Application of D-mannose in preparation of medicine for treating postoperative incision pain

Abstract

The invention discloses an application of D-mannose in preparation of a medicine for treating postoperative incision pain, and particularly relates to an application of D-mannose as an effective component in preparation of a local external gel preparation for relieving postoperative pain and promoting wound healing. The invention discloses that D-mannose reduces the expression of proinflammatory factors (such as TNF-alpha, IL-1beta and IL-6) by inhibiting NF-kappa B and MAPK inflammation signal pathways for the first time, so that the dual effects of analgesia and healing promotion are realized. Experiments show that the D-mannose has the optimal effect under the specific concentration (such as 3% w / v), and the D-mannose is high in safety and free of addiction through a local external administration mode. The invention provides a novel, safe and effective non-opioid local treatment choice for postoperative pain management, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a new pharmaceutical use of a known compound, D-mannose, and particularly to the use of D-mannose in the preparation of a drug for treating postoperative incision pain. Background Technology

[0002] Post-operative pain (POP) is one of the most common types of acute pain in clinical practice, severely impacting patients' postoperative recovery and quality of life. Currently, commonly used analgesics include opioids (such as morphine) and nonsteroidal anti-inflammatory drugs (NSAIDs). Opioids are the primary analgesics for moderate to severe postoperative pain, but they have serious side effects such as addiction, respiratory depression, nausea, and vomiting. NSAIDs may cause gastrointestinal damage and cardiovascular risks, and they only relieve pain without contributing to wound healing.

[0003] Therefore, there is an urgent clinical need for a novel topical treatment drug that can effectively relieve pain, promote wound healing, and has a high safety profile. An ideal drug should not only provide local analgesia but also actively promote wound healing. Uncontrolled local inflammation is a core cause of postoperative incision pain and poor healing. In recent years, natural products have gained attention due to their high safety and wide availability. D-Mannose, a naturally occurring hexose and an isomer of glucose, is widely found in fruits and is an important precursor for the synthesis of glycoproteins in the human body. Due to its extremely high safety profile (it is an endogenous substance in the human body), D-Mannose has been used as a dietary supplement and as an adjunct treatment for urinary tract infections. However, to date, there are no systematic studies or reports on the application of D-Mannose in the treatment of postoperative incision pain. Summary of the Invention

[0004] To address the problems mentioned above, this invention provides an application of D-mannose in the preparation of drugs for treating postoperative incision pain. This invention is the first to discover that D-mannose has significant postoperative analgesic and wound healing promoting effects at specific concentrations. The mechanism lies in inhibiting local inflammatory responses, blocking the excessive activation of NF-κB and MAPK signaling pathways, and reducing the expression of pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6).

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of D-mannose in the preparation of a drug for treating postoperative incision pain.

[0006] Through in-depth research, the inventors discovered that postoperative incision pain is mainly driven by the continuous activation of upstream signaling pathways (such as NF-κB and MAPK) caused by the excessive release of local inflammatory factors (such as IL-1β, TNF-α, IL-6).

[0007] This invention is the first to discover that the natural product D-mannose can safely and effectively inhibit these key inflammatory pathways. Its core features are anti-inflammatory and analgesic effects, while maintaining extremely high biocompatibility and actively promoting wound healing.

[0008] Furthermore, the drug is a topical preparation.

[0009] Furthermore, the topical preparation is a gel preparation.

[0010] This invention uses a topical gel formulation, which allows the drug to act directly on the wound with extremely low systemic exposure. This fundamentally avoids the systemic side effects (such as respiratory depression, addiction, and constipation) caused by opioids and the gastrointestinal damage, cardiovascular and renal risks that NSAIDs may cause.

[0011] Furthermore, the mass concentration of D-mannose in the gel formulation is 1-5%.

[0012] Preferably, the mass concentration of D-mannose in the gel formulation is 3%.

[0013] This invention not only confirms the analgesic activity of D-mannose, but also reveals through rigorous screening that its therapeutic effect exhibits significant concentration-dependent specificity. Experimental results show that a 3% concentration can exert a significant analgesic effect. This finding rules out the non-specific effects of D-mannose merely as a common humectant or sugar, and determines its precise dosage as a drug, possessing outstanding clinical guiding significance.

[0014] Furthermore, D-mannose reduced the levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, and upstream signaling pathways NF-κB and MAPK.

[0015] This invention is not merely a simple observation of phenomena, but rather the construction of a multi-layered, mutually corroborating system of scientific evidence that clearly elucidates its "anti-inflammatory-analgesic-healing" mechanism: In both cells and animal tissues, D-mannose has been shown to effectively inhibit the activation of the two core inflammatory signaling pathways, NF-κB and MAPK (manifested as a decrease in p-p65 and p-p38 phosphorylation levels). This inhibition of these pathways leads to a significant reduction in the expression and release of downstream key pro-inflammatory factors (IL-1β, TNF-α, IL-6).

[0016] The present invention also provides a pharmaceutical composition for treating postoperative incision pain, comprising an effective amount of D-mannose and a pharmaceutically acceptable topical carrier.

[0017] Furthermore, the mass concentration of the D-mannose is 3%.

[0018] Furthermore, the localized external carrier is a gel.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention not only confirmed the analgesic activity of D-mannose, but also found that its efficacy has significant concentration-dependent specificity through rigorous and systematic in vitro and in vivo dose screening. It determined the optimal therapeutic concentration (3% w / v) of D-mannose for local analgesia and healing promotion, and found that its efficacy has a "bell effect". The effect decreases when the concentration is too high (e.g., 5%). This discovery ruled out the non-specific role of D-mannose as just an ordinary "humectant" or "sugar", and provided a key basis for safe and efficient use in clinical practice.

[0020] (2) Currently, commonly used postoperative analgesics (such as opioids and NSAIDs) mainly focus on "treating the symptoms" - that is, blocking pain signal transmission or inhibiting some pain mediators, but they are not beneficial to the wound healing process and may even interfere with it. The biggest innovation of this invention is that it achieves the synergistic unity of "analgesia" and "healing promotion": on the one hand, it can quickly relieve inflammatory pain (treating the symptoms) by significantly inhibiting the release of key local inflammatory factors (TNF-α, IL-1β, IL-6); on the other hand, it can actively accelerate wound healing (treating the root cause) by inhibiting excessive inflammatory response and creating a favorable microenvironment for tissue repair. In animal behavior, this is reflected in the fact that, compared with the model group, the treatment group had significantly reduced pain sensation (increased PWT and PWL) and faster healing speed.

[0021] (3) The present invention uses a topical gel formulation, which allows the drug to act directly on the wound. The systemic exposure is extremely low and the safety is extremely high. It fundamentally avoids the systemic side effects (such as respiratory depression, addiction, and constipation) caused by opioids and the gastrointestinal damage, cardiovascular and kidney risks that NSAIDs may cause.

[0022] (4) This invention is not a simple observation of phenomena, but rather a complete scientific evidence system that is multi-layered and mutually corroborating, clearly elucidating its "anti-inflammatory-analgesic-healing" mechanism: Upstream pathway: In both cells and animal tissues, D-mannose has been shown to effectively inhibit the activation of the two core inflammatory signaling pathways, NF-κB and MAPK (p38) (manifested as a decrease in the phosphorylation levels of p-p65 and p-p38).

[0023] Midstream mediators: Inhibition of the above pathways leads to a significant reduction in the expression and release of key downstream pro-inflammatory factors (TNF-α, IL-1β, IL-6).

[0024] Downstream effects: 1. Reduced inflammation: Vascular permeability tests (reduced Evans blue exudation) and macroscopic observations (reduction of redness and swelling) directly confirmed the relief of local inflammation. 2. Pain relief: Behavioral tests (increased PWT and PWL) objectively reflected the analgesic effect. 3. Accelerated healing: Wound area and upregulated expression of healing-promoting proteins (Collagen I, α-SMA) demonstrated accelerated tissue repair from both macroscopic and microscopic perspectives. This means that the drug not only relieves pain but also actively accelerates the structural reconstruction and functional recovery of damaged soft tissue.

[0025] (5) This invention provides a novel clinical application for D-mannose (an readily available, stable, and safe compound) with huge market demand (postoperative analgesia and wound care). Its preparation is simple and easy to industrialize, and it can be developed into new postoperative analgesia and wound care gels, dressings, and other products. Attached Figure Description

[0026] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0027] Figure 1 The effect of different concentrations of mannose on the cell viability of NIH / 3T3 cells at different time points was determined by CCK-8 assay. Figure 2 To detect IL-6 expression in NIH / 3T3 cells treated with different concentrations of mannose using ELISA under IL-1β stimulation; Figure 3 To detect the expression of p-p65, p65, p-p38, p38, and β-Tubulin in NIH / 3T3 cells after treatment with 40 mmol / L D-mannose in the presence of IL-1β stimulation, West Blotting was used. Figure 4 The changes in mechanical and thermal pain in POP mice under different concentrations of D-mannose treatment: A. Changes in mechanical pain in mice; B. Changes in thermal pain in mice; Blank control group Sham, model group POP, low-dose mannose treatment group POP+Man-L, medium-dose mannose treatment group POP+Man-M, high-dose mannose treatment group POP+Man-H. Figure 5 Quantitative analysis of Evans Blue exudate in the foot tissue of mice in each group on the 3rd day after surgery and macroscopic photographs of the foot wounds of mice in each group; Figure 6To detect the expression of p-p65, p65, p-p38, p38, β-Tubulin, Collagen I, and α-SMA in mice using West Blotting, the blank control group Sham, the model group POP, and the treatment group POP + Mannose were used. Figure 7 The expression of inflammatory factors TNF-α, IL1-β, and IL-6 in mice was detected using ELISA. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The technical solution of this invention systematically verifies the efficacy of D-mannose in treating postoperative incision pain and related pain through two parts: in vitro cell experiments (Experiment 1) and in vivo animal experiments (Experiment 2), and elucidates its specific mechanism of action.

[0030] Experiment 1: Verification of Specific Mechanisms at the Cellular Level 1. Experimental Principle This experiment first established an in vitro fibroblast (NIH / 3T3) inflammation model to verify whether D-mannose can block the downstream key inflammatory pathway (NF-κB, MAPK) induced by inflammatory stimulation (IL-1β) and inhibit the expression of related inflammatory factors (IL-6). Fibroblasts are key responding cells in wound healing and the inflammatory microenvironment; stimulation with IL-1β (a major pro-inflammatory factor) can effectively mimic the amplification effect of inflammatory signals in postoperative wounds.

[0031] 2. Experimental Design and Specific Implementation Steps 2.1 Cell lines, plasmids, and main reagents Cell line: Mouse fibroblast cell line (NIH / 3T3).

[0032] Main reagents: cell culture medium (DMEM), fetal bovine serum (FBS), recombinant mouse IL-1β, D-mannose, Lipofectamine™ 3000 transfection reagent, Opti-MEM medium, cell counting kit-8 (CCK-8).

[0033] ELISA reagents: Mouse IL-6 ELISA kit (purchased from Macklin) and Mouse TNF-α ELISA kit (purchased from Macklin).

[0034] Western Blot reagents: RIPA lysis buffer, p-p65 antibody (Proteintech, 82335-1-RR), p65 antibody (Proteintech, 10745-1-AP), p-p38 antibody (Proteintech, 28796-1-AP), p38 antibody (Proteintech, 14064-1-AP), and β-Tubulin antibody (Proteintech, 10094-1-AP), the internal reference protein.

[0035] 2.2 Cytotoxicity and proliferation activity assay (CCK-8 assay) Fibroblasts in logarithmic growth phase were divided into groups of 5 × 10⁻⁶. 3 Cells were seeded at a density of 10 cells / well in 96-well plates and incubated for 24 hours to allow for complete adhesion. The old culture medium was discarded and replaced with fresh medium containing 0, 10, 20, 40, 60, and 80 mmol / L D-mannose, with three replicates for each concentration. The plates were returned to the incubator and incubated for specific time periods (24 hours, 48 ​​hours, and 72 hours). For assay, 10 µL of CCK-8 solution was added to each well, and incubation continued for 1–2 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader (Infinite M200 Pro, Tecan Group, Ltd., Männedorf, Switzerland), which reflects cell proliferation and viability.

[0036] 2.3 Mechanism Verification Experiment 2.3.1 Optimal therapeutic concentration screening (ELISA) Grouping: NIH / 3T3 cells were divided into the following groups: Group A: Control Group Group B: Model group (IL-1β) Group C: Low-dose group (IL-1β + Mannose 10mmol / L) Group D: Medium-dose group (IL-1β + Mannose 20mmol / L) Group E: High-dose group (IL-1β + Mannose 40mmol / L) ELISA assay: Collect the culture supernatant from each group of cells and centrifuge at 4°C at low speed to remove cell debris and impurities. Assay procedure: Add the standards and test supernatant samples to the pre-coated wells of an ELISA plate and incubate at room temperature. Wash the plate and add biotin-labeled detection antibody for incubation. Wash the plate and add HRP-labeled streptavidin for incubation. Wash the plate and add TMB substrate solution for color development in the dark. Finally, add stop solution to terminate the reaction. Data acquisition and analysis: Measure the absorbance (OD value) of each well at 450 nm using an ELISA reader. Plot a standard curve using the concentrations of the standards and their corresponding OD values, and calculate the IL-6 concentration (pg / mL) from the curve based on the sample OD values.

[0037] 2.3.2 Core Mechanism Verification (Western Blot) Grouping: Based on the screening results, 40 mmol / L was selected as the optimal therapeutic concentration for mechanism verification.

[0038] Group A: Control Group Group B: Model group (IL-1β) Group C: Mannose treatment group (IL-1β + Mannose 40mmol / L) Western Blot Analysis: Protein Extraction and Quantification: To extract total protein, cells were lysed on ice for 15 minutes in RIPA lysis buffer containing protease and phosphatase inhibitors, followed by sonication. The supernatant was collected after centrifugation, and the total protein concentration was determined using the BCA method. Protein Denaturation: An equal volume of protein solution was bound to loading buffer (Beyotime) and heated at 100°C for 10 minutes. Electrophoresis and Transfer: Proteins were separated using a 7.5% ExpressPlus™ PAGE gel in Tris-MOPS-SDS running buffer. After electrophoresis, protein bands were transferred to a 0.45 μm PVDF membrane using Western blotting. Blocking and Antibody Incubation: The PVDF membrane was blocked at room temperature with 5% BSA-TBST solution for 1 hour. After washing three times with TBST, diluted primary antibody solutions (p-p65, p65, p-p38, p38, β-Tubulin antibody) were added, and the membrane was incubated overnight at 4°C. Secondary antibody incubation and development: After washing the membrane again with TBST, the membrane was incubated with the corresponding HRP-conjugated secondary antibody at room temperature for 1 hour. After washing, the membrane was treated with ultrasensitive enhanced chemiluminescence detection solution, and the chemiluminescence signal was detected and acquired using a Bio-Rad imaging system. Data analysis: The gray values ​​of the bands were analyzed using ImageJ software. Standardization was performed using β-Tubulin as an internal control protein, and the relative expression levels of the target proteins (p-p65, p65, p-p38, p38) were calculated.

[0039] Experiment 2: Animal In vivo pharmacodynamics experiment 1. Experimental Principle In this experiment, D-mannose was prepared into a thermosensitive gel (Pluronic F-127) for topical application to wounds to evaluate its comprehensive efficacy in analgesia, anti-inflammation, and accelerating wound healing, and to explore its core mechanism of inhibiting the NF-κB and MAPK pathways.

[0040] 2. Experimental Design and Specific Implementation Steps 2.1 Laboratory animals, drugs and reagents Experimental animals: 6-8 week old male C57BL / 6J mice, weighing 20-25g.

[0041] Model construction: isoflurane (used for anesthesia).

[0042] Test drug: D-mannose (purity >99%).

[0043] Gel matrix: Pluronic F-127 (Beyotime, ST501-10g).

[0044] 2.2 Gel Preparation Placebo gel (Vehicle Gel): Pluronic F-127 powder was slowly dissolved in pre-cooled sterile saline solution on an ice bath to prepare a 20% (w / v) homogeneous solution, which was then refrigerated at 4°C overnight to ensure complete dissolution and liquid state.

[0045] D-Mannose Gel: Following the method described above, when preparing a 20% Pluronic F-127 solution, simultaneously add D-mannose powder to achieve final concentrations of 1%, 3%, and 5% (w / v, i.e., 0.01, 0.03, and 0.05 g / mL). Refrigerate at 4°C for later use.

[0046] 2.3 Animal model preparation, grouping, and administration Model preparation: Mice were anesthetized with isoflurane. The sole of the right hind paw (near the heel) was disinfected, and the skin and underlying fascia were longitudinally incised with a No. 11 scalpel blade, with a length of about 5 mm. The wound was immediately sutured (e.g., using 5-0 sutures).

[0047] Experimental grouping: The experimental animals were randomly divided into 5 groups: Group A: Control group (Sham): No plantar incision was performed; only anesthesia and grasping procedures were conducted.

[0048] Group B: Model group (POP+Vehicle): A plantar incision was made, and 50µL of placebo gel was applied to the wound immediately after the operation.

[0049] Group C: Low-dose treatment group (POP+Mannose1%, POP+Man-L): Foot incision was made, and 50µL of 1% D-mannose gel was applied topically to the wound immediately after the operation.

[0050] Group D: Medium-dose treatment group (POP+Mannose 3%, POP+Man-M): Foot incision was made, and 50µL of 3% D-mannose gel was applied topically to the wound immediately after the operation.

[0051] Group E: High-dose treatment group (POP+Mannose 5%, POP+Man-H): Foot incision was made, and 50µL of 5% D-mannose gel was applied topically to the wound immediately after the operation.

[0052] Administration regimen: The first dose was administered immediately after surgery (Day 0). Subsequent administration was once daily for 14 consecutive days. During administration, mice were briefly anesthetized, the old gel was removed, and the new gel was applied. The Pluronic gel is liquid at 4°C, but rapidly solidifies into a semi-solid gel upon contact with body temperature, covering the wound.

[0053] 2.4 Behavioral testing Perform the following tests daily before administration (Baseline) and for two weeks after administration.

[0054] The von Frey test was performed by placing mice in a transparent acrylic box above a metal grid for 30 minutes to allow them to acclimatize. A set of von Frey fibers with logarithmically increasing stiffness was used to vertically stimulate the middle of the sole of the mouse's right hind paw from bottom to top for 3-5 seconds. The minimum weight in grams (g) required to induce rapid paw withdrawal, licking, or shaking was defined as the paw withdrawal threshold (PWT). A higher PWT value indicates lower pain sensitivity.

[0055] Hargreaves Test: Mice are placed in a transparent test chamber on a glass plate and allowed to acclimatize for 30 minutes. A focused heat source is then applied to the sole of the mouse's right hind paw using a thermal radiation stimulator. The time from the start of irradiation to the appearance of avoidance behaviors such as paw withdrawal and licking is recorded; this is the paw withdrawal latency (PWL). To avoid tissue damage, a cutoff time of 20-30 seconds is typically set. A longer PWL value indicates higher tolerance to thermal pain.

[0056] 2.5 Vascular permeability testing (Evans Blue exudation test) The optimal therapeutic concentration of mannose (3%) and the time of most significant difference were determined by behavioral testing in mice.

[0057] Grouping and time points: Three additional groups of animals (Sham, POP, POP + Mannose 3%) were selected, and this test was performed 72 hours after surgery (Day 3).

[0058] method: Immediately after surgery, mice were injected with 2% Evans blue dye (5 mL / kg) via the tail vein 72 hours after the first administration (or at a fixed time point).

[0059] Thirty minutes after the dye was circulated, the mice were deeply anesthetized with Avertin and their hearts were perfused with saline to flush out any residual dye in the blood vessels.

[0060] Quickly remove the tissue around the incision on the right hind foot and weigh it.

[0061] The tissue was placed in formamide and incubated in a 60°C water bath for 24 hours to extract the leached dye.

[0062] Centrifuge and collect the supernatant. Use an ELISA reader to measure the absorbance (OD value) at a wavelength of 620 nm.

[0063] The Evans blue content (μg / g tissue) in the tissue homogenate was calculated based on the standard curve.

[0064] 2.6 Wound healing assessment Before euthanasia and tissue harvesting (on the 3rd day post-surgery), digital cameras were used to photograph the foot wounds of mice at a fixed height.

[0065] 2.7 Tissue biochemical marker detection (ELISA & WB) The optimal therapeutic concentration of mannose (3%) and the time of most significant difference were determined by behavioral testing in mice.

[0066] Grouping and time points: Three additional groups of animals (Sham, POP, POP + Mannose 3%) were selected, and this test was performed 72 hours after surgery (Day 3).

[0067] ELISA detection of inflammatory cytokine concentrations: 72 hours after model initiation, mice were euthanized by cervical dislocation, and the skin and subcutaneous fascia tissue of the right hind paw were quickly harvested and accurately weighed. Pre-chilled protein lysis buffer (containing protease inhibitors) was added for high-speed homogenization, followed by centrifugation. The supernatant was then aliquoted and stored at -80°C for later use. Total protein quantification: A portion of the tissue homogenate supernatant was used to determine the total protein concentration using the BCA method, which was used for subsequent standardization of cytokine concentrations. Finally, the concentrations of TNF-α, IL-1β, and IL-6 in the supernatant were measured.

[0068] Western Blot protein expression detection: Tissue homogenate supernatant was quantified using the BCA method, followed by gel electrophoresis, membrane transfer, and blocking. The membrane was then incubated with primary antibodies against p-p65, p65, p-p38, p38, Collagen I antibody (Proteintech, 14695-1-AP), and α-SMA antibody (Proteintech, 14395-1-AP), followed by incubation with secondary antibodies. Finally, images were acquired and developed using ECL chemiluminescence. ImageJ software was used to analyze the band grayscale values ​​to assess the relative expression levels of relevant proteins.

[0069] 2.8 Detection of tissue biochemical indicators ELISA inflammatory factor concentration detection: Standards and diluted test samples (tissue homogenate supernatant) were added to pre-coated wells of an ELISA plate and incubated. After washing, biotin-labeled detection antibody was added and incubated. After washing again, horseradish peroxidase (HRP)-labeled streptavidin was added and incubated. After washing, substrate solution (TMB) was added and the plate was developed in the dark. The reaction was terminated by adding stop solution, and the absorbance (OD value) of each well was measured at 450 nm using an ELISA reader. Data analysis: A standard curve was plotted using the concentrations of the standards and their corresponding OD values. The concentrations of TNF-α and IL-1β were calculated from the standard curve based on the OD values ​​of the samples. The final results are usually expressed as picograms (pg / mg) of cytokines per milligram of total protein.

[0070] Western Blot protein expression detection: Tissue homogenate supernatant was quantified using the BCA method, followed by gel electrophoresis, membrane transfer, and blocking. The membrane was then incubated with primary antibodies against p-p65, p65, p-p38, p38, and the internal control β-Tubulin, respectively, and then with secondary antibodies. Finally, images were acquired and developed using ECL chemiluminescence. ImageJ software was used to analyze the band grayscale values ​​to assess the relative expression levels of relevant proteins.

[0071] Experimental data and efficacy verification 1. In vitro experimental results Figure 1 The results of CCK-8 cytotoxicity assay showed that D-mannose did not significantly inhibit the proliferation and viability of NIH / 3T3 fibroblasts within the concentration range of 0-80 mmol / L, and the cell survival rate remained above 90%, demonstrating its high biocompatibility within the effective therapeutic concentration range.

[0072] In the IL-1β-induced NIH / 3T3 cell inflammation model, Figure 2ELISA results showed that the secretion of the key inflammatory factor IL-6 in the cell supernatant decreased in a dose-dependent manner with increasing D-mannose concentrations (10, 20, 40 mmol / L). The 40 mmol / L group showed the most significant inhibitory effect (p<0.01) and was therefore selected as the optimal in vitro concentration for subsequent mechanism verification.

[0073] Figure 3 Western blot results confirmed that IL-1β stimulation led to potent activation (significantly increased phosphorylation levels) of the NF-κB (p-p65) and MAPK (p-p38) signaling pathways in NIH / 3T3 cells. Treatment with 40 mmol / L D-mannose significantly inhibited the phosphorylation levels of these three key inflammatory pathways and the expression of their downstream inflammatory factors. This indicates that D-mannose possesses a broad-spectrum and potent anti-inflammatory mechanism at the cellular level, capable of blocking the cascade amplification of inflammatory signals at its source.

[0074] 2. In vivo experimental results Changes in mechanical and thermal pain sensitivity in POP mice under different concentrations of D-mannose treatment are shown below. Figure 4 D-mannose significantly relieved postoperative incision pain, and 3% was identified as the only effective concentration. Animal behavioral tests (mechanical pain von Frey and thermal pain von Hargreaves) showed the following results: 1. Day 1 post-surgery: The pain threshold of all mice in the model group decreased significantly, indicating that the model was successful.

[0075] 2. Postoperative days 2-4 (peak pain period): In the 3% D-mannose treatment group, the mechanical pain threshold (PWT) and heat withdrawal latency (PWL) of mice were significantly higher than those in the model group (POP) (p<0.05), demonstrating a significant analgesic effect. In the 1% and 5% D-mannose treatment groups, although the pain threshold showed a slight upward trend compared to the model group, the difference was not statistically significant (p>0.05). This indicates that low concentrations (1%) are insufficiently effective, while high concentrations (5%) may lead to reduced efficacy due to osmotic pressure or other factors.

[0076] Postoperative days 6-14 (self-healing period): Pain thresholds in all groups (including the model group) gradually returned to baseline levels (Sham group levels), with no statistically significant differences between groups, consistent with the self-healing characteristics of the postoperative incision pain model. Conclusion: The experiment confirms that 3% (w / v) is the optimal and specific effective concentration for topical analgesia of D-mannose.

[0077] To investigate the molecular mechanism of peak drug efficacy, tissue was examined on the third day after surgery (a critical point in the transition from inflammation to the proliferative phase).

[0078] D-mannose (3%) significantly inhibited early postoperative vascular exudation and accelerated wound closure: 1. Anti-permeability (Evans Blue): Figure 5 The quantitative results of Evans blue treatment showed that, compared with the model group, the amount of Evans blue exudate in the plantar tissue of the 3% D-mannose gel treatment group was significantly reduced, proving that this concentration can effectively inhibit acute inflammatory edema and increased capillary permeability.

[0079] 2. Promotes Healing (Wound Area, Molecular Indicators): Macroscopic observation and area analysis showed that on postoperative day 3, the model group's wound still exhibited significant redness, swelling, and dehiscence, indicating delayed healing. In contrast, the 3% D-mannose treatment group showed significant reduction in redness and swelling, with a significantly smaller wound area and better wound edge closure. This result directly demonstrates that D-mannose, through its potent early anti-inflammatory effect, successfully created a favorable environment for the wound, thereby significantly accelerating physical wound closure. Western blot results showed that compared to the Sham group, the expression level of Collagen I (collagen matrix) in the plantar tissue of the model group (POP) was low on day 3, indicating that inflammation led to severe tissue damage and delayed repair. In the 3% D-mannose treatment group, both of these key healing indicators significantly increased compared to the model group (Collagen I showed a highly significant difference, and α-SMA was significantly upregulated). This demonstrates that D-mannose, by inhibiting inflammation, enabled the wound tissue to initiate the repair process earlier, accelerating collagen synthesis and wound contraction.

[0080] Figure 6 and Figure 7 ELISA and WB results showed that 3% D-mannose significantly reduced the levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and upstream pathways (p-p65, p-p38).

[0081] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of D-mannose in the preparation of a drug for treating postoperative incision pain.

2. The application according to claim 1, characterized in that, The drug is a topical preparation.

3. The application according to claim 2, characterized in that, The topical preparation is a gel preparation.

4. The application according to claim 3, characterized in that, The mass concentration of D-mannose in the gel formulation is 1-5%.

5. The application according to claim 4, characterized in that, The mass concentration of D-mannose in the gel formulation is 3%.

6. The application according to any one of claims 1-5, characterized in that, D-mannose reduced the levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, and upstream signaling pathways NF-κB and MAPK.

7. A pharmaceutical composition for treating postoperative incision pain, characterized in that, It contains an effective amount of D-mannose and a pharmaceutically acceptable topical carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The mass concentration of the D-mannose is 3%.

9. The pharmaceutical composition according to claim 7, characterized in that, The localized external carrier is a gel.

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