Composition containing epigallocatechin gallate and use thereof

Abstract

The present application relates to the technical field of biological medicine and molecular biology, and particularly relates to a composition containing epigallocatechin gallate and application thereof, and preparation of the epigallocatechin gallate into a hydrogel composition, wherein the composition comprises a first reagent containing methacrylated gelatin, a second reagent containing a photoinitiator, and a third reagent containing epigallocatechin gallate; the first reagent, the second reagent, and the third reagent are all in liquid state. The present application can inhibit the formation of scars, promote the repair of spinal cord, and thus can quickly complete the functional recovery of spinal cord nerves.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and molecular biology, and in particular to compositions containing epigallocatechin gallate and their applications. Background Technology

[0002] Spinal cord injury is one of the most disabling and destructive neurological diseases. According to the World Health Organization, 250,000 to 500,000 people worldwide are affected by spinal cord injury each year, most of whom are caused by traumatic injuries such as traffic accidents, falls, or violence. Spinal cord injury not only leads to severe sensory, motor, and autonomic dysfunction below the level of injury, but also causes multi-system complications, including muscle atrophy, neuropathic pain, respiratory dysfunction, pressure sores, and urinary tract infections. Furthermore, it can cause psychological, emotional, and social impairments. Spinal cord injury not only severely impacts patients' quality of life and mental and physical health, but also imposes a huge economic burden on families and society; therefore, treating spinal cord injury has significant social importance.

[0003] Because the central nervous system has poor plasticity and limited neuronal regeneration capacity, the regeneration and repair after spinal cord injury is extremely weak, the prognosis of the injury is extremely poor, and nerve cells are terminal cells with poor regeneration capacity, which leads to poor functional recovery of spinal cord nerves. Furthermore, the regeneration of scar tissue during the spinal cord nerve repair process causes significant damage to nerve function, resulting in unsatisfactory recovery of sensory and motor functions. Summary of the Invention

[0004] The features and advantages of the present invention are set forth in part in the description which follows, or may be apparent from the description, or may be learned by practicing the invention.

[0005] To overcome the problems of existing technologies, inhibit the regeneration of scar tissue in spinal cord nerves, suppress oxidative stress in nerve cells, and promote functional repair of spinal cord nerves, this invention provides a composition containing epigallocatechin gallate. Epigallocatechin gallate is prepared into a hydrogel-like composition. The composition comprises a first reagent containing methacrylamide gelatin, a second reagent containing a photoinitiator, and a third reagent containing epigallocatechin gallate. The first, second, and third reagents are all in liquid form. The first, second, and third reagents are mixed in a mass ratio of 2:1:1. The composition takes effect after exposure to ultraviolet light. The concentration of epigallocatechin gallate in the third reagent is 0.004-0.04%.

[0006] Preferably, the solvents for the first reagent, the second reagent, and the third reagent are all water or phosphate buffer solutions.

[0007] Preferably, the composition further includes sodium hyaluronate.

[0008] Preferably, the sodium hyaluronate is mixed in the first reagent.

[0009] Preferably, the concentration of sodium hyaluronate in the first reagent is 0.2-0.6%.

[0010] Preferably, the concentration of methacrylamide gelatin in the first reagent is 9-11%.

[0011] Preferably, the concentration of the photoinitiator in the second reagent is 1.6-2.4%.

[0012] Preferably, the elastic modulus of the composition is 72.4~93.8 kPa.

[0013] Preferably, the degree of crosslinking of the methacrylamide gelatin is 68%; and / or the ultraviolet light irradiation time is ≥5 minutes.

[0014] Preferably, the composition is used in the preparation of a spinal cord injury drug.

[0015] The beneficial effects of this invention are: The composition in this application is prepared into a hydrogel composition by adding epigallocatechin gallate to methacrylamide gelatin (GelMA) and a photoinitiator, thereby enabling epigallocatechin gallate to be stably and continuously administered to the spinal cord nerves. At the same time, the hydrogel can provide good support for the spinal cord nerves to promote axonal regeneration, thereby increasing the speed of spinal cord regeneration and promoting the repair of sensory and motor functions of the spinal cord.

[0016] The hydrogel composition of this application allows epigallocatechin gallate to be directly applied in the treatment of spinal cord injury. It acts directly on the spinal nerves, exerting its anti-oxidative stress and anti-fibrotic effects by participating in the anti-oxidative stress and TGF-β / Smad signaling pathway, thereby promoting the repair of spinal cord injury. Epigallocatechin gallate can effectively inhibit the oxidative stress response of spinal nerves and inhibit the formation of scars during the spinal nerve repair process, thus promoting the repair of the spinal cord and achieving better and faster functional recovery of spinal nerves within the same time frame. Attached Figure Description

[0017] The present invention will be described in detail below with reference to the accompanying drawings and examples. The advantages and implementation methods of the present invention will become more apparent from this description. The accompanying drawings are for illustrative purposes only and do not constitute any limitation on the present invention. In the accompanying drawings: Figure 1The chemiluminescence assay results are shown in the specific embodiment of the present invention for the application of epigallocatechin gallate (EGCG) in the treatment of spinal cord injury. Figure 2 The fluorescent microscopy image shows the effect of epigallocatechin gallate (EGCG) on scar formation after spinal cord injury in a specific embodiment of the present invention. Figure 3 This is a real-time quantitative PCR result of epigallocatechin gallate (EGCG) on fibrosis after spinal cord injury in a specific embodiment of the present invention; Figure 4 This is the result of an experiment on the effect of epigallocatechin gallate (EGCG) on the resistance of nerve cells to oxidative stress after spinal cord injury, as described in a specific embodiment of the present invention. Figure 5 The results of experiments on the effect of the concentration of the composition on the anti-oxidative stress after spinal cord injury in specific embodiments of the present invention; Figure 6 These are experimental results showing the antioxidant and anti-scarring effects of the composition at preferred concentrations in specific embodiments of the present invention. Figure 7 The results of the elastic modulus test of the composition in a specific embodiment of the present invention are shown. Figure 8 The results are experimental results of cyclic fatigue performance testing of the composition in a specific embodiment of the present invention. Detailed Implementation

[0018] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0019] In the description of this invention, unless otherwise stated, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0020] Example 1 This invention provides the application of epigallocatechin gallate (EGCG) in the treatment of spinal cord injury.

[0021] Rats were divided into three groups and weighed. The first group was the spinal cord injury group, also known as the control group (SCI group). Rats were anesthetized via intraperitoneal injection, and their back hair was shaved with an electric shaver. The rats were fixed in a prone position on the operating table, and the surgical area was disinfected with alcohol. A longitudinal incision was made in the skin at the T10 segment with a scalpel, and the mucosa was bluntly dissected using hemostatic forceps to expose the surgical field. Longitudinal incisions were made on both sides of the spinous process with the scalpel, and excess tissue was removed with ophthalmic scissors. Intraoperative bleeding was cleared with cotton swabs. After exposing the lamina of the T9-T10 segments, the T9 spinous process was clamped with a hemostat in the left hand, and the T10 lamina was crushed with bone forceps in the right hand to expose the spinal cord. A transverse incision was made against the transverse process with a scalpel to complete the spinal cord hemisection injury model. The second group was the SCI+EGCG group, the experimental group. After the spinal cord hemisection injury model was established, EGCG solution was injected intraperitoneally into the rats at a dose of 200 mg / kg for treatment. The third group, the sham surgery group (hereinafter referred to as the Sham group), underwent only laminectomy without damaging the spinal cord. After the laminectomy, the rats were injected intraperitoneally with the same volume of physiological saline. All three groups of rats received a small amount of penicillin intramuscularly post-surgery to prevent infection. The incisions were sutured layer by layer, and blood was wiped away. Artificial assisted urination was performed one week post-surgery.

[0022] Fourteen days after modeling, spinal cord tissue from the injured rat site was collected and mixed with RIPA lysis buffer (purchased from Thermo Fisher Scientific (China) Co., Ltd.), protease inhibitor (purchased from MedChemExpress), and grinding beads. The mixture was then ground in a grinder, centrifuged at 12,000 rpm, and the filtrate was collected. Total tissue protein was collected and protein concentration was determined. Tissue proteins were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel (purchased from Beijing Solarbio Science & Technology Co., Ltd.). 10 μg of protein was loaded into each well, and electrophoresis was performed at a constant voltage of 120 V for 120 min. The separated proteins were then electrophoresed onto a polyvinylidene fluoride membrane (hereinafter referred to as PVDF membrane) and electrophoresed at a constant current of 200 mA for 120 min. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4°C with different antibodies. These antibodies included anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH antibody, purchased from Abogen (Shanghai) Trading Co., Ltd., mouse-derived), anti-phosphorylated-Smad2 / 3 antibody (P-Smad2 / 3 antibody, purchased from CellSignal Technology, rabbit-derived), recombinant anti-Snail protein antibody (Snail antibody, purchased from Abogen (Shanghai) Trading Co., Ltd., rabbit-derived), and anti-Vimentin antibody (recombinant anti-Vimentin antibody, purchased from Abogen (Shanghai) Trading Co., Ltd., rabbit-derived). The three antibodies were diluted 1:1000. After incubation, the PVDF membrane was washed three times with TBST buffer (purchased from Beijing Solarbio Science & Technology Co., Ltd.), 10 min each time. After washing, the cells were incubated at room temperature for 2 hours with the appropriate species-derived secondary antibody reagent, which was either goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody (both purchased from Abogen (Shanghai) Trading Co., Ltd.), diluted at a ratio of 1:5000. After incubation, the cells were washed three times with TBST buffer, 10 minutes each time. After washing, the cells were treated with a chemiluminescence assay kit (purchased from Thermo Fisher Scientific (China) Co., Ltd.) and exposed under a chemiluminescence analyzer. The results are as follows: Figure 1 As shown.

[0023] Experimental results Figure 1 The results show that EGCG can reduce the expression of phosphorylated Smad2 / 3 protein (P-Smad2 / 3), Snail protein, and vimentin, thus indicating that EGCG can participate in the TGF-β / Smad signaling pathway, inhibit scar formation, and therefore EGCG can be directly used in the treatment of spinal cord injury and promote spinal cord repair. Example 2

[0024] The effect of epigallocatechin gallate (EGCG) on scar formation after spinal cord injury: Fourteen days after establishing the spinal cord hemisection injury model in Example 1, three rats from each of the corresponding groups were taken. After intraperitoneal anesthesia, the hair on the backs of the rats was shaved with an electric shaver. The rats were fixed in a supine position on the operating table. The skin, muscles, and ribs of the rat's chest were cut open to expose the heart. A needle with a flattened tip was inserted into the left ventricle of the rat, and the right atrial appendage was cut open with ophthalmic scissors. The blood was quickly flushed out with physiological saline, and then perfused and fixed with 4% paraformaldehyde (purchased from Beijing Solarbio Science & Technology Co., Ltd.). After fixation, the spinal cord at the injured segment was removed and fixed in paraformaldehyde. After fixation, it was sequentially transferred to 20% and 30% sucrose solutions for gradient sugar deposition. After sugar deposition, frozen sections were prepared using a cryostat to a thickness of 20 μm.

[0025] The excised spinal cord sections were mounted and washed twice with TBS solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.), 5 min each time. Blocking was performed for 2 h at room temperature using blocking buffer + 0.3% Triton-100 (purchased from Beijing Solarbio Science & Technology Co., Ltd.). This blocking buffer was prepared by adding 10% fetal bovine serum (purchased from Ecosai Biotechnology (Taicang) Co., Ltd.) + 1% bovine serum albumin (BSA, purchased from MedChemExpress). The sections were then washed three times with TBS solution, 5 min each time. Incubation was performed overnight at 4°C using anti-Vimentin antibody. In this example, Vimentin primary antibody (purchased from Abogen (Shanghai) Trading Co., Ltd.) was used. After incubation, the sections were washed three more times with TBS solution, 5 min each time. Finally, the sections were incubated with goat anti-rabbit IgG secondary antibody at room temperature for 2 h, and then washed three more times with TBS solution, 5 min each time. After the tissue slides had dried, they were mounted in a darkroom using glycerin (purchased from Beijing Solarbio Science & Technology Co., Ltd.). A coverslip was then placed over the slides, and nail polish was applied around the edges for fixation. The tissue slides were then observed under a fluorescence microscope. The results are as follows: Figure 2 As shown.

[0026] Experimental results Figure 2 EGCG has been shown to effectively reduce the expression of scar markers, inhibit scar formation, and promote spinal cord injury repair. Example 3

[0027] Rats were divided into groups and weighed. The first group, the SCI group (control group), was anesthetized by intraperitoneal injection, and the hair on their backs was shaved with an electric shaver. The rats were fixed in a prone position on the operating table, and the skin of the surgical area was disinfected with alcohol. The skin at the T10 segment was longitudinally incised with a scalpel, and the mucosa was bluntly dissected with hemostats to expose the surgical field. Longitudinal incisions were made on both sides of the spinous process with a scalpel, and excess tissue was removed with ophthalmic scissors. Intraoperative bleeding was cleared with cotton swabs. After exposing the lamina of the T9-T10 segment, the T9 spinous process was clamped with a hemostat in the left hand, and the T10 lamina was crushed with a bone forceps in the right hand to expose the spinal cord. A transverse incision was made against the transverse process with a scalpel to complete the spinal cord hemisection injury model.

[0028] The second group, the SCI+EGCG group (experimental group), received an intraperitoneal injection of EGCG solution at a dose of 25 mg / kg / day after model establishment. The third group, the Sham group (sham surgery group), underwent only laminectomy without damaging the spinal cord; similarly, the same volume of physiological saline was injected into the intraperitoneal cavity of the rats after model establishment. Postoperatively, all groups received a small amount of penicillin intramuscularly to prevent infection, and the incisions were sutured layer by layer, with blood wiped away. Artificial assisted urination was performed one week postoperatively.

[0029] Finally, 7 days after modeling, spinal cord tissue was harvested from the injury sites of the three groups of rats. Trizol lysis buffer (purchased from Thermo Fisher Scientific (China) Co., Ltd.) and grinding beads were added, and the tissue was ground in a grinder to extract RNA using the Trizol method. The extracted tissue RNA was reverse transcribed into cDNA using a PCR instrument and a reverse transcription kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The cDNA was then subjected to real-time quantitative PCR using a qPCR instrument and a qPCR kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The results are as follows: Figure 3 As shown.

[0030] Experimental results Figure 3 The study confirmed that in a spinal cord injury model treated with epigallocatechin gallate (EGCG), the expression levels of α-smooth muscle actin (α-SMA), type 1 collagen α1 (COL1A1), Snail protein, and transforming growth factor-β1 (TGF-β1) in rats were all downregulated, indicating a downregulation of fibrosis indicators. This further suggests that EGCG has the function of inhibiting scar formation after spinal cord injury and can promote spinal cord repair. Example 4

[0031] To investigate the effect of epigallocatechin gallate (EGCG) on the survival rate of nerve cells after oxidative stress and to understand the role of different concentrations of EGCG in oxidative stress after spinal cord injury, the following experiments were conducted in this application: ND7 / 23 is a rat neuroblastoma cell line capable of being passaged. It was cultured in DMEM high-glucose medium (purchased from Beijing Solarbio Science & Technology Co., Ltd., containing 10% fetal bovine serum (purchased from Ecosai Biotechnology (Taicang) Co., Ltd.) and 1% penicillin and streptomycin, with antibiotics included. After reaching optimal cell condition (i.e., few dead cells and clearly visible star-shaped cells), the cells were divided into 32 wells, with 1*10 cells seeded per well. 4 indivual.

[0032] After 24 hours, the culture medium in each well was aspirated, washed with PBS solution, and then 200 μL of complete culture medium containing 100 μM H2O2 was added to each well and incubated for 2 hours. After incubation, the culture medium in each well was aspirated again, washed with PBS solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.), and then 200 μL of complete culture medium containing gradient concentrations of EGCG was added to each well; the gradient concentrations of EGCG were 0 mg / mL, 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL. The wells were incubated for 24 hours.

[0033] After 24 hours, the original culture medium was aspirated, and the cells were washed with PBS solution. Then, 200 μL of PBS solution and 10 μL of cell counting reagent were added to each well. In this example, the cell counting reagent used was CCK-8 reagent purchased from MedChemExpress. The cells were incubated for 2 hours. After incubation, the absorbance of each well at 450 nm was measured using a microplate reader. The results are as follows: Figure 4 As shown.

[0034] Experimental results Figure 4 It has been confirmed that epigallocatechin gallate (EGCG) at concentrations of 0.001–0.01 mg / ml can improve the survival rate of spinal cord nerve cells after oxidative stress, with the highest survival rate observed at a concentration of 0.005 mg / ml. This means that epigallocatechin gallate (EGCG) at concentrations of 0.001–0.01 mg / ml can significantly enhance the antioxidant capacity of spinal cord nerve cells. Example 5

[0035] Six compositions with different proportions were prepared using the scheme provided by this invention: Each composition comprises a first reagent prepared from an aqueous solution of methacrylamide gelatin (GelMA), a second reagent prepared from an aqueous solution of a photoinitiator, and a third reagent prepared from an aqueous solution of epigallocatechin gallate. Sodium hyaluronate was added to the first reagent, and the first, second, and third reagents were mixed in a silicone bag at a mass ratio of 2:1:1. The prepared composition is a hydrogel, and it becomes effective after exposure to ultraviolet light. The photoinitiator is preferably Irgacure 2959. Methacrylamide gelatin (GelMA) is prepared by mixing gelatin solution with methacrylic acid, followed by a crosslinking reaction with a crosslinking degree of 68%, and then dialysis to obtain GelMA hydrogel. The component proportions of each composition are shown in Table 1.

[0036] Rats were divided into 8 groups of 3 each and weighed. One group was the SCI group, i.e., the control group. Rats were anesthetized by intraperitoneal injection, and the hair on their backs was shaved with an electric shaver. The rats were fixed in a prone position on the operating table, and the skin of the surgical area was disinfected with alcohol. The skin at the T10 segment was longitudinally incised with a scalpel, and the mucosa was bluntly dissected with hemostatic forceps to expose the surgical field. Longitudinal incisions were made on both sides of the spinous process with a scalpel, and excess tissue was removed with ophthalmic scissors. Intraoperative bleeding was cleared with cotton swabs. After exposing the lamina of the T9-T10 segment, the T9 spinous process was clamped with a hemostat in the left hand, and the T10 lamina was crushed with bone forceps in the right hand to expose the spinal cord. A transverse incision was made with a scalpel against the transverse process to complete the spinal cord hemisection injury model. Afterward, 0.5 ml of physiological saline was injected into the rat's peritoneum. Groups two through seven were the SCI+ hydrogel composition groups, i.e., the experimental groups. After establishing the spinal cord hemisection injury model, 0.5 ml of the corresponding hydrogel composition was injected into the spinal cord injury site, and cured under ultraviolet light for 5 minutes. Group eight was the Sham group, i.e., the sham surgery group, in which only laminectomy was performed without damaging the spinal cord. After the laminectomy, the same volume of physiological saline was injected into the peritoneal cavity of the rats. The composition ratio of the therapeutic agents in each experimental group is shown in Table 1: Table 1. Composition of the therapeutic agents in the first to eighth experimental groups.

[0037] All eight groups of rats received an intramuscular injection of a small amount of penicillin postoperatively to prevent infection. The incisions were sutured layer by layer, and bloodstains were wiped away. Artificial assisted urination was performed one week postoperatively.

[0038] Five days after modeling, spinal cord tissue RNA was extracted from the injury sites of rats in each group using the Trizol method. The extracted tissue RNA was reverse transcribed into cDNA using a PCR instrument and a reverse transcription kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The cDNA was then subjected to real-time quantitative PCR using a qPCR instrument and a qPCR kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The results are as follows: Figure 5 As shown.

[0039] Figure 5 In the figure, the vertical axis represents the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after adjustment compared to the control group, and the horizontal axis represents the experimental group. The experimental results show that the hydrogel composition of GelMA-sodium hyaluronate loaded with 0.004% EGCG can effectively increase the expression level of superoxide dismutase 1 (SOD1). The fifth group of data shows the SOD1 expression level of rats loaded with GelMA-sodium hyaluronate hydrogel composition with 0.04% EGCG. The SOD1 expression levels of each rat in this group are 1.1 times, 1.2 times, and 1.45 times that of the control group, respectively. Therefore, the hydrogel containing 0.04% EGCG of GelMA-sodium hyaluronate... While the EGCG-based hydrogel composition can increase SOD1 expression, some data points are already approaching those of the control group. Furthermore, due to significant differences between groups, there is no statistically significant difference compared to the control group. Additionally, further increases in EGCG concentration have shown obvious toxic effects; specifically, higher concentrations of EGCG worsen SOD1 expression, hindering spinal cord repair. Therefore, further increases in EGCG concentration will be detrimental to spinal cord injury repair. The GelMA-sodium hyaluronate hydrogel composition loaded with 0.001% EGCG did not statistically increase SOD1 expression. The GelMA-sodium hyaluronate hydrogel composition loaded with 0.08% EGCG has shown toxic effects. In conclusion, the preferred mass concentration of epigallocatechin gallate (EGCG) is 0.004–0.04%.

[0040] In addition, the present invention found that the hydrogel composition without sodium hyaluronate has poorer elasticity than the hydrogel composition with sodium hyaluronate, which is not conducive to the elongation of spinal nerve axons. Example 6

[0041] The present invention provides a hydrogel-like composition: the composition comprises a first reagent containing methacrylamide gelatin (GelMA), a second reagent containing a photoinitiator, and a third reagent containing epigallocatechin gallate; the first, second, and third reagents are all in liquid form; the solvent for the first, second, and third reagents is phosphate buffered saline solution (hereinafter referred to as PBS solution); sodium hyaluronate is added to the first reagent, and the first, second, and third reagents are mixed in a silicone bag at a mass ratio of 2:1:1; the photoinitiator is preferably Irgacure 2959. In this embodiment, the concentration of EGCG in the third reagent is 0.02%, and a final solution containing 5% methacrylamide gelatin (GelMA), 0.2% sodium hyaluronate, 0.5% photoinitiator, and 0.005% EGCG is finally prepared, and the composition becomes effective after ultraviolet light irradiation.

[0042] Rats were divided into three groups and weighed. The first group was the control group, i.e., the SCI group. Rats were anesthetized by intraperitoneal injection, and their back hair was shaved with an electric shaver. The rats were fixed in a prone position on the operating table, and the skin of the surgical area was disinfected with alcohol. The skin at the T10 segment was longitudinally incised with a scalpel, and the mucosa was bluntly dissected with hemostatic forceps to expose the surgical field. Longitudinal incisions were made on both sides of the spinous process with a scalpel, and excess tissue was removed with ophthalmic scissors. Intraoperative bleeding was cleared with cotton swabs. After exposing the lamina of the T9-T10 segment, the T9 spinous process was clamped with a hemostat in the left hand, and the T10 lamina was crushed with a bone forceps in the right hand to expose the spinal cord. A transverse incision was made with a scalpel against the transverse process to complete the spinal cord hemisection injury model, and 0.5 ml of physiological saline was injected into the injury site. The second group was the experimental group, i.e., the SCI + hydrogel composition group. After the spinal cord hemisection injury model was completed, 0.5 ml of the prepared hydrogel composition was injected into the spinal cord injury site, and it was cured under ultraviolet light for 5 minutes. The third group was the sham surgery group, also known as the Sham group, which involved only laminectomy without damaging the spinal cord, followed by direct suturing after the resection.

[0043] All three groups of rats received an intramuscular injection of a small amount of penicillin postoperatively to prevent infection. The incisions were sutured layer by layer, and blood was wiped away. Artificial assisted urination was performed one week postoperatively.

[0044] Fourteen days after modeling, spinal cord samples were taken from the injury sites of rats in each group. RIPA lysis buffer (purchased from Thermo Fisher Scientific (China) Co., Ltd.), protease inhibitor (purchased from MedChemExpress), and grinding beads were added, and the samples were ground in a grinder. After centrifugation at 12,000 rpm, the filtrate was collected, and total tissue protein was collected and its concentration was determined. Tissue proteins were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel (purchased from Beijing Solarbio Science & Technology Co., Ltd.), with 10 μg of protein loaded into each well. Electrophoresis was then performed at a constant voltage of 120 V for 120 min. The separated proteins were then transferred to a PVDF membrane (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and electrophoresed at a constant current of 200 mA for 120 min. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4°C with different antibodies, including anti-brain-derived nerve growth factor antibody (BDNF antibody), anti-matrix metalloproteinase 3 antibody (MMP-3 antibody), and anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH antibody). All three antibodies were purchased from Abogen (Shanghai) Trading Co., Ltd., and were diluted 1:1000. After incubation, the PVDF membrane was washed three times with TBST buffer (purchased from Beijing Solarbio Science & Technology Co., Ltd.), 10 min each time. After washing, it was incubated at room temperature for 2 hours with goat anti-mouse IgG secondary antibody or goat anti-rabbit IgG secondary antibody (both purchased from Abogen (Shanghai) Trading Co., Ltd.), diluted 1:5000. After incubation, it was washed three times with TBST buffer, 10 min each time. After washing, it was treated with a chemiluminescence kit and exposed under a chemiluminescence analyzer. The results are as follows. Figure 6 As shown.

[0045] Experimental results demonstrate that the hydrogel composition can increase the expression of brain-derived nerve growth factor (BDNF) and decrease the expression of matrix metalloproteinase 3 (MMP-3), thereby inhibiting oxidative stress, suppressing scar formation, and exerting neuroprotective effects to promote spinal cord injury repair. Rats treated with the hydrogel composition of this invention showed better spinal cord injury repair, improved the repair speed of spinal nerve injury, and facilitated the rapid recovery of partial spinal cord motor function. Example 7

[0046] Elastic properties and cyclic fatigue properties of the hydrogel prepared using the method of the present invention were tested: According to the scheme of the present invention, the composition was prepared by dissolving methacrylamide gelatin (GelMA), EGCG, and sodium hyaluronate (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) in PBS solution containing 0.5% I2959 photoinitiator, and finally preparing a final solution containing 5% methacrylamide gelatin (GelMA), 0.2% sodium hyaluronate, and 0.005% EGCG. The solution was then irradiated under 365nm ultraviolet light for 5min to prepare two rectangular hydrogels with a length of 2cm, a width of 2cm, and a height of 1cm.

[0047] One portion of the prepared hydrogel was placed on the clamps of a tensile testing machine, with a load unit of 50 N and a constant head speed of 10 mm / min, until the methacrylamide gelatin (GelMA) was crushed. The experimental results are as follows: Figure 7 As shown.

[0048] The second hydrogel was placed on the clamps of a tensile testing machine. The machine head speed was 600 mm / min, and unloading began when the compression reached 5 mm. This was repeated 10 times. The results are as follows: Figure 8 As shown.

[0049] Experimental results Figure 7 The hydrogel composition prepared using the scheme of the present invention has an elastic modulus of 72.4-93.8 kPa, which is similar to that of nerve tissue. It can simulate normal nerve tissue, which is beneficial for axon elongation and promotes repair and functional recovery after spinal cord injury.

[0050] Experimental results Figure 8 The hydrogel composition prepared using the method of this invention demonstrates that it maintains its original elastic properties after 10 compression-unloading tests, exhibiting excellent fatigue resistance. It maintains the material's elastic properties under repeated pressure. Through the rational proportioning of each component, the hydrogel composition can simulate normal nerve tissue and maintain good elastic properties for a long period, thereby providing stable support for axonal regeneration of spinal nerves and promoting their elongation.

[0051] The preferred embodiments of the present invention have been described above with reference to the accompanying drawings. Those skilled in the art can implement the present invention in various modifications without departing from its scope and spirit. For example, a feature shown or described in one embodiment can be used in another embodiment to obtain yet another embodiment. The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. All equivalent changes made based on the description and drawings of the present invention are included within the scope of the present invention.

Claims

1. The use of a composition containing epigallocatechin gallate in the preparation of a spinal cord injury drug, characterized in that, A hydrogel composition is prepared from epigallocatechin gallate. The composition comprises a first reagent containing methacrylamide gelatin, a second reagent containing a photoinitiator, a third reagent containing epigallocatechin gallate, and sodium hyaluronate. The first, second, and third reagents are all in liquid form. The first, second, and third reagents are mixed in a mass ratio of 2:1:

1. The composition is effective upon exposure to ultraviolet light. The concentration of epigallocatechin gallate in the third reagent is 0.004-0.04%, and the concentration of methacrylamide gelatin in the first reagent is 9-11%.

2. The use of the composition containing epigallocatechin gallate according to claim 1 in the preparation of a spinal cord injury drug, characterized in that, The solvents for the first reagent, the second reagent, and the third reagent are all water or phosphate buffer solutions.

3. The use of the composition containing epigallocatechin gallate according to claim 2 in the preparation of a spinal cord injury drug, characterized in that, The sodium hyaluronate is mixed in the first reagent.

4. The use of the composition containing epigallocatechin gallate according to claim 3 in the preparation of a spinal cord injury drug, characterized in that, The concentration of sodium hyaluronate in the first reagent is 0.2-0.6%.

5. The use of the composition containing epigallocatechin gallate according to claim 1 in the preparation of a spinal cord injury drug, characterized in that, The concentration of the photoinitiator in the second reagent is 1.6-2.4%.

6. The use of the composition containing epigallocatechin gallate according to claim 1 in the preparation of a spinal cord injury drug, characterized in that, The elastic modulus of the composition is 72.4~93.8 kPa.

7. The use of the composition containing epigallocatechin gallate according to claim 1 in the preparation of a spinal cord injury drug, characterized in that, The degree of crosslinking of the methacrylamide gelatin is 68%; and / or the UV irradiation time is ≥5 minutes.

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