Zn-MOF for treating spinal cord injury and its preparation method and application

Abstract

The present invention relates to a Zn-MOF for treating spinal cord injury and a preparation method and application thereof. The Zn-MOF provided by the present invention can effectively reduce the level of intracellular reactive oxygen, reduce the levels of IL-6 and IL-1β after spinal cord injury, inhibit the expression of MMP9, achieve significant antioxidant capacity, repair hippocampal neuronal cells, resist damage induced by oxidative stress, and promote neuronal branch formation and neurite growth. At the same time, the Zn-MOF of the present invention can significantly promote oxygen free radicals in the body of spinal cord injured mice, promote the repair of spinal cord injury and the recovery of motor function. The Zn-MO prepared by the present invention has no obvious toxic and side effects on the main tissues and organs of the body, and its safety is significantly improved, thereby providing a new research idea and drug source for the treatment of oxidative stress-related diseases including spinal cord injury, and has significant clinical application prospects and social value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to Zn-MOF for treating spinal cord injury, and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) is a serious complication of spinal cord injury, typically secondary to spinal fractures caused by direct or indirect external forces, such as violence, exercise, falls, traffic accidents, and falls from height. SCI can cause dysfunction in the limbs, intestines, bladder, and sexual function, resulting in severe physical and mental harm and a significant reduction in quality of life. According to statistics, there are approximately 20 million patients with SCI worldwide, with the number increasing by approximately 700,000 each year. The male-to-female ratio is approximately 2:1, and adults outnumber children. Currently, treatments for SCI primarily focus on surgery, medication, and stem cell therapy, but no definitive and effective treatment is available. Therefore, further research on SCI is urgently needed.

[0003] Spinal cord injury (SCI) involves an extremely complex pathophysiology, including cell loss and death, lack of neurotrophic factors, inflammation, axonal rupture, demyelination, and glial scar formation, which inhibit regeneration and recovery of damaged nerves. Following SCI, a series of complex pathophysiological changes occur within the injured area. These changes can be categorized as primary and secondary injuries based on the timing and pathophysiological changes. Primary injury is the initial stage of injury, occurring immediately after injury. It includes spinal cord concussion, contusion, and fracture, as well as rupture of spinal cord blood vessels, loss of red blood cells in the injured area, axonal rupture, and myelin sheath disruption. The severity of injury depends primarily on the extent of the initial damage and the duration of spinal cord compression. Secondary injury occurs after the primary injury, triggering a complex cascade of secondary damage, inflicting further chemical and mechanical damage to spinal cord tissue. This includes the recruitment of activated inflammatory cells to the injured area, leading to a series of inflammatory reactions, lipid peroxidation, free radical formation, apoptosis, Wallerian degeneration, tissue edema, and glial scar formation. Spinal cord injury is divided into acute, subacute, and chronic phases. The acute phase occurs within 48 hours of injury, the subacute phase occurs between 48 hours and 14 days, the intermediate phase occurs between 14 days and 6 months, and the chronic phase occurs after more than 6 months. During the acute phase of spinal cord injury, severe bleeding due to ruptured spinal blood vessels and a significant increase in the levels of inflammatory factors such as IL-6, IL-1β, and TNF-α can lead to cell dysfunction and death. During the acute and subacute phases of spinal cord injury, disruption of the blood-spinal cord barrier, a vigorous inflammatory response, and the release of numerous cytotoxic substances, including free radicals (such as superoxide anions and hydrogen peroxide), by inflammatory cells can further exacerbate spinal cord damage. As the acute inflammatory response subsides, the intermediate and chronic phases of spinal cord injury begin, during which vascular remodeling, myelin formation, and neural circuit remodeling can occur.

[0004] Metal-organic frameworks (MOFs), also known as porous coordination polymers, are a class of porous crystalline materials composed of metal ions / clusters and functional organic ligands assembled through strong coordination bonds. MOFs possess diverse structures and advantages, including simple synthesis, easy surface functionalization, high porosity, low density, high specific surface area and loading capacity, regular pore structure, tunable pore size, diverse and customizable topologies, good biocompatibility, and biodegradability. They have been widely used in drug delivery, bioimaging and sensing, gas adsorption / separation / storage, desalination, and biomedical applications. MOFs have demonstrated excellent drug loading and controlled release capabilities in drug transport and release. However, the metals commonly used for loading are manganese and chromium, which are highly toxic and therefore unsuitable for biomedical applications. To successfully apply MOFs in biological applications, such as intracellular imaging and drug delivery, their non-toxicity must be ensured.

[0005] Currently, treatments for spinal cord injury (SCI) remain very limited. Therefore, in-depth research into the pathogenesis of SCI is crucial for its treatment. Studies have shown that oxidative stress-mediated inflammation is a key pathogenesis of SCI, and alleviating oxidative stress can reduce secondary injury during SCI treatment. Furthermore, numerous studies have demonstrated that metal-organic frameworks (MOFs) can stably and non-toxicly encapsulate drugs within their pores and slowly release them. Therefore, MOFs can serve as effective drug carriers for drug delivery. Zinc, a low-toxic transition metal cation, has become an ideal candidate for the preparation of Zn-MOFs, which can be used in biological systems, particularly as drug carriers. However, limited research on Zn-MOFs in SCI exists, and no studies have reported their repair effects after SCI. This study aimed to investigate whether Zn-MOFs possess antioxidant properties and whether they can promote SCI repair. Furthermore, the mechanisms by which Zn-MOFs promote SCI repair were investigated, providing a basis for clinical treatment of SCI. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide a Zn-MOF for the treatment of spinal cord injury, its preparation method, and its application. The Zn-MOF compound provided by the present invention can effectively reduce the level of intracellular reactive oxygen species, achieve significant antioxidant capacity, and promote neuronal growth and recovery of motor function after spinal cord injury. At the same time, the Zn-MOF compound of the present invention is safer than conventional metal framework compounds such as manganese and chromium, and has no obvious toxic side effects on either neuronal cells or major organs and tissues of the body, providing a new source of drugs for the treatment of spinal cord injury.

[0007] In order to solve the above technical problems, the present invention is achieved through the following technical solutions.

[0008] The first aspect of the present invention provides a Zn-MOF compound, which is prepared by the following preparation method:

[0009] (1) Place ZnCl2, gallic acid and water in a round-bottom flask and reflux and stir evenly;

[0010] (2) adjusting the pH value to 7-9 using NaOH solution and heating in a muffle furnace to obtain a mixture;

[0011] (3) Centrifuging the mixture obtained in step (2), discarding the supernatant and taking the precipitate to obtain a light gray solid;

[0012] (4) The light grey solid obtained in step (3) is washed with water and dried to obtain the product.

[0013] Preferably, the mass ratio of ZnCl2 to gallic acid in step (1) is 1:2-6.

[0014] Preferably, the concentration of NaOH in step (2) is 10 mol / L.

[0015] Preferably, the heating temperature in step (2) is 110-170°C, and the heating time is 12-36 hours; most preferably, the heating temperature is 140°C, and the heating time is 24 hours.

[0016] Preferably, the centrifugal speed in step (3) is 10,000-15,000 rpm, the centrifugal time is 10-20 min, and the centrifugal temperature is 2-6°C; most preferably, the centrifugal speed is 10,000-15,000 rpm, the centrifugal time is 15 min, and the centrifugal temperature is 4°C.

[0017] Preferably, the number of washings in step (4) is 1-3 times; most preferably, the number of washings is 2 times.

[0018] A second aspect of the present invention provides a method for preparing a Zn-MOF compound, comprising the following steps:

[0019] (1) Place ZnCl2, gallic acid and water in a round-bottom flask and reflux and stir evenly;

[0020] (2) adjusting the pH value to 7-9 using NaOH solution and heating in a muffle furnace to obtain a mixture;

[0021] (3) Centrifuging the mixture obtained in step (2), discarding the supernatant and taking the precipitate to obtain a light gray solid;

[0022] (4) The light grey solid obtained in step (3) is washed with water and dried to obtain the product.

[0023] Preferably, the mass ratio of ZnCl2 to gallic acid in step (1) is 1:2-6.

[0024] Preferably, the concentration of NaOH in step (2) is 10 mol / L.

[0025] Preferably, the heating temperature in step (2) is 110-170°C, and the heating time is 12-36 hours; most preferably, the heating temperature is 140°C, and the heating time is 24 hours.

[0026] Preferably, the centrifugal speed in step (3) is 10,000-15,000 rpm, the centrifugal time is 10-20 min, and the centrifugal temperature is 2-6°C; most preferably, the centrifugal speed is 10,000-15,000 rpm, the centrifugal time is 15 min, and the centrifugal temperature is 4°C.

[0027] Preferably, the number of washings in step (4) is 1-3 times; most preferably, the number of washings is 2 times.

[0028] A third aspect of the present invention provides use of the above-mentioned Zn-MOF compound or the Zn-MOF compound prepared according to the above-mentioned preparation method in the preparation of a drug for treating diseases associated with oxidative stress.

[0029] Preferably, the disease associated with oxidative stress is selected from spinal cord injury.

[0030] A fourth aspect of the present invention provides a pharmaceutical composition for treating diseases associated with oxidative stress, comprising the above-mentioned Zn-MOF compound or the Zn-MOF compound prepared according to the above-mentioned preparation method, and a pharmaceutically acceptable carrier.

[0031] Preferably, the disease associated with oxidative stress is selected from spinal cord injury.

[0032] Preferably, the pharmaceutically acceptable carrier is selected from one or more of a filler, a disintegrant, a lubricant, a binder, an antioxidant, an antibacterial agent, a flavoring agent, an aromatic agent, and a chelating agent.

[0033] Spinal cord injury involves a complex inflammatory response coordinated by numerous inflammatory factors, including IL-1β and IL-6. Following SCI, the expression of IL-1β and IL-6 increases dramatically. Studies have shown that overexpression of IL-6 can promote glial cell proliferation. Therefore, reducing IL-6 levels in the acute phase of SCI can inhibit glial scar formation. Furthermore, high levels of IL-6 after SCI inhibit axonal growth. IL-1β is one of the most extensively studied proinflammatory cytokines. It is primarily produced by microglia, but neurons and astrocytes can also produce IL-1β. IL-1β expression increases dramatically in the injured central nervous system. IL-1β can activate the immune system, promote the production of inflammatory factors and other cytokines, and activate neurotoxic mediators. IL-1β has a significant negative impact on glial cell formation and axonal plasticity after SCI and is associated with poor functional outcomes. Studies have shown that knocking out IL-1β can reduce the proliferation of astrocytes and inhibit the inflammatory response after spinal cord injury.

[0034] Matrix metalloproteinase 9 (MMP9) is one of the most extensively studied members of the matrix metalloproteinase (MMP) family. It is a gelatinase. MMP9 is involved in the degradation of the extracellular matrix, promoting the degradation of gelatin, collagen types IV, V, and XI, myelin basic protein, and elastin. MMP9 expression is increased after spinal cord injury and is associated with inflammation and disruption of the blood-spinal cord barrier. Neutrophils, macrophages, and monocytes involved in the inflammatory response to spinal cord injury all express MMP9. MMP9 is upregulated and reaches maximum activity 12-24 hours after spinal cord injury, with neutrophils at the site of spinal cord injury being the primary source of MMP9. Inhibiting MMP9 activity during the first three days after spinal cord injury can reduce vascular permeability and alleviate inflammation. MMP9 is expressed throughout the development of the nervous system and is involved in axonal growth during development.

[0035] The synthesized Zn-MOF prepared by this invention can effectively reduce intracellular reactive oxygen species (ROS) levels, achieving significant antioxidant capacity. It can also reduce IL-6 and IL-1β levels after spinal cord injury and inhibit MMP9 expression. Zn-MOF treatment promotes branch formation and neurite outgrowth, indicating that Zn-MOF can repair neuronal cells and combat oxidative stress-induced damage. Furthermore, in vivo studies have shown that Zn-MOF treatment significantly promotes the recovery of motor function in mice with spinal cord injury, with significant improvements in BMS scores. Furthermore, the ROS bioluminescence intensity in mice with spinal cord injury after Zn-MOF treatment was significantly reduced, fully confirming the significant oxygen free radical scavenging and spinal cord injury repair effects of Zn-MOF in vivo. Furthermore, methods such as the step-in-the-air test, the rotarod test, the footprint test, the CatWalk gait analysis, and electrophysiological assessments showed that the hindlimb motor function of mice treated with Zn-MOF was significantly better than that of the SCI model group, further demonstrating that Zn-MOF can significantly improve spinal cord injury repair and promote the recovery of limb function.

[0036] The present invention has the following beneficial effects compared to the prior art:

[0037] (1) The Zn-MOF compound provided by the present invention can effectively reduce the level of intracellular reactive oxygen species, reduce the levels of IL-6 and IL-1β after spinal cord injury, inhibit the expression of MMP9, achieve significant antioxidant capacity, repair hippocampal neuronal cells, combat damage induced by oxidative stress, and promote neuronal branch formation and neurite growth. At the same time, the Zn-MOF of the present invention can significantly promote the removal of oxygen free radicals in the body of spinal cord injured mice, promote the repair of spinal cord injury and the recovery of motor function.

[0038] (2) The Zn-MOF compound prepared by the present invention has no obvious toxic side effects on HT22 cells, hippocampal neuronal cells or major tissues and organs of the body. Compared with the metal framework compounds such as manganese and chromium commonly used in the prior art, its safety is significantly improved, thereby providing a new research idea and drug source for the treatment of oxidative stress-related diseases including spinal cord injury, and has significant clinical application prospects and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of transmission electron microscopy characterization of Zn-MOF.

[0040] Figure 2 Schematic diagram of the distribution of C, O, and Zn elements in Zn-MOF.

[0041] Figure 3 Schematic diagram of the analysis results of Zn-MOF using Fourier infrared spectrometer and nanoparticle size analyzer.

[0042] Figure 4 Schematic diagram of the free radical scavenging ability of Zn-MOF.

[0043] Figure 5 Schematic diagram of the results of DCFH-DA staining analysis of the effects of different concentrations of Zn-MOF on ROS levels in HT22 cells.

[0044] Figure 6 Schematic diagram of the flow cytometry analysis of the effects of different concentrations of Zn-MOF on ROS levels in HT22 cells.

[0045] Figure 7 Schematic diagram of the effects of different concentrations of Zn-MOF on HT22 cell viability.

[0046] Figure 8 Schematic diagram of the effects of different concentrations of Zn-MOF on the viability of hippocampal neurons.

[0047] Figure 9 Schematic diagram of the effect of Zn-MOF on axon extension of primary hippocampal neurons.

[0048] Figure 10 Schematic diagram of BMS scoring results for different groups.

[0049] Figure 11 These are morphological images of mice in different groups 60 days after spinal cord injury.

[0050] Figure 12 Schematic diagram of the reactive oxygen species levels in mice of different groups.

[0051] Figure 13 Schematic diagram of the foot fault rate results of mice in different groups.

[0052] Figure 14 Schematic diagram of the falling latency results of mice in different groups.

[0053] Figure 15 Schematic diagram of the stride length and stride width results of mice in different groups.

[0054] Figure 16 Schematic diagram of the average maximum contact area and average maximum contact strength of the left hind limb of mice in different groups.

[0055] Figure 17 Schematic diagram of the average maximum contact area and average maximum contact strength of the right hind limb of mice in different groups.

[0056] Figure 18 Schematic diagram of the normal gait index results of mice in different groups.

[0057] Figure 19 Schematic diagram of the motor evoked potential results of mice in different groups.

[0058] Figure 20 Schematic diagram of HE staining results of spinal cord tissues of mice in different groups 60 days after spinal cord injury.

[0059] Figure 21 Schematic diagram of HE staining results of major organs of mice in different groups.

[0060] Figure 22 Schematic diagram of the detection results of serum IL-6 and IL-1β levels in mice of different groups.

[0061] Figure 23 Schematic diagram of the MMP9 content detection results in different groups of mice. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0063] Unless otherwise specified, all reagents used in the present invention were commercially available. For animal experiments, the relevant procedures and methods complied with medical ethics requirements. The experimental methods used in the present invention were all conventional methods and techniques in the art.

[0064] Representative results from replicate experiments are presented in the accompanying figures. Data are presented as mean ± SD and mean ± SEM as indicated in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 22.0 software. Standard medical statistical methods, such as t-tests, chi-square tests, and analysis of variance, were used to compare mean values ​​between two or more groups. p A difference of <0.05 was considered significant.

[0065] Example 1 Preparation and characterization of Zn-MOF

[0066] (1) Place 1.0 g ZnCl2, 3.8 g gallic acid and 50 g water in a round-bottom flask and reflux with stirring.

[0067] (2) The pH value was adjusted to 8 using 10 mol / L NaOH solution and heated at 140 °C for 24 h in a muffle furnace (Sxi-8-10, Shinhan Instruments Co., Ltd.) to obtain a mixture.

[0068] (3) The mixture obtained in step (2) was centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was discarded to obtain a light gray solid.

[0069] (4) The light gray solid obtained in step (3) was washed twice with water and dried to obtain a Zn-MOF compound.

[0070] The Zn-MOF compound prepared above was taken and its morphology and structure were observed using a transmission electron microscope. The morphology and structure information of the sample was obtained by magnifying and imaging the electron beam carrying the internal structure information of the sample. The results are as follows Figure 1 The elements on the surface of the Zn-MOF compound were then characterized using an X-ray photoelectron spectroscopy. The results are shown in Figure 2 The results show that the prepared Zn-MOF compound is spherical with a diameter of about 100nm. Figure 2 The distribution of Zn, C, and O elements is shown in the figure. The Zn-MOF compound was further tested by a nanoparticle size analyzer. The results are as follows: Figure 3 Nanoparticle size analyzer test results showed that the diameter of the Zn-MOF was approximately 100 nm, consistent with the transmission electron microscopy results mentioned above. These test results indicate that the Zn-MOF compound was successfully synthesized.

[0071] Example 2 Study on the effect of Zn-MOF on oxidative stress in vitro

[0072] The Zn-MOF compound prepared in Example 1 was used to test its free radical scavenging ability using the ABTS method. The specific steps are as follows:

[0073] (1) Take 20 mL of PBS solution and place it in a 50 mL centrifuge tube. Then weigh 54.87 mg of ABTS and add it to the centrifuge tube containing PBS solution. Mix thoroughly to prepare ABTS stock solution.

[0074] (2) Prepare a piece of filter paper and place a sufficient amount of MnO2 on the filter paper. Then transfer the filter paper and MnO2 to the top of the centrifuge tube. Then, add 10 mL of the ABTS stock solution prepared in step (1) onto the MnO2. Filter in the dark for 5 minutes, and then continue to avoid light for 12 hours. The reaction will form an ABTS free radical mixed solution containing ABTS free radicals (ABTS•+), which will be stored at -20°C for future use.

[0075] (3) 100 μL of Zn-MOF with different concentrations (6 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) was mixed with 100 μL of ABTS free radical mixed solution. A blank group and a control group were set up. Only 200 μL of PBS solution was added to the blank group, and 100 μL of PBS solution and 100 μL of ABTS free radical mixed solution were added to the control group.

[0076] (3) The absorbance at 734 nm was measured at 10 min, 20 min, 40 min, 60 min, 90 min, and 120 min using a cell imaging multimode reader (Cytation 5, BioTek Instruments Inc.).

[0077] Test results such as Figure 4 The results showed that Zn-MOF can significantly inhibit the formation of ABTS free radicals in a time- and dose-dependent manner. It can significantly inhibit ABTS free radicals at a concentration of 6 μg / mL, and can achieve an ABTS free radical inhibition rate of nearly 100% at a concentration of 12.5 μg / mL (*** p <0.001, vs 0 μg / mL group (control group). The above experiments show that Zn-MOF has a high activity in scavenging ABTS and can efficiently scavenge free radicals outside cells, thereby playing an antioxidant role. Therefore, it can serve as an effective free radical scavenger to prevent ROS damage to neurons.

[0078] Subsequently, HT22 cells were tested using a reactive oxygen species fluorescent probe (DCFH-DA) and flow cytometry to verify whether Zn-MOF also has significant free radical scavenging activity in cells. The specific steps of the DCFH-DA experiment are as follows:

[0079] (1) Pipette 9 mL of serum-free DMEM medium into a 15 mL centrifuge tube wrapped in tin foil and protected from light. Add 9 μL of DCFH-DA stock solution, mix thoroughly to prepare DCFH-DA solution, and store in a dark environment.

[0080] (2) 120 mmol / L sodium L-glutamate was added to HT22 cells to construct a cell injury model, and then the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 12 h.

[0081] (3) After the cell damage model was successfully constructed, the cells were treated with different concentrations of Zn-MOF (12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL) for 12 h. A blank group and a control group were also set up. The blank group (Control group) consisted of normal HT22 cells, and the control group consisted of HT22 cells damaged by sodium L-glutamate and with a Zn-MOF addition amount of 0 μg / mL.

[0082] (4) Take the HT22 cells (cultured in a 24-well plate) out of the cell culture incubator, remove the original culture medium, and gently add 200 μL of the DCFH-DA solution prepared in step (1) to each well along the wall of the well. Then wrap the 24-well plate with tin foil to prevent light transmission, and then place it in a cell culture incubator at 37°C and 5% CO2 for 30 minutes.

[0083] (5) Take the cells out of the incubator and place them in a dark room. Remove the DCFH-DA solution and wash them twice with PBS. Then add 500 μL of TBST (containing Triton×-100) solution to each well and punch them three times, each time for 5 minutes. Then, use ICC technology to seal the slides and take pictures under a laser confocal microscope.

[0084] The results are as follows Figure 5 The results showed that the fluorescence intensity of the control group (injured without drug addition) was significantly increased compared to the blank group (control group), indicating that the L-glutamate sodium injury HT22 cell model was successfully established. Furthermore, the fluorescence intensity of the Zn-MOF group was significantly decreased compared to the control group, indicating that Zn-MOF effectively inhibited intracellular ROS and alleviated oxidative stress.

[0085] The specific steps for detecting reactive oxygen species by flow cytometry are as follows:

[0086] (1) HT22 cells in the logarithmic growth phase were taken, the original culture medium was removed, 120 mmol / L sodium L-glutamate solution was added, and then placed in a cell culture incubator at 37°C and 5% CO2 for 12 hours to construct a cell injury model.

[0087] (2) After the cell damage model was successfully constructed, the cells were treated with different concentrations of Zn-MOF (12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL) for 12 h. A blank group and a control group were set up. The blank group (Control group) consisted of normal HT22 cells, and the control group consisted of HT22 cells damaged by sodium L-glutamate and with a Zn-MOF addition amount of 0 μg / mL.

[0088] (3) Remove the cell supernatant, add trypsin to the culture plate to digest the cells and resuspend them. Then, mix the cell resuspension and cell supernatant and place them in a 5 mL centrifuge tube. Centrifuge at 2000 rpm for 5 minutes and discard the supernatant. Add 500 μL of PBS solution to resuspend the cells and transfer them to a 1.5 mL centrifuge tube. Centrifuge at 2000 rpm for 5 minutes.

[0089] (7) Add 300 μL of active oxygen DCFH-DA dye solution (Biyuntian), mix gently and incubate in the dark for 20 minutes; then centrifuge at 2000 rpm for 5 minutes, discard the supernatant, add 500 μL of PBS solution, mix gently and centrifuge again at 2000 rpm for 5 minutes, discard the supernatant; add 300 μL of PBS solution, mix gently and transfer to a flow tube for detection.

[0090] The results are as follows Figure 6 The results were similar to those of the DCFH-DA experiment, showing a significant increase in ROS levels in the control group (damaged without drug addition) compared to the blank group (control group). However, ROS levels in the Zn-MOF group were significantly decreased compared to the control group, indicating that Zn-MOF effectively inhibited intracellular ROS and alleviated oxidative stress. The ROS levels in HT22 cells measured using the aforementioned reactive oxygen species fluorescence probe and flow cytometry were fully demonstrated to be effective in inhibiting intracellular ROS and alleviating oxidative stress.

[0091] Furthermore, HT22 cells were selected for the toxicity test of Zn-MOF. The specific steps are as follows:

[0092] (1) Using a cell counter, seed HT22 cells at a cell count of 5000 cells / 100 μL / well in a 96-well plate.

[0093] (2) After cell adhesion, different concentrations of Zn-MOF (1.57, 3.13, 6.25, 12.5, 25, 50, 100 μg / mL) were added to each well and incubated for another 12 hours. A blank group and a control group were set up. The blank group only added an equal volume of culture medium without inoculating cells, while the control group was inoculated with an equal number of cells without Zn-MOF treatment.

[0094] (3) After 12 hours, add 110 μL of prepared CCK-8 solution (10 μL of MEM solution + 10 μL of CCK-8 stock solution, mix well and add), wrap the 96-well plate with tin foil to protect it from light, and then continue incubation for 1 hour.

[0095] (4) After incubation, the OD value (absorbance) of each group of cells at a wavelength of 450 nm was detected using a microplate reader, and then the cell viability was calculated according to the formula: Cell viability (%) = (drug group - blank group) / (Control group - blank group) × 100%.

[0096] The results are as follows Figure 7 The results showed that at the concentrations of 1.57-100 μg / mL studied, the survival rate of hippocampal neuronal cells after Zn-MOF treatment was close to 100%, and there was no significant difference in survival rate compared with the control group (Control group). p >0.05). This indicates that Zn-MOF has excellent safety and no obvious cytotoxicity to HT22 cells.

[0097] Subsequently, rat hippocampal cells were selected to further verify the toxicity of Zn-MOF. The specific steps are as follows:

[0098] (1) The hippocampus tissue of 1-day-old Sprague-Dawley (SD) rats was minced with ophthalmic scissors and added to a 15 mL centrifuge tube (Corning). Papain (Sigma, Cat# P4762) was added and digested at 37°C for 25 min.

[0099] (2) Use bovine serum albumin to terminate the action of protease and then wash the tissue.

[0100] (3) The isolated hippocampal neurons were seeded at a concentration of 20,000 cells / mL in a 96-well plate (Corning), with 200 μL per well, and cultured in DMEM medium (with glutamine, Gibco, Carlsbad, CA, Cat#12430054) containing 10% fetal bovine serum and 10% nutrient mixture F-12 in a 37°C, 95% O2, 5% CO2 incubator for 6 h.

[0101] (4) Replace the original culture medium with Neurobasal medium containing 5% B-27 (Gibco) and culture in an incubator at 37°C, 95% O2, and 5% CO2 for 12 h.

[0102] (5) After cell adhesion, different concentrations of Zn-MOF (0.40, 0.79, 1.57, 3.13, 6.25, 12.5, 25, 50, 100 μg / mL) were added to each well and incubated for another 24 hours. A blank group and a control group were set up. The blank group only added an equal volume of culture medium without inoculating cells, while the control group was inoculated with an equal number of cells without Zn-MOF treatment.

[0103] (6) After 12 hours, add 110 μL of prepared CCK-8 solution (10 μL of MEM solution + 10 μL of CCK-8 stock solution, mix well and add), wrap the 96-well plate with tin foil to protect it from light, and then continue incubation for 1 hour.

[0104] (7) After incubation, the OD value (absorbance) of each group of cells at a wavelength of 450 nm was detected using an enzyme-labeled instrument, and then the cell viability was calculated according to the formula: cell viability (%) = (drug group - blank group) / (control group - blank group) × 100%.

[0105] The results are as follows Figure 8 The results showed that at the concentrations of 0.40-100 μg / mL studied, the survival rate of hippocampal neuronal cells after Zn-MOF treatment was close to 100%, and there was no significant difference in survival rate compared with the blank control group ( p >0.05). This further confirmed that Zn-MOF has excellent safety and no obvious cytotoxicity to hippocampal neurons.

[0106] Example 3 Repair effect of Zn-MOF on neurons

[0107] Rat hippocampus tissue was selected for Zn-MOF neuron damage repair detection. The specific steps are as follows:

[0108] (1) Hippocampal tissue from one-day-old Sprague-Dawley (SD) rats was minced with ophthalmic scissors and added to a 15 mL centrifuge tube (Corning). Papain (Sigma, Cat# P4762) was added and digested at 37°C for 25 min. The tissue was washed after the protease action was terminated with bovine serum albumin.

[0109] (2) Isolated hippocampal neurons were seeded at a concentration of 20,000 cells / mL in 24-well plates (Corning), with 500 μL per well, and cultured in DMEM medium (with glutamine, Gibco, Carlsbad, CA, Cat#12430054) containing 10% fetal bovine serum and 10% nutrient mixture F-12 in a 37°C, 95% O2, 5% CO2 incubator for 24 h.

[0110] (3) The original culture medium was replaced with a culture medium containing 120 μM L-glutamate, and the cells were cultured in an incubator at 37°C, 95% O2, and 5% CO2 for 12 h to form a hippocampal neuron injury model;

[0111] (4) After cell adhesion, different concentrations of Zn-MOF (12.5, 25, 50, 100 μg / mL) were added to each well and incubated for 24 hours. The control group was a blank control group, and the hippocampal neurons were not damaged. A blank group (Control group) and a control group (Injury group) were also set up. The blank group was normal hippocampal neurons, and the control group was hippocampal neurons that had been treated with injury modeling and were not treated with Zn-MOF. The hippocampal neurons were stained and sealed using ICC technology. The sealed slides were photographed using a laser confocal microscope. Finally, Image J software was used to depict and count the number and length of primary and secondary processes, as well as the total process of neurons.

[0112] The effect of Zn-MOF on neuronal injury repair was evaluated by quantifying the length and total number of neuronal processes. Figure 9 The results showed that compared with the control group, the number and length of primary and secondary processes and total processes in the injury group were significantly reduced, indicating that the establishment of the L-glutamate sodium injury model for primary hippocampal neurons was successful. In the Zn-MOF treated group, the length of primary and secondary branches and the total length of processes were significantly longer than those in the model group (* p <0.05,** p <0.01,*** p <0.001, vs Injury group (0μg / mL group). After Zn-MOF treatment, the length of the primary branches and the total length of the neurites in the neurons were not significantly different from those in the blank control group (Control group). It was also found that the number of primary and secondary branches and the total number of neurites in the neurons treated with Zn-MOF were significantly greater than those in the model group (** p <0.01,*** p <0.001, vs. the injury group (0 μg / mL group). Furthermore, after Zn-MOF treatment, the number of primary branches and total neurites in neurons recovered to levels not significantly different from those in the blank control group. In summary, the addition of Zn-MOF significantly promoted neurite outgrowth in injured neurons, demonstrating that the Zn-MOF compound prepared in this invention can promote axonal extension in damaged neurons by inhibiting ROS production, demonstrating its significant protective and recovery-promoting effects against ROS-induced neuronal oxidative stress damage.

[0113] Example 4 In vivo functional study of Zn-MOF

[0114] (1) Twenty-four 6-week-old female mice were selected and divided into four groups, with 6 mice in each group, including a blank control group (Control group, 6 mice, no treatment), a sham control group (sham group, 6 mice, only the lamina was removed without spinal cord injury), a model group (Injury group, 6 mice), and a Zn-MOF group (Injury+Zn-MOF, 6 mice). The drugs were injected intraperitoneally starting from the second day after spinal cord injury, with an injection volume of 100 μL / mouse and an injection dose of 50 μg. The sham group and the injury group were injected with an equal volume of PBS once a day for three consecutive days. The method for constructing a spinal cord injury mouse model is as follows:

[0115] a. Remove the hair on the back of the mouse to expose the skin on its back.

[0116] b. Anesthesia: Intraperitoneally inject 1.25% tribromoethanol at a dose of 200 μg / 20 g. Successful anesthesia is indicated by stable breathing, significantly weakened limb muscle strength, and disappearance of pain and corneal reflexes.

[0117] c. Expose the spinal cord: Disinfect the skin by wiping it twice with an iodine-containing cotton ball. Identify the level of thoracic vertebrae 9-11 (T9-T11) and make a 2.5 cm longitudinal incision mid-back at the T9-T11 level. Bluntly dissect the paraspinal muscles, remove the spinous processes and lamina with rongeurs, and completely expose the spinal cord at the T9-T11 level. Use a fixator to secure the T10 level, and resect the T10 lamina, taking care to avoid additional tissue damage during the procedure. Then, use a fixator to distract and secure the spinal cord to fully expose the spinal cord.

[0118] d. The mice were secured in the Louisville Injury System Apparatus (LISA). The spinal cord was positioned under the impactor, and the impact position was confirmed by a laser beam monitor. The depth of the impact was used to determine the extent of spinal cord injury. In this example, the impactor tip pressure was 18 psi, the impact depth was 0.8 mm, and the injury duration was 0.5 s. After the impact, the mice were removed from the injury apparatus and the fixator. After adequate hemostasis, the muscles and skin of the mice were sutured. Mice in the sham-operated group underwent laminectomy at T10 without spinal cord impact.

[0119] e. Close the incision and inject gentamicin 2000U subcutaneously once a day for 3 consecutive days. Perform artificial bladder emptying every 8 hours until spontaneous urination is achieved.

[0120] (2) Monitoring: Starting from the establishment of the model, the movement status of each group of mice was monitored every day, and the BMS score was performed every 3 days. The results were as follows: Figure 10As shown. The results showed that, at the beginning, except for the blank control group, all SCI mice completely lost their hind limb movement. Subsequently, with the passage of time, the hind limb motor function of each group of SCI mice began to gradually recover. Among them, on the 6th day after injury, the recovery rate of the Zn-MOF treatment group was significantly higher than that of the model group (Injury group), and the BMS score gradually improved. During the entire experiment, the BMS score of the Control group and the Sham group mice remained at 9 points. Although the BMS score of the Injury group mice showed a slow increasing trend, it still did not exceed 1 point on the 60th day, and the rate of increase in the score was very slow. However, compared with the Injury group, the BMS score of the Zn-MOF group mice increased faster and had higher scores. Until the 60th day, the score was close to 4 points, indicating that Zn-MOF can promote the recovery of motor function in mice with spinal cord injury. After 60 days after injury, the morphology of mice in each group was as shown below. Figure 11 As shown, the hindlimb morphology of mice in the control and sham groups was normal, while the hindlimbs of mice in the injury group were paralyzed. The hindlimb morphology of mice in the Zn-MOF group was improved compared to that of the injury group. These results indicate that Zn-MOF treatment significantly enhances behavioral recovery in mice after SCI.

[0121] (3) In vivo imaging of small animals: In vivo imaging of mice was performed on day 3 after injury. First, 0.1 mL / 20 g of L-012 (chemiluminescent (CHL) probe) was intraperitoneally injected at a concentration of 4 mg / ml. Five minutes later, 1.25% tribromoethanol was injected intraperitoneally at a dose of 200 µg / 20 g to induce anesthesia. After successful anesthesia, in vivo imaging of the small animals was performed.

[0122] The results are as follows Figure 12 The results showed that ROS bioluminescence was rare in the spinal cord of normal mice, while the bioluminescence intensity of ROS in the spinal cord of mice with spinal cord injury was significantly increased and increased over time. After Zn-MOF treatment, the bioluminescence intensity of ROS was significantly reduced, significantly lower than that of the model group (Injury group) and even lower than that of the Sham group (* p <0.05,** p The quantitative value of ROS bioluminescence intensity indicated that Zn-MOF promoted the repair of spinal cord injury by reducing ROS in the spinal cord of mice. This further confirmed that Zn-MOF has significant oxygen free radical scavenging properties in vivo, which can reduce the high level of reactive oxygen species in mice after spinal cord injury, thereby playing an antioxidant role and promoting the repair of spinal cord injury.

[0123] On day 60 after spinal cord injury, mice underwent the air-step test and the rotarod test. The air-step test involved placing the mouse on parallel bars, enclosing it in a square frame, and allowing it to move freely within a defined area. The mouse was then videotaped for 3-5 minutes. During the crawling process, if a lower limb failed to step onto the parallel bars, a sensor automatically recorded it as a foot fault. The total number of foot faults recorded for each mouse during the 60 steps on the parallel bars was then calculated. The foot fault rate was calculated as the number of times both lower limbs fell off the parallel bars divided by the total number of steps (60 steps).

[0124] The experimental results are as follows Figure 13 The results showed that compared with the control and sham groups, the foot fault rate of mice in the injury group was significantly increased, indicating that the hindlimb motor function of the injury mice was very poor. However, compared with the injury group, the foot fault rate of mice in the Zn-MOF group was significantly reduced, indicating that Zn-MOF can promote the recovery of hindlimb motor function in mice with spinal cord injury.

[0125] The specific steps of the rotarod experiment are as follows: turn on the rotarod switch to make it rotate continuously; grab a mouse and place it on the rotarod so that the direction of the mouse's movement is opposite to the direction of rotation of the rotarod. Start timing from the moment the mouse is placed on the rotarod and end timing when the mouse falls off the rotarod. Record the time each mouse spends on the rotarod; collect data and analyze them.

[0126] The experimental results are as follows Figure 14 The results showed that the fall latency of mice in the injury group was significantly shorter than that of the control and sham groups. However, the fall latency of mice in the Zn-MOF group was significantly longer than that of the injury group, further demonstrating that Zn-MOF can promote the recovery of motor function in mice with spinal cord injury.

[0127] In the early stages of injury (7 days), mice with hemisection injuries experienced paralysis of the right hind limb, with dragging and minimal joint movement, making assessment impossible. Subsequently, between 7 and 14 days, lower limb function gradually recovered, with less dragging and significantly increased knee and hip joint movement. Therefore, footprint analysis was performed 14 days after injury. The evaluation parameters in the footprint analysis experiment included stride length and stride width. The specific steps are as follows:

[0128] Experimental mice were randomly divided into four groups, designated Groups 1 to 4, and a spinal cord injury model was established as described above. Group 1 served as the blank control group (Control), Group 2 as the sham group (Sham), Group 3 as the model group (Injury), and Group 4 as the Zn-MOF group. All mice underwent right spinal cord hemisection at T10 and were simultaneously treated with Zn-MOF, using the same dosing method as described above. Before the experiment, a narrow tunnel was prepared and lined with white paper. The hind paws of mice in each group were stained with non-toxic red ink. The mice were then placed in the pre-prepared tunnel and allowed to walk freely along it. Hind paw prints were visible on the paper, and stride length or width was analyzed by calculating the average of three consecutive prints.

[0129] The experimental results are as follows Figure 15 The results showed that the spinal cord hemisection model caused partial loss of hind limb motor function in mice. Compared with the Sham group (Group 2), the hind limb movement of mice in the Injury group (Group 3) was dragging ( Figure 15 A), while the hind limb motor function of mice treated with Zn-MOF (Group 4) was significantly better than that of the Injury group. The results of quantitative statistical analysis of footprints showed that the stride length and step width of the experimental group were better than those of the SCI group 14 days after surgery ( Figure 15 B. Figure 15 C) (n=6 in all groups, * p <0.05,** p <0.01,*** p <0.001, ns means not significant). This further proves that Zn-MOF can significantly improve the repair of spinal cord injury and promote the recovery of limb function.

[0130] Subsequently, CatWalk gait analysis was performed on mice in different groups. The specific steps are as follows: place the mice in a dark room to adapt for a while; then grab the mice and place them at the end of the plexiglass floor of the CatWalk analysis system, cover it with a lid, and let them freely cross from one end of the floor to the other. At the same time, observe the process of the mice freely crossing the plexiglass floor on the computer. Note that the mice cannot stop while crossing the plexiglass floor, otherwise it must be repeated. When the mice cross the plexiglass floor very smoothly, the computer will automatically save the mouse's movement video; repeat the above steps until the gait-related data of all experimental mice are collected.

[0131] The experimental results are as follows Figure 16-18The results showed that compared with the control and sham groups, the average maximum contact area and average maximum contact strength of the hind limbs of the mice in the injury group were very small, indicating that the motor function of the hind limbs of the mice in the injury group was very poor. However, compared with the injury group, the average maximum contact area and average maximum contact strength of the hind limbs of the mice in the Zn-MOF group were significantly increased ( Figure 16-17 Compared with the Control and Sham groups, the normal gait index of the mice in the Injury group was very small, indicating that the hind limb motor function of the mice in the Injury group was very poor. Compared with the Injury group, the normal gait index of the mice in the Zn-MOF group was significantly increased ( Figure 18 CatWalk gait analysis results indicate that Zn-MOF can promote the recovery of motor function in mice with spinal cord injury.

[0132] The above behavioral tests demonstrated that Zn-MOF can promote motor function recovery in mice with spinal cord injury. Subsequently, electrophysiological testing was performed to verify whether Zn-MOF can promote the recovery of nerve conduction function in mice with spinal cord injury. The specific steps were as follows: Mice were anesthetized with 1.25% 2,2,2-tribromoethanol (dose 0.2 mL / 10 g). Hair on the head and thighs was removed, and the motor area of ​​the cerebral cortex and bilateral gastrocnemius muscles were dissected. A neurophysiological monitor was activated with parameters set to 0.05 mV voltage and 0.05 mA current. All signal processing was performed on a BL-420N biosignal acquisition and analysis system (Chengdu Taimeng). Stimulating electrodes were inserted into the motor area of ​​the cerebral cortex and receiving electrodes were inserted into the bilateral gastrocnemius muscles. Motor evoked potentials (MEPs) were collected from the gastrocnemius muscles of the mice. This MEP acquisition was repeated three times for each mouse. After data collection from all mice was completed, they were analyzed.

[0133] The experimental results are as follows Figure 19 The results showed that the amplitude of the motor evoked potential of mice in the injury group was significantly lower than that of the control group and the sham group, while the amplitude of the motor evoked potential of mice in the Zn-MOF group was significantly higher than that of the injury group, indicating that Zn-MOF can effectively promote the recovery of nerve conduction function in mice with spinal cord injury.

[0134] To more intuitively evaluate the repair effect of Zn-MOF on injured spinal cord in mice, HE staining was performed on the spinal cord tissue of each group of mice on day 60 after spinal cord injury to observe the morphology of the spinal cord tissue. The results showed that no obvious damage was observed in the spinal cord tissue of the control group and the sham group; obvious structural damage was observed in the spinal cord tissue of the injury group; and the extent of spinal cord tissue damage in the Zn-MOF group was significantly reduced compared to the injury group, indicating that Zn-MOF can indeed promote the repair of injured spinal cord in mice (see Figure 20 ).

[0135] The above experiments verified that Zn-MOF had no obvious toxic side effects on HT22 cells and hippocampal neurons in vitro. To further evaluate its safety, HE staining was performed on the main organs of each group of mice on the 60th day after spinal cord injury to observe the pathological changes in the heart, liver, spleen, lung, and kidney. The results are as follows: Figure 21 The results showed that there were no significant differences in the brain, heart, liver, spleen, lung, and kidney of mice in different groups, indicating that Zn-MOF had no obvious toxic side effects on the brain, heart, liver, spleen, lung, and kidney of mice, further confirming the high safety of Zn-MOF.

[0136] Example 5 Study on the mechanism of action of Zn-MOF

[0137] To clarify the mechanism of action of Zn-MOF and its relationship with the inflammatory response after spinal cord injury, Zn-MOF was injected into mice with spinal cord injury for three consecutive days. The serum of mice in different groups was then subjected to enzyme-linked immunosorbent assay (ELISA) to detect the levels of inflammatory factors IL-6 and IL-1β in the serum of mice in different groups. The results are as follows: Figure 22 The results showed that compared with the control group and the sham group, the levels of serum IL-6 and IL-1β in the injury group were significantly increased, while compared with the injury group, the levels of serum IL-6 and IL-1β in the Zn-MOF group were significantly decreased, indicating that Zn-MOF can inhibit IL-6 and IL-1β and thus reduce the inflammatory response after spinal cord injury.

[0138] Since the expression level of MMP9 usually increases after spinal cord injury, MMP9, as an extracellular enzyme, can degrade a series of extracellular matrix proteins and participate in the process of spinal cord injury. To this end, we further extracted proteins from the cortical neurons and spinal cord tissues of mice with spinal cord injury in different groups and performed immunoblotting (Western blot), and then used MMP9 antibodies for detection. The results are as follows Figure 23The results showed that the content of MMP9 in cortical neurons of different groups was compared: compared with the control group, the expression of MMP9 in the injury group increased, while compared with the injury group, the expression of MMP9 in the Zn-MOF group decreased ( Figure 23 A). Comparison of MMP9 content in spinal cord tissue of mice in different groups: Compared with the Control group and Sham group, the expression of MMP9 in the Injury group increased, while compared with the Injury group, the expression of MMP9 in the Zn-MOF group decreased significantly ( Figure 23 B). This indicates that Zn-MOF can reduce the expression of MMP9 after spinal cord injury.

[0139] Spinal cord injury involves complex pathophysiological mechanisms, one of which is oxidative stress-mediated inflammation. This study explores the relationship between oxidative stress and spinal cord injury, investigating the effects of Zn-MOF on axonal extension in damaged neurons at the cellular level and on spinal cord repair in mice. Furthermore, the study investigates the mechanism of action of Zn-MOF in spinal cord repair.

[0140] First, the present invention demonstrated the extracellular antioxidant activity of Zn-MOF through ABTS+ free radical scavenging experiments. Furthermore, at the cellular level, Zn-MOF was verified to inhibit intracellular ROS, thereby exerting an antioxidant effect and promoting axonal extension in damaged neurons. Then, by constructing a mouse spinal cord injury model and studying the effects of Zn-MOF after spinal cord injury, it was found that Zn-MOF has an antioxidant effect in mice with spinal cord injury, reducing the levels of IL-6 and IL-1β and the expression of MMP9 after spinal cord injury, promoting the recovery of motor function and nerve conduction function in mice with spinal cord injury, and thus promoting the repair of spinal cord injury in mice.

[0141] Studies have shown that the pathogenesis of neurodegenerative diseases may be related to oxidative stress and excitotoxicity. The excitatory neurotransmitter glutamate may cause certain neurological diseases. It can mediate oxidative stress damage and excitotoxicity, a phenomenon known as glutamate neurotoxicity. It can cause damage to many cellular components, including mitochondria, and ultimately lead to cell death. During the cell death process, a large amount of reactive oxygen species (ROS) such as superoxide anions (O2· -Excessive production of ROS, including glutamate, mediates the excitotoxicity of glutamate. This study used sodium L-glutamate to damage HT22 cells, causing them to produce excessive ROS. DCFH-DA staining and flow cytometry were then used to measure ROS levels within HT22 cells. The results showed that Zn-MOF can inhibit intracellular ROS and alleviate oxidative stress.

[0142] Neurons are the cell type most susceptible to oxidative stress in the central nervous system and are particularly susceptible to ROS. ROS can damage cellular nucleic acids, proteins, and lipids, leading to neuronal dysfunction and even neuronal death. ROS are involved in the development and progression of many neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. It was previously believed that endogenously produced ROS can act as signaling molecules to regulate a range of physiological processes in the nervous system, such as neuronal development, neuronal polarity, synaptic plasticity, growth cone pathfinding, and regulation of neural circuits. However, excessive ROS production can lead to an imbalance between oxidative and antioxidant mechanisms, causing oxidative stress. The present invention measured the number and length of neuronal processes to verify whether Zn-MOF promotes the repair of damaged neurons by inhibiting ROS. The results showed that compared with the injury group, the number and length of primary and secondary processes, as well as the total process, of neurons in the Zn-MOF group were significantly increased. This suggests that Zn-MOF can promote axonal extension in damaged neurons by inhibiting ROS. Furthermore, the researchers used L-012 to measure the levels of reactive oxygen species in mice and conducted in vivo imaging to verify whether Zn-MOF could exert its antioxidant effect in mice with spinal cord injury. The results showed that Zn-MOF could reduce the high levels of reactive oxygen species in mice after spinal cord injury, thereby alleviating oxidative stress.

[0143] To investigate whether Zn-MOF can promote the repair of spinal cord injury in mice, different groups of mice were tested using methods such as the BMS score, air step test, rotarod test, footprint test, CatWalk gait analysis, and electrophysiological assessment. Results showed that 60 days after spinal cord injury, the Zn-MOF group showed faster recovery of hindlimb motor function, with a BMS score approaching 4 and a significantly lower foot error rate compared with the injury group. Rotarod test results showed a significantly longer fall latency in the Zn-MOF group compared with the injury group. Footprint test results showed a significant increase in stride length and width in the Zn-MOF group compared with the injury group. CatWalk gait analysis showed that the mean maximum contact area, mean maximum contact strength, and step sequence normality index of the Zn-MOF group were significantly improved compared with the injury group. Furthermore, at 60 days after spinal cord injury, spinal cord tissue from the different groups was stained with hematoxylin and eosin to observe spinal cord morphology. The results showed that the extent of spinal cord tissue damage in the Zn-MOF group was significantly reduced compared to the Injury group. These results together indicate that Zn-MOF can promote the recovery of motor function in mice with spinal cord injury.

[0144] Furthermore, the present invention also conducted an in-depth study of the safety of Zn-MOF. The results showed that Zn-MOF had no significant toxicity to HT22 cells or primary hippocampal neurons. Furthermore, an analysis of the major organs of different mice 60 days after spinal cord injury revealed no significant differences in the brain, heart, liver, spleen, lungs, and kidneys among the different groups of mice. This indicates that Zn-MOF has no significant toxic side effects on the brain, heart, liver, spleen, lungs, and kidneys of mice, demonstrating the high safety of Zn-MOF.

[0145] The above detailed description of the analytical methods involved in the present invention provides a detailed introduction. It should be noted that the above description is intended solely to help those skilled in the art better understand the methods and concepts of the present invention, and is not intended to limit the relevant content. Without departing from the principles of the present invention, those skilled in the art may make appropriate adjustments or modifications to the present invention, and such adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Use of a Zn-MOF compound in the preparation of a drug for treating a disease associated with oxidative stress, characterized in that: The Zn-MOF compound is prepared by the following method: (1) Place ZnCl2, gallic acid and water in a round-bottom flask and reflux and stir evenly; (2) adjusting the pH value to 7-9 using a NaOH solution and heating the mixture in a muffle furnace to obtain a mixture; (3) centrifuging the mixture obtained in step (2), discarding the supernatant and taking the precipitate to obtain a light gray solid; (4) The light grey solid obtained in step (3) is washed with water and dried to obtain.

2. The use according to claim 1, characterized in that: The disease associated with oxidative stress is selected from spinal cord injury.

3. The use according to claim 1, characterized in that: The concentration of NaOH in step (2) is 10 mol / L.

4. The use according to claim 1, characterized in that: The heating temperature in step (2) is 110-170° C., and the heating time is 12-36 hours.

5. The use according to claim 1, characterized in that: The centrifugal speed in step (3) is 10000-15000 rpm, the centrifugal time is 10-20 min, and the centrifugal temperature is 2-6°C.

6. The use according to claim 1, characterized in that: The number of washings in step (4) is 1-3 times.

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