Application of magnesium hydride in improvement of abdominal cavity postoperative rehabilitation and medicine and medical instrument thereof

Abstract

The invention relates to application of magnesium hydride in preparation of drugs and medical instruments for improving abdominal cavity postoperative rehabilitation. Through verification, the magnesium hydride powder disclosed by the invention is applied to postoperative wounds, has remarkable antithrombin activity, can inhibit the transformation of fibrinogen to fibrin and prevent the formation of fiber clots, and has the effect of inhibiting the aggregation of fibrinogen after abdominal operation. Further experiments prove that no obvious peritoneal adhesion occurs around the suture, local inflammation and peritoneal adhesion hyperplasia are effectively inhibited, the occurrence of peritoneal adhesion (PAs) can be effectively reduced or avoided, and wound healing is promoted.

Description

Technical Field

[0001] The invention relates to application of an inorganic material in medical treatment, and in particular to application of a powder containing magnesium and hydrogen elements in improving recovery after abdominal surgery, and to medicine and medical equipment thereof. Background Art

[0002] Peritoneal Adhesions (PAs) can be caused by a variety of factors, such as surgery, trauma, chronic peritonitis, peritoneal dialysis, and endometriosis. Among them, surgery is the most common cause, with the incidence of postoperative abdominal adhesions as high as 93%, and often leads to serious complications such as chronic abdominal pain, intestinal obstruction, and infertility.

[0003] Current clinical management strategies rely mainly on repeated surgeries to separate adhesion tissues, supplemented by anticoagulants, anti-inflammatory drugs, fibrinolytic drugs, and cell-targeted drug therapy. At the same time, solid or liquid physical barrier materials have also been used in clinical practice. In addition, combination therapies integrating these methods have also been explored. However, these treatments have significant limitations: even after multiple surgical separations, the recurrence rate of adhesions is still higher than 53%.

[0004] Hydrogen (H2) therapy has attracted attention due to its many therapeutic advantages in the biomedical field. Hydrogen exhibits powerful antioxidant, anti-inflammatory and anti-apoptotic effects, and can exert its therapeutic effect by reducing the generation of reactive oxygen species (ROS) and relieving inflammation. As a small molecule gas with high diffusivity, hydrogen has a strong ability to penetrate tissues and cells, can deeply penetrate tissues and cells, expand its therapeutic range, and participate in multiple links of cell repair and regeneration.

[0005] Magnesium (Mg) is a strong reducing agent that has been widely used as a degradable implant material in the clinic due to its excellent performance in tissue repair and regeneration. Although Mg is recognized for its degradability and mechanical support properties, its potential for disease treatment by spontaneously reacting with water to generate hydrogen (H2) has only been explored in recent years.

[0006] CN110559467 provides a micro-nano magnesium hydride antibacterial and anti-inflammatory wound dressing, which is used for antibacterial, anti-inflammatory and infection prevention of surface wounds, and is beneficial to wound repair. It uses magnesium hydride micro-nano powder with a particle size of 10nm to 5μm, which enters the bacteria under endocytosis, releases OH radicals, changes the pH microenvironment of the bacteria, causes nucleic acid and protein denaturation, and achieves the purpose of antibacterial. In addition, the released hydrogen forms a low-oxygen environment near the wound, inhibiting the growth of aerobic bacteria.

[0007] It has been verified that when these micro-nano magnesium hydrides are used for (surgical) wound anticoagulation and anti-adhesion of tissues in the abdominal cavity and other body tissues, they cannot be achieved. On the one hand, hydrogen is released too quickly, local overheating of the tissue occurs, causing secondary damage to the wound tissue, and local magnesium ion excess occurs, causing hypermagnesemia. On the other hand, all animals (mice) to which these micro-nano magnesium hydrides are applied intraperitoneally died, as many as dozens. Therefore, the scheme of the prior art does not really achieve the purpose of anticoagulation and anti-adhesion.

[0008] In addition, the understanding of preventing wound adhesion in the prior art seems to be different from the medical understanding. The prior art believes that the purpose of preventing wound adhesion is achieved due to the physical effect of 0.0018 to 0.17 mL of hydrogen produced per square centimeter of dressing. In other words, anticoagulation and anti-adhesion belong to physical isolation, not biological effects.

[0009] Generally, adhesion refers to abnormal fibrous connections between tissues or organs caused by surgery, trauma, infection or chronic inflammation. This phenomenon is usually accompanied by the deposition of fibrin and the proliferation of fibroblasts, and may be gradually aggravated by acute inflammation or chronic stimulation. Summary of the invention

[0010] One object of the present invention is to provide an application of magnesium hydride powder in preparing a medicament for inhibiting fibrinogen aggregation after abdominal surgery.

[0011] Another object of the present invention is to provide an application of magnesium hydride powder in the preparation of a medical device for inhibiting fibrinogen aggregation after abdominal surgery.

[0012] Another object of the present invention is to provide an application of magnesium hydride powder in the preparation of a medical device for preventing adhesion after abdominal surgery.

[0013] Another object of the present invention is to provide an application of magnesium hydride powder in the preparation of a drug for anti-adhesion after abdominal surgery.

[0014] A magnesium hydride powder, wherein particles with a particle size of 10 microns or more account for 70% or more.

[0015] Another magnesium hydride powder, wherein particles with a particle size of 10 microns or more account for 80% or more.

[0016] Another magnesium hydride powder, wherein particles having a particle size of 13 microns or more account for 45% or more.

[0017] Another magnesium hydride powder, wherein particles with a particle size of 13 microns or more account for 50% or more.

[0018] Another magnesium hydride powder, wherein particles having a particle size of 13 microns or more account for 55% or more.

[0019] Another magnesium hydride powder, wherein particles with a particle size of 15 microns or more account for 30% or more.

[0020] Another magnesium hydride powder, wherein particles with a particle size of 15 microns or more account for 35% or more.

[0021] Another magnesium hydride powder has particles with a particle size of 15 microns or more accounting for 40% or more.

[0022] Another magnesium hydride powder has an average particle size of 14 microns ± 1 micron.

[0023] After the magnesium hydride powder provided by the present invention absorbs liquid, a magnesium hydroxide (Mg(OH)2) film is formed on the particle surface as a passivation layer, which has the effect of inhibiting or delaying the reaction between magnesium hydride and water. This reduces the degradation rate of the MgH2 particles and realizes the reaction between hydrogen and Mg 2+ Slower controlled release.

[0024] The magnesium hydride powder provided by the present invention is applied to the postoperative wound surface. It has been verified that it has significant antithrombin activity, can inhibit the conversion of fibrinogen to fibrin, and prevent the formation of fiber clots, and has the effect of inhibiting the aggregation of fibrinogen after abdominal surgery. Further experiments have shown that no obvious peritoneal adhesions occur around the sutures, and local inflammation and peritoneal adhesion hyperplasia are effectively inhibited, which can effectively reduce or avoid the occurrence of peritoneal adhesions (PAs) and promote wound healing.

[0025] MgH2 releases Mg 2+ It has a strong anti-inflammatory and antioxidant effect synergistically with H2, effectively reducing ROS and alleviating excessive inflammatory response. 2+ It can induce macrophages to switch from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, which is essential for tissue repair and prevention of fibrosis. 2+ By regulating local pH levels to neutralize acidic metabolites and inhibit fibroblast activation, further reducing collagen deposition and preventing excessive fibrosis. The release of H2 enhances this effect by alleviating oxidative stress, while Mg 2+ It promotes angiogenesis and accelerates tissue regeneration.

[0026] Comprehensive in vivo and in vitro experimental results showed that MgH2 not only prevented intra-abdominal adhesion formation, but also significantly improved wound healing by supporting tissue regeneration and reducing fibrous scars.

[0027] Therefore, the MgH2 powder of the present invention can be used as an active ingredient to prepare drugs or medical devices, and can be used to reduce or avoid the inhibition of fibrinogen aggregation after abdominal surgery. In particular, it can reduce or avoid the occurrence of adhesion after abdominal surgery, improve the patient's recovery process, and promote postoperative care and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The characterization results of MgH2 are shown in Figure 1; A is the MgH2 powder and its SEM image and particle size distribution, B is the XRD result, C is the hydrogen release curve of MgH2, D is the CCK-8 test result of MgH2 cytotoxicity, E is the Mg 2+ Release curve;

[0029] Figure 2 Figure 1 is a graph showing the verification of the anticoagulant properties of MgH2; A1 is a photo of the results of the 5-minute in vitro hemolysis experiment of each group, and from left to right in the figure are: blank control group (Control), Celox group, PLGA group, MgH2 group and Triton X group; A2 is a photo of the results of the 10-minute in vitro hemolysis experiment of each group, and from left to right in the figure are: blank control group (Control), Celox group, PLGA group, MgH2 group and Triton X group; B is a photo of the rat tail amputation hemostasis experiment, and from left to right in the figure are: MgH2 group, Celox group, blank control group (Control) and PLGA group; C is a statistical graph of the in vitro hemostasis time of each group; and D is the pH-controlled release curve of magnesium hydride (MgH2) and magnesium hydroxide (Mg(OH)2);

[0030] Figure 3 It is a verification result diagram of MgH2 reducing ROS in vitro; wherein, A is a fluorescence image of ROS contained in each group, B is a statistical result diagram of ROS fluorescence intensity in each group, C is a flow cytometric analysis of ROS content in each group, D is a quantitative statistical result diagram of ROS consumption in each group, E is a flow cytometric diagram and immunofluorescence staining of double staining of CD86 and CD206 in each group, F is a statistical result diagram of M1 macrophage polarization in flow cytometry in each group, G is a statistical result diagram of M2 macrophage polarization in flow cytometry in each group, H is a flow cytometric diagram of macrophages, I is a flow cytometric analysis result diagram of FITC phenotype of macrophages in each group, and J is a flow cytometric analysis result diagram of PE phenotype of macrophages;

[0031] Figure 4 The results of MgH2 promoting wound healing and inhibiting adhesion formation in vivo are shown in Figure 1, where A is the photos of rats in each group on the 0th, 5th and 10th days after surgery, and B is the comprehensive score statistical chart of the cecum-abdominal wall suture model in each group.

[0032] Figure 5 It is a structural diagram for verifying that MgH2 promotes macrophage polarization; wherein, A is the staining diagram of M1 macrophages in each group on the 5th day (the scale bar in the figure indicates 200 μm), B is the quantitative statistical diagram of the staining results of Figure A, C is the staining diagram of M1 macrophages in each group on the 10th day (the scale bar in the figure indicates 200 μm), D is the quantitative statistical diagram of the staining results of Figure C, E is the staining diagram of M2 macrophages in each group on the 5th day (the scale bar in the figure indicates 200 μm), F is the quantitative statistical diagram of the staining results of Figure E, G is the staining diagram of M2 macrophages in each group on the 10th day (the scale bar in the figure indicates 200 μm), and H is the quantitative statistical diagram of the staining results of Figure G;

[0033] Figure 6 The results of H&E and Masson staining are shown in Figure 1; A is the HE staining and Masson staining of each group on the 5th day (the scale bar in the figure represents 300 μm), and B is the HE staining and Masson staining of each group on the 10th day (the scale bar in the figure represents 300 μm);

[0034] Figure 7 The figures are the verification results of MgH2 promoting tissue repair in vivo; wherein, A is the CD31 immunofluorescence staining image of each group (the scale bar in the figure indicates 200 μm), B is the Ki67 immunofluorescence staining image of each group (the scale bar in the figure indicates 200 μm), C is the α-SMA immunohistochemistry staining image (the scale bar in the figure indicates 200 μm), D is the quantitative statistical graph of CD31 immunofluorescence staining of each group, and E is the quantitative statistical graph of Ki67 immunofluorescence staining of each group. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0036] The various test methods used in the following examples of the present invention are specifically described as follows:

[0037] 1) Materials and cell culture

[0038] The anti-adhesion polylactic-glycolic acid (PLGA) membrane used in this study was provided by Chongqing Yongtong Information Engineering Co., Ltd. MgH2 was synthesized by reacting magnesium powder with hydrogen under controlled high pressure and high temperature conditions. The reaction process lasted for 6 hours at 400°C and 12MPa to ensure optimal crystallinity and consistency. The size of each PLGA membrane was 10mm×20mm to ensure its consistency and applicability in anti-adhesion applications.

[0039] High-glucose Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (UK). Endothelial cells and RAW264.7 macrophages were cultured in complete growth medium consisting of high-glucose DMEM and 10% FBS. They were cultured in a cell culture incubator at 37°C and 5% CO2 to maintain optimal growth conditions.

[0040] 2) In vitro release test

[0041] The in vitro hydrogen release profile of MgH2 was evaluated by placing 3 mg of MgH2 powder in a sealed vial (total volume of 15 mL) filled with 10 mL of deionized water. 1 mL of hydrogen samples were collected at specific time points (0 h, 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 1 day, 2 days, 3 days, 5 days, 7 days, and 10 days) and the amount of hydrogen released was analyzed using a gas chromatograph (Agilent 7890 B, USA).

[0042] 30 mg of MgH2 powder was soaked in 10 mL of pure water and placed in a dialysis bag. The dialysis bag was then placed in a beaker filled with 100 mL of pure water. ICP-OES tests (Optima 7000DV, PerkinElmer, USA) were performed at different time points (0 h, 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 1 day, 2 days, 3 days, 5 days, 7 days and 10 days) to obtain the magnesium ion release curve of MgH2 powder in pure water (pH = 7.0) to quantify the released Mg 2+ content.

[0043] 30 mg of MgH2 powder and 30 mg of Mg(OH)2 powder were soaked in 10 mL of pure water and placed in separate dialysis bags. Each dialysis bag was then placed in a beaker filled with 100 mL of pure water. The pH of the solution was measured at room temperature at 0 hours, 5 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 7 days and 10 days to evaluate the pH changes of MgH2 and Mg(OH)2 in pure water.

[0044] 3) CCK-8 assay

[0045] In order to evaluate the cell compatibility of MgH2 and its effect on cell proliferation, a concentration-dependent experiment was conducted in this example, using an extract obtained by immersing each PLGA membrane in a mixture of 5 mL of phosphate buffered saline (PBS) and MgH2 for 3 days.

[0046] Human umbilical vein endothelial cells (HUVECs) were seeded in 96-well plates at a density of 3,000 cells / well and pre-cultured in high-glucose DMEM for 12 hours to ensure initial cell attachment (Arch Surg. 1985; 120(8): 949-951). Subsequently, the cells were co-cultured with a mixture of 90 μL complete medium and 10 μL PLGA extract for 3 days. Next, different concentrations of MgH2 solution (dissolved in complete medium) (0 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL and 5 μg / mL) were added to each well and the cells were cultured for another 3 days.

[0047] After treatment, 10% CCK-8 solution (prepared in complete medium) was added to each well to assess cell viability. The culture plate was then incubated at 37°C for 2 hours, and the absorbance was measured at 450 nm using a microplate reader (SpectraMAX iD3, Molecular Devices, LLC, USA) to quantify cell proliferation and viability.

[0048] 4) Flow cytometric analysis of macrophage polarization in vitro

[0049] RAW264.7 macrophages were seeded in 12-well plates and cultured to about 80% confluence in standard growth medium. Subsequently, the cells were co-cultured with PLGA membranes, MgH2 extracts, or normal culture medium (control group) for 24 hours.

[0050] After treatment, cells were collected and stained with antibodies against CD206 (a marker of M2 macrophages) and CD86 (a marker of M1 macrophages) to assess the polarization state of macrophages. The expression levels of CD206 and CD86 on the cell surface were quantified by flow cytometry analysis to evaluate the effects of different treatments on macrophage polarization in detail.

[0051] 5) Inhibition of ROS generation and in vitro flow cytometry analysis

[0052] To evaluate the ROS inhibition effect, we seeded RAW264.7 macrophages in 6-well plates and pretreated them with 2 μL lipopolysaccharide (LPS, Sigma-Aldrich, USA) to induce inflammation. Subsequently, the cells were divided into four groups: a control group (2 mL normal cell culture medium), a MgH2-treated group (1 mL MgH2 extract mixed with 1 mL normal cell culture medium), and two other groups for comparative analysis (Arch Surg. 1985; 120(8): 949-951).

[0053] After 24 h of co-culture under these conditions, the cells were stained using a ROS detection kit (Biyuntian, China) according to the manufacturer's instructions. The stained cells were first observed and images were taken by fluorescence microscopy to qualitatively assess the ROS level. Next, we collected the cells and quantitatively analyzed ROS generation by flow cytometry (Beckman Coulter, USA) to determine the extent of ROS inhibition in each group.

[0054] 6) Animal experiments

[0055] Sprague-Dawley (SD) rats (weight 220-250 g) were purchased from Wukong Experimental Animal Co., Ltd. (Jiangsu, China). Before the experiment, the animals were acclimatized for one week, during which they had free access to water and food. The rats were kept in a room with a temperature of 24°C and a relative humidity of 65%.

[0056] Under sterile conditions, a midline laparotomy was performed to open the abdominal cavity of the rat, expose the cecum, and place it on sterile gauze until the serosal surface became dry. Subsequently, the cecum surface was scratched using a surgical brush through a 1 cm × 2 cm rectangular window in the mold until punctate bleeding occurred. The mesenteric artery was clamped with a hemostat for 2 minutes to induce transient ischemia. In addition, a 1 cm × 2 cm area was excised along the superficial abdominal wall muscle more than 1 cm from the midline of the abdomen to create a rough bleeding surface.

[0057] Subsequently, the cecum was returned to the abdominal cavity, and the rats were divided into different groups: no treatment (control group), PLGA membrane treatment group, or MgH2 particle spreading group. The cecum and abdominal wall were sutured with two stitches about 1 cm away from the wound edge to maintain close contact between the wound site. After taking photos to record the experimental process, the abdominal cavity was sutured closed layer by layer, and the surgical area was disinfected. Penicillin was injected intramuscularly every day for 3 days after surgery to prevent infection. The rats were housed individually to facilitate postoperative care and monitoring.

[0058] 7) In vivo wound healing analysis and in vitro anticoagulation assay

[0059] A rat peritoneal adhesion model was established by making a 1 cm × 2 cm circular postoperative bleeding wound on the surface of the rat cecum. After surgery, the rats were randomly divided into three groups: a blank control group, a MgH2-treated group, and a PLGA membrane group. The wound sites were photographed on days 0, 5, and 10 to record the progress of wound healing.

[0060] The severity of peritoneal adhesions was assessed using the CSSM comprehensive scoring system. This system quantifies adhesions based on the following three criteria:

[0061] A: Ratio of adhesion area to abrasion area

[0062] B: Number of adhesions

[0063] C: Toughness of each bond

[0064] The score range for each criterion is 0 (no adhesion) to 4 (severe adhesion). The total score is calculated as: A×CA+B×CB, where CA and CB represent the toughness scores of type A and type B adhesion, respectively (Biomaterials. Jul 2016; 96: 33-46).

[0065] This scoring system provides a comprehensive and quantitative assessment of adhesion severity.

[0066] After the in vivo experiment, we prepared sample extracts for further analysis. Briefly, 5 mL of fresh anticoagulated rabbit blood was mixed with 10 mL of phosphate buffered saline (PBS, pH 7.4). The microsphere sample was dissolved in PBS to prepare a 1 mg / mL suspension and kept at 37°C for 24 hours. Subsequently, 100 μL of the blood-PBS mixture was added to 1 mL of the sample extract and incubated at 37°C. The reaction was photographed at 5 and 10 minutes to qualitatively evaluate the anticoagulation effect, PBS was used as a negative control, and Triton X was used as a positive control.

[0067] To further evaluate the hemostatic and anticoagulant properties of MgH2, we performed tail bleeding time tests on adult rats. After anesthesia, the rats were amputated and treated according to the group: MgH2 powder group, Celox hemostatic powder group, untreated group (blank control) and PLGA membrane group. The bleeding time of each group was recorded, and statistical analysis was performed to determine the significance of the differences between the groups.

[0068] This experiment provides a controllable and reproducible method for evaluating the hemostatic and anticoagulant properties of MgH2 in vivo.

[0069] 8) Analysis of wound healing, tissue repair and macrophage differentiation in vivo

[0070] On days 5 and 10 after treatment, the rats were killed by cervical dislocation. The intestinal tissues surrounding the wound area were carefully collected and immediately immersed in 4% paraformaldehyde for 24 h to preserve the tissue morphology. Subsequently, the tissue samples were embedded in paraffin blocks, sectioned at a thickness of 5 μm, and subjected to various histological and immunohistochemical analyses.

[0071] Hematoxylin-eosin (H&E) staining was used to assess the overall structure and inflammatory response of the tissue.

[0072] Masson's trichrome staining was used to observe collagen deposition and fibrosis.

[0073] To evaluate tissue regeneration and angiogenesis, immunofluorescence staining was performed using anti-CD31 (1:500) and anti-Ki67 (1:500) antibodies.

[0074] In the control and MgH2-treated groups, the polarization status of macrophages was analyzed by immunohistochemical staining, and the staining markers included:

[0075] CD68 (broad-spectrum macrophage marker)

[0076] CD206 (M2 phenotype marker)

[0077] iNOS (M1 phenotype marker)

[0078] In addition, α-SMA (α-smooth muscle actin) immunostaining was performed to assess the extent of fibrosis and the presence of myofibroblasts, which are key indicators of tissue repair and fibrotic response.

[0079] 9) Statistical analysis

[0080] To evaluate the effects of MgH2 on wound healing, tissue repair, and macrophage differentiation, we performed statistical analyses. Quantitative measurements of tissue staining results were performed using ImageJ software to ensure accurate assessment of tissue morphology and fibrosis. Fluorescence microscopy and flow cytometry were used for data acquisition of cell staining and macrophage polarization.

[0081] In the cell proliferation assay, cells were counted using a hemocytometer to determine cell growth and proliferation rates. According to the requirements of the study design, we applied one-way analysis of variance (ANOVA) and post hoc Tukey test for multiple group comparisons; at the same time, for pairwise comparisons, a two-tailed Student's t test was used. Data were plotted and analyzed using GraphPad Prism software.

[0082] Each experiment was repeated three times, and the results are presented as mean ± standard error (SEM). A significance level of p < 0.05 was used to indicate that the results were statistically significant. "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001.

[0083] Example 1 Preparation and Characterization of MgH2

[0084] The reaction was carried out at 400°C for 6 hours with an applied hydrogen pressure of 12 MPa. After the synthesis, the morphology of the MgH2 particles was characterized using scanning electron microscopy (SEM). The SEM images showed relatively uniform spherical particles, with particles larger than 10 μm accounting for more than 80%, particles larger than 13 μm accounting for 56.18%, particles larger than 15 μm accounting for 39.94%, and an average particle size of 14 μm. This result was confirmed by image analysis using ImageJ software and presented in a graph using GraphPad Prism software ( Figure 1 A and 1B).

[0085] To further investigate its structural properties, we performed X-ray diffraction (XRD) analysis. The results showed that the synthesized MgH2 was highly crystalline and had a very low residual magnesium content ( Figure 1 C). These results verify the successful synthesis of MgH2 under the specified conditions and ensure the quality of the material and its suitability for subsequent experiments.

[0086] After synthesizing the MgH2 particles, we plotted the release curves of hydrogen and magnesium ions to evaluate the release characteristics of MgH2 ( Figure 1 C and 1E). When MgH2 pellets were immersed in pure water, hydrogen release peaked on the third day, which is consistent with the hydrolysis reaction of MgH2 with water. The chemical equation of this reaction is as follows:

[0087] MgH2+2H2O→Mg(OH)2+H2↑ (1)

[0088] Mg(OH)2+2H + →Mg 2+ +2H2O (2)

[0089] During the reaction of MgH2 with water, a thin film of magnesium hydroxide (Mg(OH)2) is formed on its surface, which acts as a passivation layer and temporarily inhibits its further reaction with water. This phenomenon reduces the degradation rate of MgH2 and realizes the hydrogen and Mg 2+ Under neutral conditions, the dissolution rate of Mg(OH)2 is extremely low and it is almost insoluble in water. However, even in the presence of this passivating Mg(OH)2 film, MgH2 can still react directly and quickly with water to continuously release hydrogen, such as Figure 2 This highlights its potential for stable and long-term hydrogen release.

[0090] The effect of MgH2 on endothelial cell viability and proliferation was evaluated by CCK-8 assay. Figure 1As shown in D, after 72 hours of treatment, the proliferation of endothelial cells reached a peak at a concentration of 0.5 μg / mL, while at higher concentrations (such as above 3 μg / mL), cell proliferation gradually decreased, showing a certain inhibitory effect.

[0091] These results indicate that MgH2 is non-cytotoxic to endothelial cells at low concentrations and can promote cell proliferation at the optimal concentration. The optimal concentration for promoting proliferation was determined to be 0.5 μg / mL. Therefore, unless otherwise stated, all subsequent experiments in this example were treated with this concentration.

[0092] Example 2 Effect of MgH2 on reducing ROS in vitro

[0093] Previous studies have shown that ROS (reactive oxygen species) is one of the factors associated with the formation of peritoneal adhesions, which highlights the importance of regulating oxidative stress during the healing process. In this embodiment, the magnesium hydroxide film formed on the surface of MgH2 enables it to continuously and controllably release hydrogen, which is known to have potent antioxidant properties. This slow-release property enables MgH2 to more effectively inhibit the generation of ROS during the critical period of peritoneal adhesion formation.

[0094] To simulate the production of excessive ROS (reactive oxygen species), lipopolysaccharide (LPS) was added to wells containing Raw264.7 cells (a classic cell line commonly used to study inflammation and oxidative stress). These cells were seeded in 6-well plates and co-cultured with MgH2 solution or standard cell culture medium (as a control). After 24 hours of co-culture, the intracellular ROS levels were visualized using a fluorescent ROS staining kit and imaged using a fluorescent microscope. The experimental images showed that the ROS level in the MgH2 group was reduced by 52.27% compared with the control group, and was reduced by 20.16% compared with the PLGA group (see Figure 3 A and 3B).

[0095] To further quantify the extent of ROS reduction, flow cytometry analysis was also performed on the stained Raw264.7 cells. The results showed that the ROS level in the MgH2 group was reduced by 25.73% compared with the control group (see Figure 3 C and 3D), which further confirmed the effectiveness of MgH2 in alleviating oxidative stress.

[0096] This enhanced ROS-inhibiting effect is achieved due to the slow and sustained release of hydrogen from MgH2, which neutralizes ROS and helps maintain intracellular redox homeostasis during the critical phase of healing and tissue repair.

[0097] Example 3 MgH2 promotes wound healing and inhibits adhesion formation in vivo

[0098] The effects of MgH2 on wound healing and adhesion formation prevention were evaluated in Sprague-Dawley (SD) rats using the cecum-abdominal wall suture model. Visible adhesions appeared in both the control group and the polylactic-co-glycolic acid (PLGA) membrane group on postoperative day 5, but these adhesions could be undone by blunt dissection. In contrast, only a few scattered bleeding spots appeared on the tissue surface of the MgH2-treated group, indicating that it has significant antithrombin activity, which can inhibit the conversion of fibrinogen to fibrin and prevent the formation of fibrin clots.

[0099] Of particular note, no visible adhesions were observed in the MgH2 group. By day 10, the control group showed complete adhesions between the abdominal wall and the adjacent intestinal wall, while the PLGA group showed mild to moderate adhesions. However, no adhesions to surrounding tissues or organs were found in the MgH2 group, and tissue healing was good (see Figure 4 A).

[0100] Quantitative evaluation using a comprehensive adhesion scoring system further confirmed these results. The MgH2 group had the smallest adhesion area, the fewest adhesions, and the lowest adhesion strength score among all groups (see Table 2). Figure 4 B). This indicates that MgH2 has significant advantages over traditional treatments in preventing postoperative adhesions.

[0101] The ability of MgH2 to inhibit adhesion proliferation was further demonstrated by analyzing indirect evidence reflecting the changes in peritoneal tissue around the suture site. Sutures were used for wound closure in all groups (Control, PLGA, and MgH2). In the Control and PLGA groups, peritoneal adhesions proliferated around the sutures, increasing the contact area between the intestinal wall and the abdominal wall. In the MgH2 group, very little peritoneal adhesions formed around the sutures, indicating that the hydrogen continuously released by MgH2 effectively inhibited local inflammation and peritoneal adhesion proliferation.

[0102] The percentage of wound adhesion area was quantified using ImageJ software, and the effect of adhesion prevention was evaluated by the cecum-abdominal wall suture model (CSSM) scoring table. The results showed that the MgH2 group had significant improvements in wound healing and inflammatory hyperplasia inhibition compared with the control group and PLGA group.

[0103] Further analysis of the anticoagulant properties of MgH2 showed that it has the potential to inhibit fibrinogen aggregation, thereby reducing fibroblast adhesion and inhibiting the formation of peritoneal adhesions. Blood compatibility testing showed that at 5 minutes, the blood compatibility of MgH2 was similar to that of the Triton X group, indicating that it has strong anti-cell adhesion properties (see Figure 2 A1 and Figure 2 A2).

[0104] To further verify these findings, we performed a rat tail amputation hemostasis experiment (see Figure 2 B). The results showed that the bleeding time in the MgH2 group was significantly prolonged and the amount of bleeding increased 1.61 times compared with the control group. These results highlight the potent anticoagulant and anti-adhesion properties of MgH2, supporting its use as an effective strategy for preventing abdominal adhesions (see Figure 2 C).

[0105] The anticoagulant effect of MgH2 is mainly attributed to magnesium ions (Mg 2+ ) is released, which regulates the coagulation process through two main pathways:

[0106] 1. Inhibit platelet activation and aggregation: Mg 2+ By using Ca 2+ Compete for binding sites and reduce intracellular calcium mobilization.

[0107] 2. Destroy the key enzyme activity in the coagulation cascade reaction: such as the activity of thrombin and factor Xa is disturbed.

[0108] These mechanisms may explain the prolonged bleeding time and reduced thrombosis observed in this study.

[0109] Example 4 MgH2 promotes macrophage polarization

[0110] RAW264.7 cells were polarized and cultured for 3 days, and then double immunofluorescence staining was performed, using CD86 (FITC) to label M1 macrophages and CD206 (PE) to label M2 macrophages (see Figure 3 E) to investigate the effect of MgH2 on macrophage polarization.

[0111] Flow cytometry analysis showed that the M1 polarization in the MgH2-treated group was reduced by 4.22-fold and 3.73-fold compared with the blank control group and the PLGA group, respectively. This significant reduction suggests that MgH2 effectively inhibits the polarization of M1 macrophages in vitro (see Figure 3 F).

[0112] In addition, MgH2 treatment increased the repolarization of M2 macrophages by 56.05-fold and 52.09-fold compared with the blank control group and PLGA group, respectively, indicating that MgH2 is significantly superior to the clinical standard PLGA membrane in promoting the repolarization of M2 macrophages (see Figure 3 G).

[0113] Taken together, these results indicate that MgH2 is able to reduce M1 polarization while enhancing M2 repolarization, highlighting its therapeutic potential in modulating macrophage activity.

[0114] Immunofluorescence staining was performed on tissue samples from multiple groups, using CD68 (red) to mark all macrophages, iNOS (green) to mark M1 macrophages, and CD206 (green) to mark M2 macrophages to further verify the role of MgH2 in promoting macrophage polarization.

[0115] During the first week of wound healing, it is critical to shorten the acute inflammatory phase and accelerate the entry into the inflammatory resolution phase. Initially, neutrophils rapidly infiltrate the wound site within a few minutes (Journal of Gastrointestinal Surgery. 2023; 27(6): 1216-1227) and peak within 24 to 48 hours to clear bacteria and debris. Subsequently, macrophages begin to migrate to the wound site, initially appearing as an M1 phenotype, secreting proinflammatory cytokines (such as IL-1β, IL-6, and TNF-α), and then transforming into an M2 phenotype, promoting tissue repair by secreting anti-inflammatory cytokines (such as IL-10, TGF-β). This dynamic polarization is critical for effective wound healing. Recent studies have shown that hydrogen and magnesium ions (Mg 2+ ) can accelerate this polarization process, reduce inflammation, and promote the early appearance of M2 macrophages, thereby improving the healing effect.

[0116] On day 5, CD68 / iNOS double staining images and quantitative cell counts of CD68 and iNOS showed that MgH2 treatment reduced M1 macrophage polarization by 37.36% and 34.72% compared with the control group and PLGA group, respectively (see Figure 5 B). By day 10, the number of M1 macrophages in the MgH2-treated group was reduced by 36.85% and 25.88% compared with the control group and PLGA group, respectively (see Figure 5 D). These results indicate that MgH2 is superior to the control and PLGA groups in reducing M1 macrophages.

[0117] CD68 / CD206 double staining analysis was also performed to evaluate the repolarization of M2 macrophages. The results showed that on day 5, the M2 repolarization level in the MgH2-treated group increased by 1.56-fold and 0.44-fold compared with the control group and PLGA group, respectively (see Figure 5 F). By day 10, the M2 repolarization level in the MgH2-treated group increased by 41.03% and 21.68% compared with the control group and PLGA group, respectively (see Figure 5 H).

[0118] In summary, the application of MgH2 effectively reduces M1 polarization and promotes M2 repolarization both in vivo (peritoneal) and in vitro, and its effect in regulating macrophage polarization and promoting wound healing is significantly better than PLGA membrane.

[0119] Example 5 MgH2 promotes tissue repair in vivo

[0120] Hematoxylin-eosin (H&E) staining provides a detailed view of tissue architecture and cell morphology, helping to identify different tissue types and assess inflammatory responses. Masson's trichrome staining distinguishes collagen from muscle fibers, highlighting areas of fibrosis and overall tissue architecture, while α-SMA staining is used to identify myofibroblasts as an indicator of fibrosis severity. For clarity, adhesion sites are marked with black dashed lines (see Figure 6 A, 6B and Figure 7 C).

[0121] On day 5, the mucosal epithelium in the MgH2-treated group recovered well, with no obvious adhesions and minimal fibrosis. In contrast, the control group showed severe adhesions between the intestinal wall and the abdominal wall, accompanied by extensive fibrosis. The fibrosis in the PLGA group was milder, but diffuse fibrosis was still observed. In addition, a large number of fat lacunae were present in both the control and PLGA groups, indicating incomplete healing and a more severe inflammatory response. By day 10, the MgH2 group still showed minimal fibrosis, while the PLGA group had some residual diffuse fibrosis and the control group showed extensive fibrosis. Notably, the number of fat lacunae was smaller in the MgH2 group, reflecting a better healing effect.

[0122] Masson trichrome staining further showed that collagen deposition in the MgH2 group was significantly reduced, and tissue arrangement was more orderly, indicating that it had a better effect in anti-adhesion remodeling. Similarly, α-SMA staining showed that the expression of α-SMA in the adhesion site in the MgH2 group was significantly reduced compared with the PLGA group and the control group, indicating that it effectively reduced the severity of fibrosis.

[0123] Subsequently, immunofluorescence staining using anti-CD31 and anti-Ki67 antibodies was also performed to evaluate in vivo cell proliferation and angiogenesis after different treatments (see Figure 7 A and 7B). In terms of angiogenesis, the vascular expression level in the MgH2 group was significantly higher, increasing by 25.59% compared with the PLGA group and 50.63% compared with the control group (see Figure 7 D). In terms of cell proliferation, the Ki67 expression level in the MgH2 group was also significantly higher than that in the PLGA group and the control group (see Figure 7 E).

[0124] These results indicate that MgH2 treatment is superior to conventional treatments in promoting tissue repair, reducing fibrosis, and preventing adhesion formation, suggesting its potential as a novel therapeutic strategy for the clinical management of intra-abdominal adhesions.

Claims

1. Use of magnesium hydride powder in the preparation of a medicament for inhibiting fibrinogen aggregation after abdominal surgery, wherein the magnesium hydride powder is at least one selected from the following: The magnesium hydride powder particles with a particle size of 10 microns or more account for 70% or more, The magnesium hydride powder particles with a particle size of 10 microns or more account for 80% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 45% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 50% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 55% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 30% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 35% or more, The magnesium hydride powder particles having a particle size of 15 microns or more account for 40% or more, and The average particle size of magnesium hydride powder is 14 microns ± 1 micron.

2. Use of magnesium hydride powder in the preparation of a medical device for inhibiting fibrinogen aggregation after abdominal surgery, wherein the magnesium hydride powder is at least one selected from the following: The magnesium hydride powder particles with a particle size of 10 microns or more account for 70% or more, The magnesium hydride powder particles with a particle size of 10 microns or more account for 80% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 45% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 50% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 55% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 30% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 35% or more, The magnesium hydride powder particles having a particle size of 15 microns or more account for 40% or more, and The average particle size of magnesium hydride powder is 14 microns ± 1 micron.

3. Use of magnesium hydride powder in the preparation of a drug for anti-adhesion after abdominal surgery, wherein the magnesium hydride powder is at least one selected from the following: The magnesium hydride powder particles with a particle size of 10 microns or more account for 70% or more, The magnesium hydride powder particles with a particle size of 10 microns or more account for 80% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 45% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 50% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 55% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 30% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 35% or more, The magnesium hydride powder particles having a particle size of 15 microns or more account for 40% or more, and The average particle size of magnesium hydride powder is 14 microns ± 1 micron.

4. Use of magnesium hydride powder in the preparation of a medical device for anti-adhesion after abdominal surgery, wherein the magnesium hydride powder is at least one selected from the following: The magnesium hydride powder particles with a particle size of 10 microns or more account for 70% or more, The magnesium hydride powder particles with a particle size of 10 microns or more account for 80% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 45% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 50% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 55% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 30% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 35% or more, The magnesium hydride powder particles having a particle size of 15 microns or more account for 40% or more, and The average particle size of magnesium hydride powder is 14 microns ± 1 micron.

5. The use according to claim 4, characterized in that The medical device is bagged powder.

6. The use according to claim 4, characterized in that The medical device is powder and its conveying device.

7. The use according to claim 6, characterized in that The medical device is a powder and a delivery device packaged separately.

8. The use according to claim 6, characterized in that The medical device is a powder placed in a conveying device.

9. A medical device for anti-adhesion after abdominal surgery, characterized in that The magnesium hydride powder is used as the active ingredient, and the magnesium hydride powder is at least selected from the following: The magnesium hydride powder particles with a particle size of 10 microns or more account for 70% or more, The magnesium hydride powder particles with a particle size of 10 microns or more account for 80% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 45% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 50% or more, The magnesium hydride powder particles with a particle size of 13 microns or more account for 55% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 30% or more, The magnesium hydride powder particles with a particle size of 15 microns or more account for 35% or more, The magnesium hydride powder particles having a particle size of 15 microns or more account for 40% or more, and The average particle size of magnesium hydride powder is 14 microns ± 1 micron.

10. A drug for anti-adhesion after abdominal surgery, characterized in that The magnesium hydride powder is used as the active ingredient, and the magnesium hydride powder is at least selected from the following: The magnesium hydride powder particles with a particle size of 10 microns or more account for 70% or more, The magnesium hydride powder particles with a particle size of 10 microns or more account for 80% or more, 45% or more of the magnesium hydride powder particles have a particle size of 13 microns or more, 50% or more of the magnesium hydride powder particles have a particle size of 13 microns or more, 55% or more of the magnesium hydride powder particles have a particle size of 13 microns or more, 30% or more of the magnesium hydride powder particles have a particle size of 15 microns or more, 35% or more of the magnesium hydride powder particles have a particle size of 15 microns or more, 40% or more of the magnesium hydride powder particles have a particle size of 15 microns or more, and The average particle size of magnesium hydride powder is 14 microns ± 1 micron.

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