Use of macrophage pyroptosis inhibitor in preparation of diabetic wound healing preparation

Abstract

The application provides an application of a macrophage pyroptosis inhibitor in preparation of a diabetic wound healing preparation. It belongs to the technical field of biological medicine. The macrophage pyroptosis inhibitor can effectively inhibit dissociation of hexokinase 1 from mitochondria, reduce the level of hexokinase 1 in the cytoplasm of macrophages, and reduce the activation of NLRP3 inflammasome through regulation of hexokinase 1, thereby reducing macrophage pyroptosis, promoting diabetic wound healing and relieving inflammation of wound tissue. The technical scheme has the advantages of clear mechanism, high safety and low treatment cost, and has important significance for the development of the diabetic wound healing preparation and the development of the diabetic complication treatment drug, and has an extremely wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a macrophage pyroptosis inhibitor in the preparation of diabetic wound healing agents. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by elevated blood glucose levels, its pathological basis being a relative or insufficient secretion of insulin. Diabetes is mainly divided into type 1 and type 2 diabetes. Type 1 diabetes is caused by autoimmune destruction of pancreatic beta cells, leading to insufficient insulin production, while type 2 diabetes is mainly characterized by insulin resistance or relative insulin deficiency. Typically, diabetic patients experience long-term, persistent hyperglycemia, which can lead to various serious systemic complications. Vascular disease is one of the most common complications of diabetes, often accompanied by increased blood viscosity and microvascular flow disturbances, leading to common complications such as retinopathy, diabetic nephropathy, and diabetic foot. Vascular disease significantly slows the healing of skin wounds, so even small wounds can develop into deep ulcers and are prone to infection. Intensive treatment to control blood glucose can prevent or delay many of these complications, but it cannot reverse existing lesions.

[0003] Besides insufficient blood perfusion due to vascular lesions, diabetic patients also experience wound healing impairment at the cellular and molecular levels. Normally, wound healing involves multiple stages, including hemostasis, inflammation, proliferation, contraction, and remodeling. However, in diabetes, patients experience a chronic inflammatory state, disrupting the fine-tuned regulation of wound healing. For example, normally, the M1 phenotype of macrophages kills microorganisms and releases pro-inflammatory factors in the early stages of inflammation, then switches to the M2 phenotype in the later stages, releasing anti-inflammatory and pro-healing factors to facilitate the transition from inflammation to proliferation. However, diabetes induces a pro-inflammatory phenotype in macrophages, and the transition from pro-inflammatory to anti-inflammatory is difficult, resulting in extremely slow wound healing in diabetic patients. Currently, clinical treatments for chronic wounds in diabetic patients mainly include growth factors, anti-inflammatory factors, blood products, and skin replacement therapy. However, these treatments are less effective at promoting wound healing and are costly.

[0004] In view of this, there is an urgent clinical need to develop a novel drug that can effectively intervene in the abnormal inflammatory microenvironment of diabetic wounds, so as to provide an effective treatment plan for solving this clinical problem. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to promote the healing of diabetic wounds.

[0006] To address the aforementioned technical problems, this invention provides the application of a macrophage pyroptosis inhibitor in the preparation of a diabetic wound healing agent.

[0007] Preferably, the macrophage pyroptosis inhibitor is neogamica or a pharmaceutically acceptable salt thereof.

[0008] Preferably, the content of macrophage pyroptosis inhibitor in the diabetic wound healing preparation is 0.1~10 mg / mL.

[0009] Preferably, the dosage form of the diabetic wound healing preparation is selected from any one of oral, injectable, or topical preparations.

[0010] Preferably, the topical preparation is selected from any one of ointments, gels, creams, sprays, and dressings.

[0011] Preferably, the injectable is a subcutaneous injectable.

[0012] Preferably, the diabetic wound healing preparation also includes pharmaceutically acceptable excipients.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention demonstrates that under high glucose conditions, substances such as gambogeylic acid can reduce the level of hexokinase 1 (HK1) in the cytoplasm of macrophages by inhibiting the dissociation of HK1 from mitochondria, thereby reducing the activation of the NLRP3 inflammasome and alleviating macrophage pyroptosis. This, in turn, promotes the healing of diabetic wounds and alleviates wound tissue inflammation. Furthermore, gambogeylic acid is a known plant extract, and its safety as an injection, oral, or topical preparation has been confirmed. Therefore, the technical solution provided by this invention has the advantages of a clear mechanism, high safety, and low treatment cost. It is of great significance for the development of diabetic wound healing agents and drugs for the treatment of diabetic complications, and has extremely broad application prospects. Attached Figure Description

[0014] Figure 1 These are wound healing photographs and closure rate calculation results from Example 1 of the present invention; Figure 2 HE staining results of mouse wound tissues in each group in Example 1 of this invention on day 14; Figure 3 The results of Masson staining of mouse wound tissues in each group in Example 1 of this invention on day 14; Figure 4 The results show the calculated mRNA expression level of TNF-α in Example 1 of this invention; Figure 5This refers to the calculated mRNA expression level of IL-6 in Example 1 of the present invention; Figure 6 The results are the calculated mRNA expression levels of IL-1β in Example 1 of this invention; Figure 7 The result is the calculated mRNA expression level of iNOS in Example 1 of this invention; Figure 8 The results of PI staining in Example 2 of this invention; Figure 9 The results of Western blot analysis of GSDMD and GSDMD-NT protein levels in the wound tissues of mice in each group in Example 2 of this invention at 7 days. Figure 10 The results of Western blot analysis of pyroptosis-related protein levels in the wound tissues of mice in Example 2 of this invention at 7 days. Figure 11 The results of quantitative analysis of the protein levels of NLRP3, cleaved caspase-1, cleaved caspase-1 / caspase-1 ratio, and ASC in Example 2 of this invention are as follows: Figure 12 The results of IL-1β and IL-18 level detection in the wound tissue of each group of mice in Example 2 of the present invention on day 7; Figure 13 The results of cell viability detection in Example 3 of this invention; Figure 14 This is the result of RT-qPCR detection of the mRNA expression level of pro-inflammatory mediators in macrophages in Example 3 of the present invention; Figure 15 The results of PI staining in Example 4 of this invention; Figure 16 The results of Western blot analysis of GSDMD and GSDMD-NT protein levels in macrophages of each group in Example 4 of this invention after 24 hours of culture. Figure 17 The results of Western blot analysis of protein expression related to NLRP3 inflammasome in each group of macrophages in Example 4 of this invention; Figure 18 The results of ELISA detection of IL-1β and IL-18 levels in macrophages in each group in Example 4 of this invention; Figure 19 This is the result of Western blot analysis of the effect of GNA treatment on mitochondrial dissociation of HK1 in macrophages and the level of HK1 in the cytoplasm in Example 5 of the present invention; Figure 20The results of RT-qPCR and Western blot detection of the relative expression level of HK1 24 h after siRNA transfection in Example 6 of this invention; Figure 21 This is the result of Western blot analysis of GSDMD and GSDMD-NT protein levels in macrophages of each group after 24 hours of culture, as described in Example 6 of this invention. Figure 22 This is the result of Western blot analysis of protein expression related to the NLRP3 inflammasome in each group of macrophages in Example 6 of this invention; Figure 23 The results of the ELISA detection of IL-1β and IL-18 levels in macrophages in each group in Example 6 of this invention are shown. Detailed Implementation

[0015] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0016] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0017] Pyroptosis, an inflammatory form of cell death, plays a crucial role in diabetes-related pathology. Primarily mediated by the NLRP3 inflammasome, pyroptosis activates caspase-1, leading to the cleavage of gasdermin D (GSDMD) and the release of inflammatory mediators. Studies have shown that hyperglycemia can induce abnormal activation of the NLRP3 inflammasome in macrophages, thereby amplifying the inflammatory response and delaying wound healing. Therefore, inhibiting NLRP3 inflammasome activation is considered a potential therapeutic approach for diabetic wounds.

[0018] Based on this, a specific embodiment of the present invention provides the application of a macrophage pyroptosis inhibitor in the preparation of a diabetic wound healing agent.

[0019] In the above embodiments, the macrophage pyroptosis inhibitor is neogamica or a pharmaceutically acceptable salt thereof.

[0020] In the above embodiments, the content of macrophage pyroptosis inhibitor in the diabetic wound healing preparation is 0.1~10 mg / mL.

[0021] In the above embodiments, the dosage form of the diabetic wound healing preparation is selected from any one of oral preparations, injections, and topical preparations.

[0022] In the above embodiments, the topical preparation is selected from any one of ointments, gels, creams, sprays, and dressings.

[0023] In the above embodiments, the injection is a subcutaneous injection.

[0024] In the above embodiments, the diabetic wound healing preparation also includes pharmaceutically acceptable excipients.

[0025] Specific embodiments of this invention demonstrate that macrophage pyroptosis inhibitors, represented by gamogenic acid (GNA), can promote wound closure and reduce inflammation in diabetic mice. Mechanistically, macrophage pyroptosis inhibitors can inhibit the activation of the NLRP3 inflammasome and macrophage pyroptosis under high glucose conditions. Furthermore, specific embodiments of this invention identify hexokinase 1 (HK1) as a key mediator in this process and demonstrate that macrophage pyroptosis inhibitors can inhibit HK1 dissociation from mitochondria, thereby regulating HK1-dependent inflammasome activation. These findings not only expand upon previous understanding of HK1-mediated metabolic regulation of inflammation but also reveal a novel mechanism by which macrophage pyroptosis inhibitors promote diabetic wound healing, highlighting the potential therapeutic value of targeting the HK1-NLRP3 axis. This has significant implications for the development of drugs for diabetic wound healing.

[0026] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0027] Unless otherwise specified, gambogeylic acid (GNA) in the following examples was purchased from MCE, China, catalog number 173932-75-7. RAW264.7 mouse macrophages were purchased from Procell, China. C57BL / 6J male mice (6–8 weeks old) were provided by the Animal Center of the Army Medical Center. Animals were housed under standard conditions: temperature 20–25°C, relative humidity 50 ± 5%, 12-hour light-dark cycle, and free access to food and water. All animal experimental procedures complied with the ARRIVE guidelines and the 8th edition (2011 revision) of the National Institutes of Health (NIH) Laboratory Animal Care and Use Guidelines. This study protocol was approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University of China (Approval No.: AMUWEC20267016). Throughout the study, every effort was made to minimize the discomfort, pain, and suffering of the animals.

[0028] In the following examples, unless otherwise specified, cell viability was assessed using the CCK-8 assay kit (Cell Counting Kit-8, CCK-8, Beyotime, China, C0037) according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates, with 10 μL of CCK-8 solution added to each well, and incubated at 37°C for 2 hours. The absorbance was then recorded at 450 nm using a Thermo Fisher microplate reader.

[0029] In the following examples, unless otherwise specified, the Western blot procedure is as follows: Total protein from tissues or cells is extracted using RIPA lysis buffer (Meilunbio, China) containing 0.1 mM benzyl sulfonyl fluoride (PMSF, Meilunbio). The lysis buffer is centrifuged at 12000 × g for 15 min at 4 °C, and the supernatant is collected for analysis.

[0030] To perform subcellular fractionation, mitochondria and cytoplasmic proteins were extracted using a mitochondrial extraction kit (Beyotime) according to the manufacturer's instructions. Briefly, cells were homogenized in separation buffer and differentially centrifuged to obtain cytoplasmic components (supernatant) and mitochondrial components (precipitate). The obtained mitochondrial precipitate was further lysed to extract proteins. SDHA (mitochondrial marker) and β-actin (cytoplasmic marker) were used to verify the purity of mitochondrial and cytoplasmic fractionation.

[0031] Protein concentration was determined using a BCA assay kit (Beyotime). Equal volumes of protein were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was then blocked in TBST with 5% skim milk for 1 hour (room temperature), followed by overnight incubation with primary antibody at 4°C. Primary antibody dilutions were all 1:1000, including: GSDMD (CST, #39754), GSDMD-NT (Abcam, ab255603), β-actin (CST, #4970), NLRP3 (Abcam, ab263899), caspase-1 (Abcam, ab179515), cleaved caspase-1 (CST, #89332), ASC (CST, #67824), HK1 (Thermo Fisher, MA5-14789), and SDHA (CST, #5839). After washing with TBST, the membrane was incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1–2 hours. The protein bands were developed using an enhanced chemiluminescence system (Bio-Rad, USA), and the intensity of the protein bands was quantitatively analyzed using ImageJ software.

[0032] Immunofluorescence staining was used to assess the infiltration of pyroptotic macrophages in wound tissues of diabetic mice. Paraffin-embedded skin sections (4 μm) were dewaxed with xylene and hydrated to the aqueous phase using a gradient of ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0) and heated for 10–15 min. After cooling to room temperature, the sections were permeabilized with 0.1% Triton X-100 (Beyotime) for 10 min, followed by blocking with 5% BSA (Beyotime) for 1 h. The sections were then incubated overnight at 4 °C with primary antibodies: CD68 (macrophage marker; Abcam, ab283654) and GSDMD-NT (pyroptosis marker; Abcam, ab255603). After washing with PBS, the sections were incubated with the corresponding fluorescent secondary antibodies at room temperature for 1 h in the dark. CD68 was labeled with Alexa Fluor 488, and GSDMD-NT with Alexa Fluor 594. The sections were then counterstained with DAPI (Yeasen) for 5 min. Finally, the wound was sealed with an anti-fluorescence quenching mounting medium and observed under a fluorescence microscope (Olympus, Japan) (×200). Co-localization analysis of CD68 and GSDMD-NT signals was performed to assess the level of macrophage pyroptosis in the wound tissue.

[0033] Pyroptosis cells were assessed using propidium iodide (PI) staining. Cells were first washed with PBS, then stained with 5 μmol / L PI (Yeasen) at 37°C for 15 min, followed by Hoechst (Yeasen) staining for 10 min. Cell images (×200) were then immediately observed under a fluorescence microscope (Olympus). The proportion of positive cells was assessed using ImageJ software.

[0034] The concentrations of inflammatory factors, including IL-1β and IL-18, in tissue homogenates or cell culture supernatants were detected using commercially available ELISA kits (Elabscience, China, E-EL-M0037 and E-EL-M0730), strictly following the instructions.

[0035] For tissue samples, wound tissue was collected and homogenized with cold physiological saline under ice bath conditions. The homogenate was then centrifuged at 5000×g for 15 min at 4°C, and the supernatant was collected for subsequent analysis. For cell experiments, cell culture supernatant was collected and centrifuged to remove cell debris.

[0036] Statistical analysis and graphing were performed using GraphPad Prism 8.0 (USA). Data are expressed as mean ± standard deviation (SD). One-way ANOVA with Tukey post-hoc test was used for multiple group comparisons; or two-way ANOVA with Bonferroni post-hoc correction for pairwise comparisons was used. A p-value less than 0.05 (p < 0.05) was considered statistically significant.

[0037] Example 1

[0038] gambogeylic acid can promote the healing of diabetic wounds and relieve wound inflammation. Mouse model preparation: Mice were fasted for 12 hours, followed by a single intraperitoneal injection of streptozotocin (STZ) (150 mg / kg). STZ was freshly prepared using citrate buffer (pH 4.2–4.5; Yeasen). Control mice received an equal volume of citrate buffer. One week later, fasting blood glucose levels were measured. Mice with blood glucose levels above 16.7 mmol / L were defined as diabetic models and used in subsequent experiments. After inducing diabetes, mice were anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection), and their dorsal hair was removed. A full-thickness excision wound (8 mm in diameter) was then created on the back of each mouse using a sterile biopsy punch.

[0039] For treatment, GNA or its carrier was administered topically around the wound. In short, GNA (2 mg / mL dissolved in PBS, 100 μL per wound) was injected subcutaneously at multiple points around the wound every two days; diabetic control mice received an equal volume of PBS. The healing process was documented by photographing the wound on days 0, 1, 4, 7, 10, and 14 post-injury. The wound area was measured using ImageJ software (USA), and the closure rate was calculated. The wound healing photographs and closure rate calculation results are shown below. Figure 1 As shown, where, Figure 1 In the figure, A represents the effect of GNA on wound healing in diabetic mice. Figure 1 In this context, B represents the calculated closure ratio. Figure 1 The results showed that GNA treatment promoted wound healing in diabetic mice. Compared with the sham group, diabetic mice exhibited delayed wound closure; however, GNA administration significantly accelerated wound healing at multiple time points. Consistently, quantitative analysis showed increased wound closure rates on days 4, 7, 10, and 14 after GNA administration.

[0040] At predetermined time points, mice were euthanized by intraperitoneal overdose anesthesia with sodium pentobarbital, and wound tissue was collected for further analysis.

[0041] Histological analysis: Wound tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm. HE staining was used to assess histological structure and inflammatory cell infiltration; Masson staining was used to assess collagen deposition and tissue remodeling. All sections were observed and imaged using an optical microscope (Leica, Germany). HE staining results of mouse wound tissue on day 14 are shown below. Figure 2 As shown, the Masson staining results of the wound tissues of mice in each group on day 14 are as follows. Figure 3 As shown. Figure 2 HE staining results showed that the skin structure of diabetic mice was disordered and accompanied by obvious inflammatory cell infiltration; while GNA treatment could restore tissue integrity and reduce inflammatory cell infiltration. Figure 3 Masson staining results showed that collagen deposition in diabetic wounds was reduced and significantly improved after GNA treatment.

[0042] RT-PCR detection: RNA extraction was performed using TRIzol reagent (Invitrogen, USA, 15596026CN), strictly following the manufacturer's instructions. The concentration and purity of the extracted RNA were determined using a Nanodrop spectrophotometer (Thermo Fisher, USA). After RNA extraction, reverse transcription was performed using a PrimeScript RT kit (Yeason, 11123ES60) to convert RNA into cDNA. Quantitative real-time PCR was performed using an Mx3000P real-time PCR system (Agilent Technologies, USA). The reaction mixture included SYBR Mixture (Yeason, 11184ES08), specific primers, and a cDNA template. Normalization was performed using the internal reference gene β-actin. Primer sequences (synthesized by Sangon Biotch, China) are as follows: β-Actin forward: 5'-CTACCTCATGAAGATCCTGACC-3' (SEQ ID No. 1); β-Actin reverse: 5'-CACAGCTTCTCTTTGATGTCAC-3' (SEQ ID No. 2); TNF-α forward: 5'-ACGCTCTTCTGTCTACTGAACTTCG-3' (SEQ ID No. 3); TNF-α reverse: 5'-TGGTTTGTGAGTGTGAGGGTCTG-3' (SEQ ID No. 4); IL-6 forward: 5'-TTCTTGGGACTGATGCTGGTGAC-3' (SEQ ID No. 5); IL-6 reverse: 5'-GTGGTATCCTCTGTGAAGTCTCCTC-3' (SEQ ID No. 6); IL-1β forward: 5'-CTCGCAGCAGCACATCAACAAG-3' (SEQ ID No. 7); IL-1β reverse: 5'-CCACGGGAAAGACACAGGTAGC-3' (SEQ ID No. 8); iNOS forward: 5'-AGCCGCACCACCCTCCTC-3' (SEQ ID No. 9); iNOS reverse: 5'-TCCACAACTCGCTCCAAGATTCC-3' (SEQ ID No. 10); HK1 forward: 5'-GTCTCCTTCCGATCAGCCAACC-3' (SEQ ID No. 11); HK1 reverse: 5'-AGAACCGTCTACGCCAACTGTG-3' (SEQ ID No. 12).

[0043] After RT-PCR, the relative mRNA expression levels of inflammatory factors in the wound tissues of mice in each group were calculated, with the mRNA expression level of TNF-α as shown below. Figure 4 As shown; the mRNA expression level of IL-6 is as follows Figure 5 As shown; the mRNA expression level of IL-1β is as follows Figure 6 As shown; the mRNA expression level of iNOS is as follows: Figure 7 As shown. By Figures 4-7 The results showed that the expression levels of pro-inflammatory cytokine mRNAs, including TNF-α, IL-6, IL-1β, and iNOS, were elevated in the wound tissue of diabetic mice on day 7. GNA treatment effectively inhibited the expression of these inflammatory mediators, suggesting that it can attenuate the local inflammatory response. In summary, these results indicate that GNA can accelerate the healing of diabetic wounds, and its potential mechanism may be related to the inhibition of inflammation.

[0044] Example 2

[0045] GNA can inhibit macrophage pyroptosis in wound tissue of diabetic mice. To clarify whether GNA can affect macrophage pyroptosis under high glucose conditions, macrophages from mouse wound tissues in each group of Example 1 were subjected to PI (propidium iodide) staining. The results are as follows: Figure 8 As shown, Figure 8 In the image, A represents a representative PI staining image (200×) of macrophages in each group at 7 days. Figure 8 In the figure, B represents the quantitative analysis result of the number of PI-positive cells, derived from... Figure 8 It is evident that high glucose stimulation significantly increased the number of PI-positive cells, suggesting exacerbated cell membrane damage accompanied by increased pyroptosis. In contrast, GNA treatment significantly reduced the proportion of PI-positive cells compared to the high glucose group.

[0046] Western blot analysis was performed on the GSDMD and GSDMD-NT protein levels in the wound tissues of mice in each group at 7 days. The results are as follows: Figure 9 As shown, Figure 9 In the figure, A represents the Western blot results of GSDMD and GSDMD-NT protein levels. Figure 9 B in the table represents the quantitative analysis results of GSDMD protein levels (n=6). Figure 9 In this context, C represents the quantitative analysis result of the GSDMD-NT / GSDMD ratio (n=6). Figure 9 It is evident that the protein level of GSDMD-NT (GSDMD activated cleavage fragment) increases under high glucose conditions, while its expression decreases after GNA administration, suggesting that GNA can inhibit pyroptosis activation.

[0047] Western blot analysis was performed on pyroptosis-related proteins in the wound tissues of mice in each group at 7 days, including NLRP3, caspase-1, cleaved caspase-1, and ASC. The results are as follows: Figure 10 As shown in the figure. After Western blot analysis, the protein levels of NLRP3, cleaved caspase-1, caspase-1, and ASC were quantitatively analyzed, and the cleaved caspase-1 / caspase-1 ratio was calculated (n=6). The results are as follows. Figure 11 As shown, where, Figure 11 In the figure, A represents the quantitative analysis of NLRP3 protein levels. Figure 11 B in the figure represents the quantitative analysis of cleaved caspase-1 protein levels. Figure 11 In the analysis, C represents the cleaved caspase-1 / caspase-1 ratio. Figure 11 In this context, D represents the quantitative analysis of ASC protein levels. Figure 10 and Figure 11 It is evident that high glucose stimulation can upregulate the levels of NLRP3, cleaved caspase-1, and ASC proteins, while also increasing the cleaved caspase-1 / caspase-1 ratio; GNA treatment can effectively inhibit these changes.

[0048] The levels of IL-1β and IL-18 in the wound tissues of mice in each group were detected by ELISA on day 7. The results are as follows: Figure 12 As shown, Figure 12 In the figure, A represents the quantitative detection result of IL-1β. Figure 12 In the figure, B represents the quantitative detection result of IL-18. (From...) Figure 12 It was observed that the levels of pro-inflammatory factors IL-1β and IL-18 were elevated in diabetic wound tissue, while these levels decreased after GNA treatment. In summary, these results indicate that GNA can alleviate macrophage pyroptosis in the wound tissue of diabetic mice.

[0049] Example 3

[0050] GNA can inhibit the inflammatory response of macrophages under high glucose conditions. RAW264.7 mouse macrophages were purchased from Procell (China) and cultured in DMEM medium (Procell) containing 10% fetal bovine serum under normoxic conditions (Thermo Fisher). To simulate diabetic conditions, cells were cultured in high-glucose medium (45 mM) for 24 h; control cells were maintained in standard medium (25 mM). Different concentration gradients of GNA (0–10 μM) were established to determine the optimal working concentration. After treatment with different concentrations of GNA for 24 h, the results were analyzed.

[0051] Cell viability was assessed using a CCK-8 assay. Results are as follows: Figure 13 As shown, Figure 13 As shown in Figure A, GNA exhibits no significant cytotoxicity to cells within the concentration range of 0–1 μM. Figure 13 Figure B shows that under high glucose conditions, macrophage viability was significantly reduced compared to the control group; while GNA treatment improved cell viability. Notably, 1 μM GNA had the most significant protective effect and was therefore selected for subsequent experiments.

[0052] To further evaluate the anti-inflammatory effect of GNA, the mRNA expression level of pro-inflammatory mediators in macrophages was detected by RT-qPCR. The relevant primers are described in Example 1. The results are as follows: Figure 14 As shown, Figure 14 In this context, A represents the result of TNF-α mRNA expression level detection; Figure 14 B in the figure represents the result of IL-6 mRNA expression level detection; Figure 14 In the figure, C represents the result of IL-1β mRNA expression level detection; Figure 14 In the figure, D represents the result of iNOS mRNA expression level detection. Figure 14 It is evident that high glucose stimulation upregulates the expression of TNF-α, IL-6, IL-1β, and iNOS in macrophages; conversely, GNA treatment significantly reduces the expression of these inflammatory factors. In summary, these results indicate that GNA can effectively alleviate high glucose-induced macrophage inflammatory responses.

[0053] Example 4

[0054] Under high glucose conditions, GNA can inhibit the activation of the macrophage NLRP3 inflammasome and reduce macrophage pyroptosis. Based on Example 3, to clarify whether GNA can affect macrophage pyroptosis under high glucose conditions, macrophages were subjected to PI (propidium iodide) staining. The results are as follows: Figure 15 As shown, Figure 15 A in the image represents a PI staining image (200×). Figure 15 In the figure, B represents the quantitative analysis of the number of PI-positive cells (n=6, scale bar: 50μm). [From...] Figure 15 It is evident that high glucose stimulation significantly increased the number of PI-positive cells, suggesting exacerbated cell membrane damage accompanied by increased pyroptosis. In contrast, GNA treatment significantly reduced the proportion of PI-positive cells compared to the high glucose group.

[0055] The levels of GSDMD and GSDMD-NT proteins in macrophages of each group after 24 h of culture were detected by Western blot. The results are as follows: Figure 16 As shown, Figure 16 In the image, A represents a Western blot image. Figure 16 B in the table represents the quantitative analysis of GSDMD protein levels (n=6). Figure 16 C in the figure represents the quantitative analysis of the GSDMD-NT / GSDMD ratio (n=6). From Figure 16 It is evident that the protein level of GSDMD-NT (GSDMD activated cleavage fragment) increases under high glucose conditions, while its expression decreases after GNA administration, suggesting that GNA can inhibit pyroptosis activation.

[0056] The expression of proteins associated with the NLRP3 inflammasome in macrophages in each group was detected by Western blot. The results are as follows: Figure 17 As shown, Figure 17 In the image, A represents a Western blot image. Figure 17 B in the figure represents the NLRP3 protein quantification result; Figure 17 In this context, C represents the quantitative detection result of caspase-1 protein; Figure 17 D in the figure represents the quantitative result of the cleaved caspase-1 / caspase-1 ratio; Figure 17 In the figure, E represents the result of ASC protein quantification. Figure 17 It is evident that high glucose stimulation can upregulate the levels of NLRP3, cleaved caspase-1, and ASC proteins, while also increasing the cleaved caspase-1 / caspase-1 ratio; GNA treatment can effectively inhibit these changes.

[0057] The levels of IL-1β and IL-18 in macrophages of each group were detected by ELISA. The results are as follows: Figure 18 As shown, Figure 18 In the figure, A represents the quantitative detection result of IL-1β. Figure 18 In the figure, B represents the quantitative detection result of IL-18. (From...) Figure 18 It was observed that the secretion levels of pro-inflammatory factors IL-1β and IL-18 were increased in the culture supernatant under high glucose conditions, while GNA treatment reduced their levels. In summary, these results indicate that under high glucose conditions, GNA can inhibit NLRP3 inflammasome activation, thereby alleviating macrophage pyroptosis.

[0058] Example 5

[0059] GNA treatment inhibits HK1 dissociation from mitochondria and reduces HK1 levels in macrophage cytoplasm. Based on Examples 1 and 3, the effects of GNA treatment on mitochondrial dissociation of HK1 cells and the level of HK1 in the cytoplasm of macrophages were analyzed by Western blot. The results are as follows: Figure 19 As shown, Figure 19 A and B in the figure represent the Western blot results of HK1 in the cytoplasmic components of macrophages in each group at 24 h. Figure 19 C and D in the table represent the Western blot results of macrophages in the mitochondrial components at 24 h. Figure 19 E and F in the figure represent the Western blot results of HK1 in the cytoplasmic components of the wound tissue of each group of mice on day 7; Figure 19 G and H in the table represent the Western blot results of HK1 in the mitochondrial components of wound tissue from each group of mice on day 7. Figure 19 It is evident that high glucose stimulation promotes the dissociation of HK1 from the mitochondrial membrane in macrophages. This is manifested by a decrease in HK1 levels in the mitochondrial fractionation and a corresponding increase in HK1 levels in the cytoplasmic fractionation. Conversely, GNA treatment reverses these changes: mitochondrial localization of HK1 is restored, and its accumulation in the cytoplasm is reduced. Consistent with in vitro results, a similar trend was observed in vivo. In diabetic mice, HK1 expression in the mitochondrial fractionation is decreased, while cytoplasmic HK1 levels are significantly increased. After GNA administration, on day 7, mitochondrial HK1 levels increased in wound tissue, while cytoplasmic HK1 levels decreased. In summary, these results indicate that under diabetic conditions, GNA can inhibit the dissociation of HK1 from mitochondria, thereby maintaining its mitochondrial localization in macrophages.

[0060] Example 6

[0061] GNA affects macrophage pyroptosis by regulating HK1-mediated NLRP3 inflammasome activation. To investigate the role of HK1 in GNA-mediated macrophage pyroptosis regulation, HK1 was knocked down using a specific siRNA. The small interfering RNA (siRNA) targeting HK1 has the following sequence: siRNA1 sense: 5'-GGCGGGUCUUCCUUUCGAAUU-3' (SEQ ID No. 13); antisense: 5'-AUUCGAAAGGAAGACCCGCCU-3' (SEQ ID No. 14); siRNA2 sense: 5'-GCAGCUUUUUGAUCA CGUCGUU-3' (SEQ ID No. 15); antisense: 5'-CGACGUGAUCAAAAAGCUGCUU-3' (SEQ ID No. 16); siRNA3 sense: 5'-GGAGCCUUUGGGGAUGAUGGUU-3' (SEQ ID No. 17); antisense: 5'-CCAUCAUCCCCAAAG GCUCCUU-3' (SEQ ID No. 18); NC sense: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID No. 19); antisense: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID No. 20).

[0062] The siRNA and vector were transfected into RAW264.7 cells using Lipofectamine 3000 (Thermo Fisher). RT-qPCR and Western blot were performed 24 h after transfection.

[0063] RT-qPCR and Western blot results are as follows Figure 20 As shown, Figure 20 In the figure, A represents the results of RT-PCR quantitative detection of HK1 mRNA expression level in macrophages after siRNA intervention (n=6). Figure 20 In the figure, B represents the Western blot analysis results of HK1 protein levels after siRNA intervention. Figure 20 C in the figure represents the quantitative analysis of HK1 protein levels after siRNA intervention (n=6).

[0064] The levels of GSDMD and GSDMD-NT proteins in macrophages of each group after 24 h of culture were detected by Western blot. The results are as follows: Figure 21 As shown, Figure 21 In the image, A represents a Western blot image. Figure 21 B in the table represents the quantitative analysis of GSDMD protein levels (n=6). Figure 21 C in the figure represents the quantitative analysis of the GSDMD-NT / GSDMD ratio (n=6). From Figure 21 It is evident that, in the presence of GNA, knocking down HK1 can further reduce the GSDMD-NT level and decrease the GSDMD-NT / GSDMD ratio, suggesting that pyrodeogenesis is further suppressed.

[0065] The expression of proteins associated with the NLRP3 inflammasome in macrophages in each group was detected by Western blot. The results are as follows: Figure 22 As shown, Figure 22 In the image, A represents a Western blot image. Figure 22 B in the figure represents the NLRP3 protein quantification result; Figure 22 In this context, C represents the quantitative detection result of caspase-1 protein. Figure 22 D in the figure represents the quantitative result of the cleaved caspase-1 / caspase-1 ratio; Figure 22 In the figure, E represents the result of ASC protein quantification. Figure 22 It is evident that, compared to using GNA alone, HK1 knockdown can further downregulate NLRP3 inflammasome-related proteins, including NLRP3, cleaved caspase-1, and ASC, while also reducing the cleaved caspase-1 / caspase-1 ratio. The levels of IL-1β and IL-18 in macrophages of each group were detected by ELISA. The results are as follows: Figure 23 As shown, Figure 23 In the figure, A represents the quantitative detection result of IL-1β. Figure 23 In the figure, B represents the quantitative detection result of IL-18. (From...) Figure 23 It is evident that under high glucose conditions and in the context of GNA treatment, HK1 knockdown can further reduce the secretion levels of IL-1β and IL-18 in macrophage culture supernatant.

[0066] In summary, these results indicate that GNA inhibits NLRP3 inflammasome activation by regulating HK1, and further reduces macrophage pyroptosis.

[0067] As demonstrated in the above examples, GNA can promote diabetic wound healing by inhibiting macrophage pyroptosis, through the mechanism of suppressing HK1-mediated NLRP3 inflammasome activation. At the mechanistic level, GNA can prevent HK1 dissociation from mitochondria, thereby alleviating the inflammatory response. These results provide new insights into the metabolic regulation of inflammation in diabetic wounds and suggest that targeting the HK1-NLRP3 axis may be a promising therapeutic strategy.

[0068] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Application of macrophage pyroptosis inhibitors in the preparation of diabetic wound healing agents.

2. The application as described in claim 1, characterized in that, The macrophage pyroptosis inhibitor is neogamica or a pharmaceutically acceptable salt thereof.

3. The application as described in claim 1, characterized in that, The content of macrophage pyroptosis inhibitor in the stated diabetic wound healing preparation is 0.1~10 mg / mL.

4. The application as described in claim 1, characterized in that, The dosage form of the diabetic wound healing preparation is selected from any one of oral, injectable, or topical preparations.

5. The application as described in claim 4, characterized in that, The topical preparation is selected from any one of ointments, gels, creams, sprays, and dressings.

6. The application as described in claim 4, characterized in that, The injection is a subcutaneous injection.

7. The application as described in claim 1, characterized in that, The diabetic wound healing preparation also includes pharmaceutically acceptable excipients.

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