Functional hydrogel loaded with diacylglycerol acyltransferase inhibitor as well as preparation method and application of functional hydrogel

Abstract

The invention discloses functional hydrogel loaded with a diacylglycerol acyltransferase inhibitor as well as a preparation method and application of the functional hydrogel. The invention reveals for the first time that the diacylglycerol acyltransferase (DGAT) inhibitor can regulate and control the triglyceride level of nerve cells so as to relieve neuron death and / or apoptosis, constructs the functional hydrogel loaded with the DGAT inhibitor, and the hydrogel has good biocompatibility, can realize 7-day local slow release of the DGAT inhibitor, and can be used for treating neuronal death and / or apoptosis. The DGAT inhibitor can realize in-vivo local administration of the DGAT inhibitor, effectively reduces nerve cell death and / or apoptosis in spinal cord injury repair, promotes regeneration of spinal cord tissues and nerves, and provides a new material and a new method for treatment of spinal cord injury repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of neurology, and relates to a functional hydrogel loaded with a diacylglycerol acyltransferase inhibitor, a preparation method thereof, and an application thereof. Background Art

[0002] Neural regeneration and functional recovery after spinal cord injury are one of the current medical problems in the world. This is mainly because neurons, as the basic units of the structure and function of the spinal cord neural network, will be greatly lost after spinal cord injury and do not have the ability of self-renewal. Spinal cord injury includes two stages: primary injury and secondary injury. The former causes truncation of nerve bundles, damage and death of neurons in the injury area, and destruction of the spinal cord neural network; the latter occurs ischemia, inflammation, formation of cavities at both ends of the injury area, insufficient neurotrophic factors, proliferation of glial cells to form scar tissue, etc. These factors promote the formation of an injury microenvironment that inhibits neural regeneration, hinder the axonal growth of surviving neurons, and induce apoptosis, pyroptosis or necrosis. Thus, the death range and degree of injury of spinal cord neurons at the injury end closely affect the bridging of neural circuits, and play a decisive role in neural function recovery and the prognosis of spinal cord injury.

[0003] Over the years, scientific researchers have carried out a large number of research works from the perspectives of regulating the inflammatory microenvironment and giving exogenous neurotrophic factors, etc., trying to reduce neuron injury and promote neural regeneration. For example, a three-dimensional printed bionic hydrogel spinal cord scaffold seeded with neural progenitor cells has been developed to support neural regeneration after spinal cord injury. However, the method of using stem cells as seeds still has great controversies in terms of cell resource availability and safety; on the other hand, in order to minimize tissue damage during the operation, in-situ formation of hydrogels by injecting synthetic polymers has been recommended as the first choice for clinical treatment of spinal cord injury. For example, based on the method of continuously delivering neurotrophic factors and reconstructing the necessary extracellular matrix (ECM), a strategy for promoting axonal regeneration in animal models of spinal cord injury with injectable materials has been proposed; but due to the very complex pathological process of spinal cord injury, the current biomaterials have very limited effects on neural repair after spinal cord injury.

[0004] Chemical inhibitors of diacylglycerol acyltransferase (DGAT) can inhibit triglyceride synthesis and are mainly used for the treatment of familial chylomicronemia syndrome and hypertriglyceridemia. In the technical field of neurology, current research only finds that DGAT inhibitors can promote axonal regeneration of neurons at the cellular level. However, its effects on the survival of neurons, etc. are still unknown, and there is no relevant report on the use of DGAT inhibitors for in vivo neural repair.

[0005] In summary, developing a new regulatory platform for promoting repair after spinal cord injury, reducing neuron death and apoptosis, and promoting axonal regeneration has very important theoretical significance. SUMMARY OF THE INVENTION

[0006] In view of the deficiencies of the prior art and the actual needs, the present invention provides a functional hydrogel loaded with diacylglycerol acyltransferase inhibitor, its preparation method and application, in order to provide new materials and new methods for the repair after spinal cord injury.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of an inhibitor of diacylglycerol acyltransferase in the preparation of an inhibitor of neuronal death and / or apoptosis.

[0009] In the present invention, it is found that an inhibitor of diacylglycerol acyltransferase can inhibit the death and / or apoptosis of neuronal cells, and has broad application prospects in alleviating neuronal death and / or apoptosis and treating spinal cord injury.

[0010] Preferably, the inhibitor includes any one or a combination of at least two of A-922500, DGAT1-IN-1, DGAT1-IN-3, DGAT-1 inhibitor2, JNJ-DGAT2-A, Obeversen, ABT-046, AZD3988, T863, Aphadilactone C, AZD7687, PF-06424439, PF-04620110, GSK2973980A or Pradigastat.

[0011] In a second aspect, the present invention provides the use of an inhibitor of diacylglycerol acyltransferase in the preparation of an inhibitor of neuronal death and / or apoptosis for non-disease diagnosis and / or treatment.

[0012] In the present invention, it is found that an inhibitor of diacylglycerol acyltransferase can inhibit the death and / or apoptosis of neuronal cells. Therefore, it can be used as an inhibitor of neuronal cell apoptosis for in vitro basic research of neuronal cells, etc.

[0013] In a third aspect, the present invention provides the use of an inhibitor of diacylglycerol acyltransferase in the preparation of a preparation for treating spinal cord injury.

[0014] In the present invention, it is found that an inhibitor of diacylglycerol acyltransferase can inhibit the death and / or apoptosis of neuronal cells, and the death range and degree of injury of spinal neurons at the injury site are closely related to the bridging of neural circuits, which play a decisive role in nerve function recovery and the prognosis of spinal cord injury. Therefore, new materials and new methods for treating spinal cord injury can be further developed.

[0015] Preferably, the inhibitor includes any one or a combination of at least two of A-922500, DGAT1-IN-1, DGAT1-IN-3, DGAT-1 inhibitor2, JNJ-DGAT2-A, Obeversen, ABT-046, AZD3988, T863, Aphadilactone C, AZD7687, PF-06424439, PF-04620110, GSK2973980A or Pradigastat.

[0016] In a fourth aspect, the present invention provides a functional hydrogel loaded with a diacylglycerol acyltransferase inhibitor, and the functional hydrogel includes an inhibitor of diacylglycerol acyltransferase, a micelle material, and a hydrogel material.

[0017] In the present invention, a functional hydrogel capable of loading an inhibitor of diacylglycerol acyltransferase is designed and can be locally transplanted to a nerve injury site to promote nerve injury repair.

[0018] Preferably, the inhibitor includes any one or a combination of at least two of A-922500, DGAT1-IN-1, DGAT1-IN-3, DGAT-1 inhibitor2, JNJ-DGAT2-A, Obeversen, ABT-046, AZD3988, T863, Aphadilactone C, AZD7687, PF-06424439, PF-04620110, GSK2973980A or Pradigastat.

[0019] Preferably, the micelle material includes phospholipid molecules and / or polyethylene glycol block copolymers.

[0020] Preferably, the polyethylene glycol block copolymer includes at least one of distearoyl phosphatidylethanolamine-polyethylene glycol, dipalmitoyl phosphatidylethanolamine-polyethylene glycol, polylactic acid-polyethylene glycol, poly(lactic-co-glycolic acid)-polyethylene glycol, polyvinyl alcohol-polyethylene glycol, polycaprolactone-polyethylene glycol, or polystyrene-polyethylene glycol, etc.

[0021] Preferably, the number average molecular weight of polyethylene glycol in the distearoyl phosphatidylethanolamine-polyethylene glycol is 2000 - 5000 Da, including but not limited to 2100 Da, 2200 Da, 2300 Da, 2500 Da, 2800 Da, 3000 Da, 3500 Da, 3800 Da, 4000 Da, 4200 Da, 4500 Da, 4600 Da, 4700 Da, 4800 Da, or 4900 Da.

[0022] Preferably, the hydrogel material includes any one or a combination of at least two of gelatin hydrogel material, collagen hydrogel material, hyaluronic acid hydrogel material, chitosan hydrogel material, silk fibroin hydrogel material, sodium alginate hydrogel material, methacrylated gelatin material, or poly(D,L-lactide) hydrogel material.

[0023] Preferably, the mass ratio of the inhibitor of diacylglycerol acyltransferase, the micelle material, and the hydrogel material in the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor is 1:(1.5 - 30):(50 - 300), including but not limited to 1:1.6:51, 1:1.8:55, 1:10:100, 1:25:260, 1:28:280, 1:5:290, 1:15:80, 1:12.5:150, 1:17.5:175, 1:27.5:275, 1:7.5:225, 1:22.5:125, 1:29:75, or 1:2.5:295, etc.

[0024] In a fifth aspect, the present invention provides a method for preparing the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor described in the fourth aspect, and the method includes:

[0025] Mix the inhibitor of diacylglycerol acyltransferase with a first solvent to obtain an inhibitor solution; mix the micelle material with a second solvent to obtain a micelle material solution; mix the hydrogel material with a third solvent and a photoinitiator to obtain a hydrogel material solution; mix the inhibitor solution and the micelle material solution, remove the solvent, and then mix with the hydrogel material solution, and irradiate with an ultraviolet light source to obtain the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor.

[0026] Preferably, the first solvent and the second solvent are each independently selected from any one or a combination of at least two of methanol, ethanol, chloroform, or dichloromethane, etc.

[0027] Preferably, the third solvent includes any one or a combination of at least two of PBS solution, deionized water, physiological saline, or HEPES buffer solution, etc.

[0028] Preferably, the photoinitiator includes any one or a combination of at least two of lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate, 2 - hydroxy - 4′-(2 - hydroxyethoxy)-2 - methylpropiophenone, or ethyl 2,4,6 - trimethylbenzoyl phenylphosphinate.

[0029] Preferably, the concentration of the inhibitor of diacylglycerol acyltransferase in the inhibitor solution is 0.1 to 2 mg / mL, including but not limited to 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, or 1.9 mg / mL, etc.

[0030] Preferably, the concentration of the micelle material in the micelle material solution is 1.5 to 30 mg / mL, including but not limited to 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 5 mg / mL, 1.5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 28 mg / mL, or 29 mg / mL, etc.

[0031] Preferably, the concentration of the hydrogel material in the hydrogel material solution is 50 to 300 mg / mL, including but not limited to 52 mg / mL, 55 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 102 mg / mL, 104 mg / mL, 106 mg / mL, 108 mg / mL, 110 mg / mL, 120 mg / mL, 150 mg / mL, 180 mg / mL, 200 mg / mL, 220 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, 295 mg / mL, or 299 mg / mL, etc.

[0032] Preferably, the mass percentage of the photoinitiator in the hydrogel material solution is 0.1% to 1%, including but not limited to 0.2%, 0.3%, 0.5%, 0.6%, 0.8%, or 0.9%, etc.

[0033] Preferably, the mixing volume ratio of the inhibitor solution and the micelle material solution is 1:(3 to 6), including but not limited to 1:3.2, 1:3.4, 1:3.25, 1:3.6, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:5, 1:5.5, 1:5.6, or 1:5.8, etc.

[0034] Preferably, the irradiation time is 30 to 180 s, including but not limited to 32 s, 35 s, 40 s, 45 s, 50 s, 60 s, 70 s, 80 s, 90 s, 100 s, 150 s, 160 s, or 170 s, etc.

[0035] Sixth aspect, the present invention provides an application of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor in the preparation of a preparation for treating spinal cord injury described in the fourth aspect.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention for the first time reveals that the use of a diacylglycerol acyltransferase (DGAT) inhibitor can regulate the triglyceride level of nerve cells so as to alleviate neuronal death and / or apoptosis, constructs a functional hydrogel loaded with a DGAT inhibitor, the hydrogel has good biocompatibility, and can achieve a local slow release of the DGAT inhibitor for up to 7 days, can realize local administration of the DGAT inhibitor in vivo, and can effectively reduce nerve cell death and / or apoptosis and promote the regeneration of spinal cord tissue and nerves during spinal cord injury repair, providing new materials and new methods for the treatment of spinal cord injury repair. Description of the Drawings

[0038] Figure 1 It is a schematic diagram of the preparation of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor and the treatment application for rats with spinal cord injury according to the present invention;

[0039] Figure 2 It is a scanning electron microscope result diagram of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor in Example 1, and the scale bar is 50 micrometers;

[0040] Figure 3 It is a rheological test result diagram of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor in Example 1;

[0041] Figure 4 It is a drug release curve diagram of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor in Example 1;

[0042] Figure 5 It is a rheological test result diagram of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor in Example 2;

[0043] Figure 6 It is a drug release curve diagram of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor in Example 2;

[0044] Figure 7 It is a rheological test result diagram of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor in Example 3;

[0045] Figure 8 It is a drug release curve diagram of the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor in Example 3;

[0046] Figure 9Results graph of DGAT inhibitor reducing neuronal death and / or apoptosis. Among them, Figure A shows the survival ratio of neurons under different glutamate (GLU) concentrations and after adding DGAT inhibitor; Figures B - D are the results graphs of neuronal apoptosis: including the representative results of flow cytometry detection of neurons under normal conditions, glutamate stimulation (GLU), and glutamate stimulation with DGAT inhibitor added (GLU + DGAT inhibitor) (Figure D), as well as the statistics of the proportion of early apoptotic cells (Figure B) and late apoptotic cells (Figure C) under different conditions;

[0047] Figure 10 Results graph of the apoptosis of nerve cells in the injured tissue on the 3rd day after complete transection of the spinal cord in rats of different treatment groups in Example 1. Among them, Figure A is the results graph of the distribution of apoptotic nerve cells in the spinal cord tissues of different treatment groups 3 days after injury, scale bar: 400 μm; Figure B is the results graph of the statistics of the proportion of apoptotic nerve cells in the injured spinal cord tissues of different treatment groups;

[0048] Figure 11 Results graph of the BBB behavioral scores of spinal cord injured rats in different treatment groups in Example 1 from week 0 to week 8 after injury;

[0049] Figure 12 Results graph of the tissue regeneration of the injured spinal cord in rats of different treatment groups in Example 1 at 8 weeks after injury, scale bar: 800 μm;

[0050] Figure 13 Results graph of the spinal nerve recovery status of spinal cord injured rats in different treatment groups in Example 1. Among them, Figure A is the results graph of the distribution of mature neurons in the spinal cord tissues of different treatment groups 8 weeks after injury, scale bar: 350 μm; Figure B is the results graph of the statistics of the immunofluorescence staining intensity of mature neurons in the injured spinal cord tissues of different treatment groups;

[0051] Figure 14 Results graph of the detection of potential toxic and side effects of different treatments on spinal cord injured rats in Example 1, scale bar: 100 μm;

[0052] Figure 15 Results graph of the apoptosis of nerve cells in the injured tissue on the 3rd day after complete transection of the spinal cord in rats of different treatment groups in Example 2, scale bar: 100 μm;

[0053] Figure 16 Results graph of the inclined plane test at 8 weeks after complete transection of the spinal cord in rats of different treatment groups in Example 2. Among them, Figure A is the schematic diagram of the inclined plane test, and Figure B is the results graph of the statistics of the inclined plane test;

[0054] Figure 17 Results graph of the inclined plane test at 8 weeks after complete transection of the spinal cord in rats of different treatment groups in Example 3;

[0055] Figure 18 This is a graph showing the results of spinal cord nerve recovery at the injured site in rats in different treatment groups in Example 3 at 8 weeks after complete spinal cord transection. Scale: 50 microns. DETAILED DESCRIPTION

[0056] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0057] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0058] In a specific embodiment of the present invention, a functional hydrogel loaded with a diacylglycerol acyltransferase inhibitor is prepared and used in the treatment of spinal cord injury in rats. The schematic diagram is shown in FIG. Figure 1 shown.

[0059] Example 1

[0060] In this example, a functional hydrogel loaded with a diacylglycerol acyltransferase inhibitor was prepared.

[0061] 1. Dissolve the diacylglycerol acyltransferase (DGAT) inhibitor (A-922500) in methanol to obtain a 1 mg / mL DGAT inhibitor methanol solution, and dissolve distearoylphosphatidylethanolamine-polyethylene glycol (2000)-amino (DSPE-PEG) in methanol to obtain a 1 mg / mL DSPE-PEG methanol solution.

[0062] 2. The DGAT inhibitor methanol solution and the DSPE-PEG methanol solution were mixed at a volume ratio of 1:3, and then the methanol solution was evaporated in vacuo to obtain a dry lipid film.

[0063] 3. Add phosphate buffered saline (PBS) to the dried lipid film to make the final concentration of DGAT inhibitor 2 mg / mL. Ultrasonicate at 37°C for 30 min to obtain a uniformly dispersed micelle suspension.

[0064] 4. Methacrylated gelatin (GelMA) was dissolved in PBS at 45°C to a concentration of 100 mg / mL. Subsequently, a photoinitiator LAP (concentration of 0.2%) was added at 45°C in the dark to obtain a 10% (g / 100 mL) GelMA precursor solution.

[0065] 5. Mix the micelle suspension and the GelMA precursor solution in equal volumes at 37 °C in the dark to obtain the hydrogel precursor solution.

[0066] 6. Irradiate the hydrogel precursor solution with an ultraviolet light source for 60 s. After the hydrogel is completely cured, the functional hydrogel loaded with the DGAT inhibitor is obtained.

[0067] The prepared functional hydrogel was detected by scanning electron microscopy, and the results are as Figure 2 shown. The functionalized hydrogel has a porous structure.

[0068] The prepared functional hydrogel was subjected to a rheological test, specifically including: detecting the storage modulus (G’) and loss modulus (G”) of the functional hydrogel at different frequencies (0.1 - 10 Hz) under a constant strain of 1%, and the results are as Figure 3 shown. The functional hydrogel has mechanical properties matching those of spinal cord tissue.

[0069] The prepared functional hydrogel was subjected to a drug release test, specifically including: soaking the functional hydrogel in PBS containing 0.5% (v / v) Tween - 80 and placing it under shaking conditions at 37 °C and 100 rpm, and detecting the concentration of the DGAT inhibitor in the PBS solution at different time points. The results are as Figure 4 shown. The functional hydrogel can slowly release the DGAT inhibitor in a physiological environment for up to 7 days.

[0070] At the same time, a blank hydrogel without loading the DGAT inhibitor was prepared by referring to the above method as a control for subsequent tests. The main difference in the preparation process is that during the preparation of the micelles, the methanol solution containing the DGAT inhibitor was not added to the methanol solution containing DSPE - PEG, so the prepared micelles are blank micelles (not encapsulating the DGAT inhibitor).

[0071] Example 2

[0072] This example prepares a functional hydrogel loaded with a diacylglycerol acyltransferase inhibitor.

[0073] 1. Dissolve the diacylglycerol acyltransferase (DGAT) inhibitor (T863) in ethanol to obtain a 2 mg / mL DGAT inhibitor ethanol solution, and dissolve dipalmitoylphosphatidylethanolamine - polyethylene glycol (2000) (DPPE - PEG) in ethanol to obtain a 3 mg / mL DPPE - PEG ethanol solution.

[0074] 2. Mix the ethanol solution containing the DGAT inhibitor and the DPPE - PEG ethanol solution in a volume ratio of 1:6, and then vacuum - evaporate the ethanol solution to obtain a dry lipid film.

[0075] 3. Add phosphate buffered saline (PBS) to the dried lipid film to make the final concentration of the DGAT inhibitor 4 mg / mL. Sonicate for 20 min at 45 °C to obtain a uniformly dispersed micelle suspension.

[0076] 4. Dissolve methacrylated sodium alginate (AlgMA) in PBS at 37 °C at a concentration of 100 mg / mL. Subsequently, add the photoinitiator LAP (at a concentration of 0.25%) at 37 °C in the dark to obtain a 10% (g / 100 mL) AlgMA precursor solution.

[0077] 5. Mix the micelle suspension and the AlgMA precursor solution in equal volumes at 37 °C in the dark to obtain a hydrogel precursor solution.

[0078] 6. Irradiate the hydrogel precursor solution with an ultraviolet light source for 120 s. After the hydrogel is completely cured, a functional hydrogel loaded with the DGAT inhibitor is obtained.

[0079] At the same time, prepare a blank hydrogel without loading the DGAT inhibitor by referring to the above method.

[0080] Perform rheological tests on the prepared functional hydrogel, specifically including: detecting the storage modulus (G') and loss modulus (G") of the functional hydrogel within the detection time of 15 - 300 s at a constant strain of 1% and a constant frequency of 1 Hz. The results are as Figure 5 shown, and the functional hydrogel has mechanical properties matching those of spinal cord tissue.

[0081] Perform drug release tests on the prepared functional hydrogel, specifically including: soaking the functional hydrogel in PBS containing 0.5% (v / v) Tween - 80 and placing it under shaking conditions at 37 °C and 100 rpm. Detect the concentration of the DGAT inhibitor in the PBS solution at different time points. The results are as Figure 6 shown, and the functional hydrogel can slowly release the DGAT inhibitor in a physiological environment for up to 7 days.

[0082] Example 3

[0083] This example prepares a functional hydrogel loaded with a diacylglycerol acyltransferase inhibitor.

[0084] 1. Dissolve the diacylglycerol acyltransferase (DGAT) inhibitor (Aphadilactone C) in dichloromethane to obtain a dichloromethane solution containing 0.1 mg / mL of the DGAT inhibitor. Dissolve poly (lactic - co - glycolic acid) (4500) - poly (ethylene glycol) (2000) (PLGA - PEG) in dichloromethane to obtain a 0.5 mg / mL PLGA - PEG dichloromethane solution.

[0085] 2. Mix the dichloromethane solution containing the DGAT inhibitor and the dichloromethane solution containing PLGA-PEG in a volume ratio of 1:3. Subsequently, evaporate the organic solution under vacuum to obtain a dry lipid film.

[0086] 3. Add phosphate-buffered saline (PBS) to the dried lipid film to make the final concentration of the DGAT inhibitor 0.1 mg / mL. Sonicate for 60 min at 37 °C to obtain a uniformly dispersed micelle suspension.

[0087] 4. Dissolve methacrylated hyaluronic acid (HaMA) in PBS at 37 °C at a concentration of 150 mg / mL. Subsequently, add a photoinitiator LAP (concentration 0.1%) at 37 °C under dark conditions to obtain a 15% (g / 100 mL) HaMA precursor solution.

[0088] 5. Mix the micelle suspension and the HaMA precursor solution in equal volumes at 37 °C under dark conditions to obtain a hydrogel precursor solution.

[0089] 6. Irradiate the hydrogel precursor solution with an ultraviolet light source for 180 s. After the hydrogel is completely cured, a functional hydrogel loaded with the DGAT inhibitor is obtained.

[0090] At the same time, prepare a blank hydrogel without loading the DGAT inhibitor by referring to the above method.

[0091] Perform rheological tests on the prepared functional hydrogel, specifically including: detecting the storage modulus (G') and loss modulus (G") of the functional hydrogel under different stresses (0.1% - 100%) at a constant frequency of 1 Hz. The results are as Figure 7 shown, and the functional hydrogel has mechanical properties matching those of spinal cord tissue.

[0092] Perform drug release tests on the prepared functional hydrogel, specifically including: Immerse the functional hydrogel in PBS containing 0.5% (v / v) Tween-80 and place it under shaking conditions at 37 °C and 100 rpm. Detect the concentration of the DGAT inhibitor in the PBS solution at different time points. The results are as Figure 8 shown, and the functional hydrogel can slowly release the DGAT inhibitor in a physiological environment for up to 7 days.

[0093] Example 4

[0094] Test the effect of the DGAT inhibitor on neurons, specifically including: Using glutamate (GLU) to simulate the injury environment, detecting the cell viability and apoptosis of neurons under normal culture, glutamate (GLU), and after injury with the addition of the DGAT inhibitor (GLU + DGAT inhibitor). The results are as Figure 9As shown, a neuronal injury environment was constructed using glutamate (GLU). The results in Figure A showed that as the concentration of GLU increased, the proportion of neuronal death increased. However, after adding the DGAT inhibitor, the proportion of neuronal death could be significantly reduced and the neuronal survival rate could be increased. Figures B - D show the results of staining with an apoptosis kit and detecting neuronal apoptosis using a flow cytometer. Among them, FITC-positive cells represent cells in the early and middle stages of apoptosis, and Propidium Iodide (PI)-positive cells represent cells in the late stage of apoptosis and dead cells. It can be seen from the figure that the addition of the DGAT inhibitor can reduce the apoptosis proportion of neurons under GLU conditions. Thus, it can be seen that the present invention discovers that the use of a DGAT inhibitor can alleviate neuronal death / apoptosis, thereby achieving the treatment of spinal cord injury.

[0095] Example 5

[0096] In this example, a functional hydrogel was used to treat spinal cord injured rats by in-situ transplantation.

[0097] 1. A rat model of complete transection spinal cord injury was constructed, with the length of spinal cord transection being 4 mm. The specific modeling process is as follows: Healthy adult female SD rats (180 - 200 g, purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were selected, and the SD rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital. The comatose rats were fixed in the prone position, and their backs were routinely disinfected, hair was shaved, and the T9 position was determined. A longitudinal incision of about 2 cm was made on the surface of the spinous processes of T8 - T10 along the spinal cord midline, the muscles on both sides were retracted with a retractor, the lamina was pried open, and the spinal cord was exposed. A transverse cut was made at the T9 segment with a micro-scissors. Gelatin sponge was used to press and stop bleeding at the injury site, and the muscles and skin were sutured in sequence. One week after the operation, sodium penicillin was intraperitoneally injected to prevent bacterial infection. The bladder was massaged twice a day after the operation to assist urination until autonomous urination was restored.

[0098] 2. The rat models of complete transection spinal cord injury were randomly divided into 9 groups, with at least 3 rats in each group. For the 3 kinds of hydrogels prepared in Examples 1 - 3, each hydrogel was tested using 3 groups of rat models, namely the SCI group (i.e., the injury control group), the Gel-M group (the blank hydrogel group without loading the DGAT inhibitor), and the Gel-MD group (the functional hydrogel group loaded with the DGAT inhibitor); the specific usage method was: The precursor solution of the hydrogel was dropped into the spinal cord injury site of each group of mice, with a volume of 0.1 mL.

[0099] 3. The ultraviolet light source was irradiated for 100 s to in-situ solidify the functionalized hydrogel at the spinal cord injury site.

[0100] 4. The wounds of the spinal cord injured rats were sutured, and postoperative care was carried out according to the relevant instructions.

[0101] 5. The rats after intervention were examined, including: characterization of apoptotic nerve cells in spinal cord tissue in the short term after injury (the third day after surgery) in rats, assessment of hind limb motor function of rats 0 - 8 weeks after injury, HE staining of spinal cord tissue 8 weeks after injury in rats to observe the regeneration of injured tissue, immunofluorescent staining of mature neurons in spinal cord tissue to evaluate nerve regeneration of injured tissue, and HE staining of major organs 8 weeks after surgery in injured rats to evaluate the toxic and side effects of the hydrogel.

[0102] (1) Results of preparing the hydrogel in Example 1

[0103] On the 3rd day after total transection of the spinal cord in rats in different treatment groups, the apoptosis of nerve cells in the injured tissue was as Figure 10 shown. The treatment groups included: SCI group (i.e., injury control group), Gel - M group (blank hydrogel group without loading DGAT inhibitor), Gel - MD (functional hydrogel group loaded with DGAT inhibitor). Figure A is the TUNEL staining of spinal cord tissue in different treatment groups 3 days after injury, and Figure B is the proportion of TUNEL - positive cells in the injured spinal cord tissue of different treatment groups, indicating that the functional hydrogel prepared by the present invention can alleviate the apoptosis of nerve cells in spinal cord injured tissue.

[0104] Two months after hydrogel transplantation, the results of the recovery of hind limb function in rats were as Figure 11 shown. Among different treatment groups, the BBB behavioral scores of spinal cord injured rats from 0 - 8 weeks after injury. The BBB score is divided into 0 - 21 levels: 0 indicates complete loss of function, and 21 indicates normal function. It can be seen that the hind limb function of rats intervened with the functional hydrogel of the present invention was significantly restored.

[0105] Among different treatment groups, the tissue regeneration of the injured spinal cord in rats 8 weeks after injury was as Figure 12 shown. It can be seen that the spinal cord tissue has good regeneration ability after being intervened with the functional hydrogel of the present invention.

[0106] The spinal cord nerve recovery status of spinal cord injured rats in different treatment groups was as Figure 13 shown. Figure A is the distribution of mature neurons in spinal cord tissue in different treatment groups 8 weeks after injury, scale bar: 350 microns; Figure B is the statistical result of the immunofluorescent staining intensity of mature neurons in the injured spinal cord tissue of different treatment groups. After being intervened with the functional hydrogel of the present invention, the number of neurons in the injured spinal cord tissue increased significantly.

[0107] The detection results of potential toxic and side effects of different treatments on spinal cord injured rats were as Figure 14 shown. It can be seen that the functional hydrogel of the present invention did not cause toxic and side effects in spinal cord injured rats.

[0108] (2) Results of preparing hydrogel in Example 2

[0109] On the 3rd day after complete transection of the spinal cord in rats in different treatment groups, the apoptosis of nerve cells in the injured tissue was as Figure 15 shown. The treatment groups included: SCI group (i.e., injury control group), Gel-M group (blank hydrogel group without loading DGAT inhibitor), Gel-MD (functional hydrogel group loaded with DGAT inhibitor), indicating that the functional hydrogel prepared by the present invention can alleviate the apoptosis of nerve cells in spinal cord injury tissue.

[0110] Two months after hydrogel transplantation, the results of the hindlimb function recovery of rats were as Figure 16 shown. The rats in different treatment groups were subjected to a flat plate test. The specific operation was as follows: The spinal cord injured rats were placed on a horizontally placed flat plate, and one end of the flat plate was slowly lifted until the rats slipped, and the angle formed by the flat plate and the horizontal plane at this time was recorded. Among them, Figure A is a schematic diagram of the flat plate test, and Figure B is a result diagram of the flat plate test of different treatment groups. It can be seen that the hindlimb support function of rats was significantly restored after intervention with the functional hydrogel of the present invention.

[0111] (3) Results of preparing hydrogel in Example 3

[0112] Two months after hydrogel transplantation, the results of the hindlimb function recovery of rats were as Figure 17 shown. The rats in different treatment groups were subjected to a flat plate test. It can be seen that the hindlimb support function of rats was significantly restored after intervention with the functional hydrogel of the present invention.

[0113] The spinal nerve recovery status of spinal cord injured rats in different treatment groups was as Figure 18 shown. It can be seen from the figure that after intervention with the functional hydrogel of the present invention, the number of neurons in the injured spinal cord tissue increased significantly.

[0114] In summary, compared with the current main methods of nerve treatment, such as administering neurotrophic factors and regulating the inflammatory microenvironment, the present invention first reveals a new method of regulating the triglyceride level of nerve cells by using DGAT inhibitors to alleviate neuron death and apoptosis, constructs a functional hydrogel loaded with DGAT inhibitors, which has good biocompatibility, and can achieve a local slow release of DGAT inhibitors for up to 7 days, can achieve local administration of DGAT inhibitors in vivo, and effectively reduce nerve cell apoptosis, promote the regeneration of spinal cord tissue and nerves, and enhance the hindlimb motor function of rats during spinal cord injury repair, providing new materials and new methods for the treatment of spinal cord injury repair.

[0115] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the present invention's product, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Use of an inhibitor of diacylglycerol acyltransferase in the preparation of an inhibitor of neuronal death and / or apoptosis.

2. The use according to claim 1, characterized in that, The inhibitor includes any one or a combination of at least two of A-922500, DGAT1-IN-1, DGAT1-IN-3, DGAT-1 inhibitor 2, JNJ-DGAT2-A, Obeversen, ABT-046, AZD3988, T863, Aphadilactone C, AZD7687, PF-06424439, PF-04620110, GSK2973980A, or Pradigastat.

3. Use of an inhibitor of diacylglycerol acyltransferase in the preparation of a preparation for treating spinal cord injury.

4. The use according to claim 3, characterized in that, The inhibitor includes any one or a combination of at least two of A-922500, DGAT1-IN-1, DGAT1-IN-3, DGAT-1 inhibitor 2, JNJ-DGAT2-A, Obeversen, ABT-046, AZD3988, T863, Aphadilactone C, AZD7687, PF-06424439, PF-04620110, GSK2973980A, or Pradigastat.

5. A functional hydrogel loaded with an inhibitor of diacylglycerol acyltransferase, characterized in that, The functional hydrogel includes an inhibitor of diacylglycerol acyltransferase, a micelle material, and a hydrogel material.

6. The functional hydrogel loaded with an inhibitor of diacylglycerol acyltransferase according to claim 5, characterized in that, The inhibitor includes any one or a combination of at least two of A-922500, DGAT1-IN-1, DGAT1-IN-3, DGAT-1 inhibitor 2, JNJ-DGAT2-A, Obeversen, ABT-046, AZD3988, T863, Aphadilactone C, AZD7687, PF-06424439, PF-04620110, GSK2973980A, or Pradigastat; Preferably, the micelle material includes phospholipid molecules and / or polyethylene glycol block copolymers; Preferably, the polyethylene glycol block copolymer includes at least one of distearoyl phosphatidylethanolamine-polyethylene glycol, dipalmitoyl phosphatidylethanolamine-polyethylene glycol, polylactic acid-polyethylene glycol, poly(lactic-co-glycolic acid)-polyethylene glycol, polyvinyl alcohol-polyethylene glycol, polycaprolactone-polyethylene glycol, or polystyrene-polyethylene glycol; Preferably, the hydrogel material includes any one or a combination of at least two of a gelatin hydrogel material, a collagen hydrogel material, a hyaluronic acid hydrogel material, a chitosan hydrogel material, a silk fibroin hydrogel material, an alginate hydrogel material, a methacrylated gelatin material, or a poly(D,L-lactide) hydrogel material.

7. The functional hydrogel loaded with an inhibitor of diacylglycerol acyltransferase according to claim 5 or 6, characterized in that, In the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor, the mass ratio of the diacylglycerol acyltransferase inhibitor, the micelle material, and the hydrogel material is 1:(1.5 - 30):(50 - 300).

8. A method for preparing the functional hydrogel loaded with an inhibitor of diacylglycerol acyltransferase according to any one of claims 5 - 7, characterized in that, The method includes: Mixing the inhibitor of diacylglycerol acyltransferase with a first solvent to obtain an inhibitor solution; mixing the micelle material with a second solvent to obtain a micelle material solution; mixing the hydrogel material with a third solvent and a photoinitiator to obtain a hydrogel material solution; Mix the inhibitor solution and the micelle material solution, remove the solvent, and then mix with the hydrogel material solution, followed by irradiation with an ultraviolet light source to obtain the functional hydrogel loaded with the diacylglycerol acyltransferase inhibitor.

9. The method for preparing the functional hydrogel loaded with an inhibitor of diacylglycerol acyltransferase according to claim 8, characterized in that, The first solvent and the second solvent are each independently selected from any one or a combination of at least two of methanol, ethanol, chloroform, or dichloromethane; Preferably, the third solvent includes any one or a combination of at least two of PBS solution, deionized water, physiological saline, or HEPES buffer solution; Preferably, the photoinitiator includes any one or a combination of at least two of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, or ethyl 2,4,6-trimethylbenzoyl phenylphosphinate; Preferably, the concentration of the diacylglycerol acyltransferase inhibitor in the inhibitor solution is 0.1 - 2 mg / mL; Preferably, the concentration of the micelle material in the micelle material solution is 1.5 - 30 mg / mL; Preferably, the concentration of the hydrogel material in the hydrogel material solution is 50 - 300 mg / mL; Preferably, the mass percentage of the photoinitiator in the hydrogel material solution is 0.1% - 1%; Preferably, the mixing volume ratio of the inhibitor solution and the micelle material solution is 1:(3 - 6); Preferably, the irradiation time is 30 - 180 s.

10. Use of the functional hydrogel loaded with an inhibitor of diacylglycerol acyltransferase according to any one of claims 5 - 7 in the preparation of a preparation for treating spinal cord injury.

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