

[0059] Example 3: Effect of inhibiting HSP90 on the pro-angiogenic function of CD93 To investigate the regulatory role of HSP90 in the pro-angiogenic function of CD93, this study examined the effect of the HSP90 inhibitor 17-AAG on CD93 function using cell budding and migration assays. The cell budding assay results showed that the budding length of cells in the ad.mock group was 322.31±27.21µm; the budding length of cells in the ad.CD93 group was 426.43±15.4µm, significantly longer than that in the ad.mock group (p<0.05), indicating that overexpression of CD93 can promote the budding ability of vascular endothelial cells. However, the budding length of cells in the 17-AAG+ad.CD93 group was 247.86±46.49µm, significantly shorter than that in the ad.CD93 group (p<0.05). Figure 9 and Figure 11 (Left middle figure) shows that inhibiting HSP90 can significantly inhibit the cell budding function of CD93.

[0060] Migration assays showed that the number of migrating cells in the ad.mock group was 248±7.53, while the number in the ad.CD93 group was 310.5±34.1, significantly higher than that in the ad.mock group (p<0.05), indicating that overexpression of CD93 can promote the migration ability of vascular endothelial cells. However, the number of migrating cells in the 17-AAG+ad.CD93 group was 224.5±13.1, significantly lower than that in the ad.CD93 group (p<0.05), indicating that inhibition of HSP90 can significantly inhibit the cell migration-promoting function of CD93. Figure 10 and Figure 11 (Right image in the middle). These results indicate that HSP90 significantly affects the pro-angiogenic function of CD93 by regulating its stability and glycosylation state. Inhibition of HSP90 significantly suppressed the cell budding and migration-promoting functions of CD93, suggesting that the interaction between CD93 and HSP90 plays an important role in angiogenesis.

[0061] Example 4: Molecular mechanism of HSP90 regulation of CD93—HSP90 regulates the glycosylation homeostasis of CD93 through the ubiquitination protease pathway. Protein degradation pathways can be broadly classified into two categories: autophagy-lysosomal degradation and ubiquitin-proteasome degradation. Studies have shown that HSP90 plays a crucial role in stabilizing client proteins by inhibiting their ubiquitination and subsequent proteasome degradation. To investigate whether HSP90 maintains CD93 glycosylation homeostasis by inhibiting the ubiquitination-proteasome degradation pathway, this study treated cells with the proteasome inhibitor bortezomib in combination with the HSP90 inhibitor 17-AAG, and detected changes in CD93 protein ubiquitination levels and expression. Western blot results showed that, compared with the 17-AAG group, the expression of glycosylated CD93 (G-CD93) and non-glycosylated CD93 (NG-CD93) was significantly increased in the 17-AAG+bortezomib group (p<0.05). Figure 12 Middle left image and Figure 13 This indicates that CD93 turnover is regulated by the proteasome pathway. Co-IP experiments further revealed that, compared to the 17-AAG group, the 17-AAG + bortezomib group showed significantly enhanced CD93 ubiquitination levels. Figure 12 The rightmost figure (marked with a red asterisk) shows that CD93 protein did indeed undergo ubiquitination modification after HSP90 inhibition. These results indicate that HSP90 protects CD93 from degradation via the ubiquitination-proteasome pathway, maintaining its glycosylation homeostasis. The reduction in CD93 expression after HSP90 inhibition is due to enhanced ubiquitination-proteasome-mediated degradation. Figure 14 ).

[0062] Example 5: HSP90-CD93 interaction promotes activation of downstream FAK signaling pathway Follicle adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that is crucial for the formation and disaggregation of focal adhesions. Its phosphorylation activation can promote endothelial cell adhesion and migration.

[65] GO analysis by mass spectrometry showed that CD93 and its interacting proteins are involved in focal adhesion and cadherin binding. Figure 15 (Middle right image, blue box) suggests that CD93 may affect endothelial cell function by regulating the FAK signaling pathway. Western blot results showed that, compared with the ad.mock group, the phosphorylation level of FAK in the ad.CD93 group was significantly increased ( p <0.05)( Figure 16 This indicates that overexpression of CD93 activates downstream FAK. Conversely, compared to the control group, the phosphorylation level of FAK in the siCD93 group was significantly reduced. p <0.05)( Figure 17 This indicates that silencing CD93 can inhibit downstream FAK. To further investigate the regulatory role of HSP90-CD93 interaction on FAK, cells were pretreated with 17-AAG. Western blot results showed that, compared with the ad.CD93 group, the phosphorylation level of FAK in the 17-AAG+ad.CD93 group was significantly reduced (…). p <0.05)( Figure 18 This indicates that inhibiting HSP90 can suppress the activation of downstream FAK pathways of CD93. In summary, HSP90 protects CD93 from ubiquitin-proteasome-mediated degradation by interacting with the extracellular domain of CD93, thereby maintaining CD93 glycosylation homeostasis and promoting the activation of downstream FAK signaling pathways.

[0063] Example 6: Overexpression of CD93 under high glucose environment To verify whether CD93 overexpression can delay the inhibition of angiogenesis in vascular endothelial cells under high glucose conditions, adenovirus overexpressing CD93 (ad.CD93) was transfected after high glucose pretreatment. G-CD93 (glycosylated CD93 group), NG-CD93 (non-glycosylated CD93 group), ad.mock (adenovirus empty vector group), and ad.CD93 (adenovirus overexpressing CD93 group) were set up. Western blot results showed that compared with the ad.mock group, the expression of CD93 protein in the ad.CD93 group was significantly increased (p<0.05), indicating successful transfection with CD93-overexpressing adenovirus; compared with the ad.mock+30 mM group, the expression of CD93 protein in the ad.CD93+30 mM group was significantly increased (p<0.05), indicating that CD93 overexpression can delay the inhibitory effect of high glucose on CD93 expression. Figure 19 ).

[0064] Example 7: Effects of CD93 overexpression on cell migration under high glucose environment To investigate the effect of CD93 overexpression on the migration ability of vascular endothelial cells under high glucose conditions, Transwell migration assay was used to detect cell migration ability. The results showed that compared with the ad.mock group (107.4 ± 4.62 cells), the number of migrating cells in the ad.CD93 group was 139.17 ± 15.8, significantly increased. p <0.05, indicating that overexpression of CD93 promotes cell migration; compared with the ad.mock group, the number of migrating cells in the ad.mock+30 mM group was 68.17±3.31, which was significantly reduced ( p <0.05, indicating that a high-glucose environment can inhibit the migration ability of vascular endothelial cells; compared with the ad.mock+30 mM group, the number of cells that migrated in the ad.CD93+30 mM group was 105.33 ± 13.82, although there was no statistically significant difference ( p >0.05), but showed an upward trend, preliminarily indicating that overexpression of CD93 can partially delay the inhibitory effect of high glucose on the migration ability of vascular endothelial cells. Figure 20 and Figure 21 The above results indicate that a high-glucose environment can significantly inhibit the migration ability of vascular endothelial cells, while overexpression of CD93 can promote cell migration and partially delay the inhibitory effect of high glucose.

[0065] Example 8: Effect of CD93 overexpression on cell budding under high glucose environment Cell budding experiments showed that the budding length of cells in the ad.CD93 group was 683.35 ± 19.49 µm, significantly longer than that in the ad.mock group (377.86 ± 76.74 µm, p<0.05), indicating that overexpression of CD93 can promote cell budding ability. The budding length of cells in the ad.mock+30 mM group was 273.41 ± 46.6 µm, significantly longer than that in the ad.mock group (p<0.05), indicating that a high-glucose environment inhibits cell budding ability. The budding length of cells in the ad.CD93+30 mM group was 651.38 ± 32.85 µm, significantly longer than that in the ad.mock+30 mM group (p<0.05), indicating that overexpression of CD93 can delay the inhibitory effect of high glucose on the budding ability of vascular endothelial cells. Figures 22-24 The above results indicate that overexpression of CD93 can not only promote cell budding, but also effectively delay the inhibitory effect of high glucose.

[0066] Example 9: Changes in HSP90-CD93 interaction under high sugar conditions To investigate the effect of a high-glucose environment on the interaction between HSP90 and CD93, this study analyzed the results using Western blot and Co-IP experiments. Western blot results showed that, compared to the control group, the expression of HSP90 protein was significantly reduced after high-glucose treatment (p<0.05). Figure 25 This indicates that a high-glucose environment can inhibit HSP90 expression. Co-IP experiments further showed that, compared to the control group, the interaction between HSP90 and CD93 was significantly reduced after high-glucose treatment. Figure 26 (marked with a red asterisk) indicates that a high-glucose environment disrupts the interaction between HSP90 and CD93. Combined with the significant decrease in the expression of glycosylated and non-glycosylated CD93 proteins after high-glucose treatment, we hypothesize that the downregulation of HSP90 expression and the reduction in HSP90-CD93 interaction under high-glucose conditions are one of the important reasons for the decreased CD93 expression. This finding provides new experimental evidence for understanding the impact of a high-glucose environment on the regulation of CD93 function.

[0067] Example 10: Effects of CD93 knockout on angiogenesis during wound healing in type 2 diabetes 1. CD93 - / - Construction of mice and type 2 diabetic mice To investigate the effect of CD93 knockout on angiogenesis during wound healing in type 2 diabetes, this study constructed four experimental animal models: wild-type (WT) group, wild-type diabetes (WT T2DM) group, CD93 gene knockout (CD93 knockout) group, and CD93 knockout group. - / - ) group, CD93 gene knockout diabetes (CD93 - / -T2DM group. Previous gene identification results from the research group confirmed CD93. - / - The gene knockout state of homozygous mice was stable and reliable. A type 2 diabetes model was induced by feeding a high-fat diet for 5 weeks followed by STZ injection. Figure 27 The results showed that on days 4, 7, and 14 after STZ injection, the fasting blood glucose level in mice remained above 11.1 mmol / L, indicating that the type 2 diabetes model was successfully established. Figure 28 Furthermore, prior to STZ injection, after 5 weeks of high-fat diet feeding, the average weight of wild-type diabetic mice was 28.88 g, which was not significantly different from the average weight of wild-type mice (26.2 g). p >0.05); CD93 - / - The average weight of diabetic mice was 29.98 g, compared to CD93... - / - There was no significant difference in the average weight of the mice in the two groups (26.18 g). p >0.05)( Figure 29 The results indicate that the weight changes of mice in each group during the high-fat diet feeding phase were relatively stable.

[0068] 2. CD93 - / - Effects on the healing of dorsal skin wounds in type 2 diabetic mice To investigate the effect of CD93 knockout on skin wound healing in type 2 diabetic mice, this study constructed a full-thickness skin wound model on the back of mice and recorded the wound healing process of each group on day 0 (D0), day 5 (D5), and day 10 (D10) after wound healing. Figure 30 And ImageJ was used to create a superimposed schematic diagram of wound healing at different time points in the same group of mice. Figure 31 Analysis of the wound area size at the same time point in the four groups of mice showed that at D0, the wound area size in the wild-type group, wild-type diabetic group, and CD93 group was significantly larger. - / - Group, CD93 - / - The wound area in the diabetic group was 23.66 ± 1.73 mm. 2 23.05 ± 0.58 mm 2 23.3 ± 1.08 mm 2 23.11 ± 3.09 mm 2 Compared with the wild-type group, the wild-type diabetic group had lower CD93 levels. - / - Group and CD93 - / - There was no significant difference in wound area among the diabetic groups. p >0.05)( Figure 32 This indicates that the initial wound conditions of the mice in each group were basically the same, and the wound model construction process was highly consistent.

[0069] At day 5, the wild-type group, the wild-type diabetic group, and CD93... - / - Group, CD93 - / - The wound area in the diabetic group was 4.4 ± 0.85 mm. 2 8.03 ± 1.52 mm 2 8.14 ± 2.17 mm 2 15.48 ± 1.86 mm 2 The wound area in the wild-type diabetes group was significantly larger than that in the wild-type group. p <0.05), indicating that type 2 diabetes significantly delayed the healing of skin wounds; wild-type diabetes group and CD93 - / - There was no significant difference in wound area between the groups. p >0.05); in addition, CD93 - / - The wound area in the diabetic group was significantly larger than that in the wild-type diabetic group. p <0.05), indicating CD93 - / - This further exacerbates the delay in healing of diabetic skin wounds. Figure 32 Middle left image and Figure 33 (Middle left image).

[0070] At day 10, the wild-type group, the wild-type diabetic group, and CD93... - / - Group, CD93 - / - The wound area in the diabetic group was 0.47 ± 0.08 mm. 2 2.62 ± 0.72 mm 2 2.44 ± 0.55 mm 2 4.57 ± 1.06 mm 2 The wild-type group showed almost complete healing of skin wounds; the wild-type diabetic group and CD93... - / - The wound area of ​​the group was significantly larger than that of the wild-type group. p <0.05); CD93 - / - The wound area in the diabetes group was significantly larger than that in the wild-type diabetes group and the CD93 group. - / - Group( p <0.05, indicating that CD93 knockout further delayed the late healing process of diabetic skin wounds. Figure 32 Middle left image and Figure 33 (Right image in the middle)

[0071] 3. CD93 - / - Effects on wound reepithelialization and collagen fiber deposition in type 2 diabetic mice To investigate the effects of CD93 knockout on wound re-epithelialization and collagen fiber deposition in type 2 diabetic mice, this study analyzed wound tissue using H&E staining and Masson staining. H&E staining results showed that at day 10, the wild-type group achieved complete re-epithelialization, granulation tissue formation, and tissue remodeling, while the epidermis of other groups exhibited discontinuity (red arrows) and poor tissue remodeling, especially in the wild-type diabetic group and the CD93 knockout group. - / - The epithelial layer in the diabetic group was significantly discontinuous. Figure 34 ).

[0072] Masson staining results showed that at day 10, the wild-type group exhibited significant collagen fiber deposition in the wound area, while the wild-type diabetic group and CD93 group showed... - / - In the diabetic group, some wound areas showed less collagen fiber deposition, and the integrity of the skin wound structure was impaired, manifesting as obvious tissue gaps. Figure 35 The above results indicate that CD93 knockout may inhibit collagen synthesis or deposition during wound healing, thereby affecting the normal wound repair process.

[0073] 4. CD93 - / - Effects on wound angiogenesis in type 2 diabetic mice To investigate CD93 - / - To investigate the effect of CD31 and CD93 on wound angiogenesis in type 2 diabetic mice, this study used dual immunofluorescence staining of CD31 and CD93 to detect angiogenesis in the wound area. At day 5, the levels of CD93 in the wild-type group, wild-type diabetic group, and other groups were [not specified in the original text]. - / - Group, CD93 - / - The CD31-positive areas (red) in the diabetes group were 6.24% ± 0.7%, 1.96% ± 0.15%, 1.87% ± 0.17%, and 0.5% ± 0.11%, respectively. Compared with the wild-type group, the wild-type diabetes group had higher CD93 levels. - / - Group, CD93 - / - The CD31-positive areas were significantly reduced in the diabetes group. p <0.05), of which CD93 - / - The CD31 positive area was the smallest in the diabetes group. Figure 36 ).

[0074] At day 10, the wild-type group, the wild-type diabetic group, and CD93... - / - Group, CD93 - / -The CD31-positive areas (red) in the diabetes groups were 11.06% ± 0.77%, 2.93% ± 0.48%, 2.43% ± 0.23%, and 0.77% ± 0.24%, respectively. Compared with day 5, the CD31-positive areas in all groups increased at day 10. Compared with the wild-type group, the wild-type diabetes group and CD93... - / - Group, CD93 - / - The number of CD31-positive areas was significantly reduced in the diabetic group. p <0.05), including the wild-type diabetes group and CD93 - / - There was no significant difference in the CD31 positive area between the groups. p >0.05); CD93 - / - The diabetic group had the fewest CD31-positive areas, indicating that CD93... - / - In the diabetic group, wound angiogenesis was significantly inhibited. Figure 37 In conclusion, CD93 - / - This further delayed the healing and angiogenesis of diabetic wounds, suggesting that CD93 plays an important role in angiogenesis in diabetic wounds.

[0075] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements 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 should 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. The application of a reagent that enhances the interaction between HSP90 and CD93 in the preparation of a drug that promotes diabetic wound healing, characterized in that: The reagent is an HSP90 agonist, and the HSP90 agonist is AMP-PCP.

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